CN101893523A - Methods and compositions for analyte detection - Google Patents

Abstract

The present invention relates to methods and devices for detecting one or more analytes. Analytes include infectious agents such as pathogenic viruses as well as reagents or components of enzymes, proteins and biomarkers.

Description

Methods and compositions for analyte detection

technical field

The present invention relates to assays for analytes (eg, antigens) in samples (eg, biological samples obtained from animals). In particular, the present invention relates to methods and devices for detecting analytes using binding moieties that specifically target selected analytes. More specifically, the analytes to be detected include infectious agents and/or their components.

Background technique

A variety of assays have been used to detect the presence of various substances, commonly referred to as analytes or ligands, in bodily samples. Typically, these assays involve antigen-antibody reactions, ligands, anti-ligands, ligand-receptors, and utilize radioactive metal-soluble labels, metalloenzyme-soluble labels, fluorescent metal-soluble labels, or visually detectable metal-soluble labels Synthetic conjugates and specially designed reaction chambers. Most current tests are designed to make a quantitative determination, but in many cases, a qualitative or positive/negative indication is desired. Assays have been utilized to detect infectious agents, such as influenza.

Since the concentration of the analyte of interest in the test fluid is usually low, even a positive/negative assay must be very sensitive. False positives can also be troublesome, especially with agglutination or other rapid detection methods (eg, dipstick and color change tests). Because of these problems, sandwich assays and other sensitive detection methods using metal sols or other types of colored particles have been developed. These techniques have not solved all the problems encountered in these rapid detection methods. Furthermore, in the presence of highly pathogenic pathogens (e.g., influenza virus), there is a need to develop efficient laboratory or point-of-care methods and systems that can efficiently and accurately detect one or Multiple infectious agents, such as influenza types or strains within subtypes.

Influenza is often seen in localized outbreaks or epidemics throughout the world. Epidemics can emerge at any time and can occur explosively with little or no warning. The number of people infected can vary from a few hundred to hundreds of thousands. Epidemics can be short-lived, lasting days or weeks, but most last for months. While epidemics are mild illnesses for most people, they can be life-threatening for the elderly, the very young, or the infirm. Epidemics lead to huge losses in productivity. Therefore, it is necessary to develop a device and method for effectively detecting the type and subtype of an existing pathogen (pathogen) (for example, influenza) in a sample obtained from a subject, thereby judging whether the infection is caused by influenza (seasonal influenza) typical or predicted subtype of the pathogen or as a subtype of an epidemic or national epidemic pathogen.

The object of the present invention is to provide a rapid and sensitive method for detecting analytes in biological samples. Another object is to provide an assay which has high sensitivity and has fewer false positives than conventional assays. Yet another object is to provide a device and system for detecting low levels of analytes present in biological samples. A further object is to provide an assay system which involves a minimum number of procedural steps and still produces reliable results even if the assay system is used by untrained persons. Yet another object is to provide a system for testing for infectious agents that provides results identifying one or more infectious agents within minutes. Yet another object is a system in which the results of the test ware are specific and sensitive to the target analyte although the results cannot be read from 1 hour to several hours after the reaction required to obtain the result is complete of. These and other objects and features of the present invention will be apparent from the following description, drawings and claims.

Contents of the invention

In a particular aspect of the invention, a sample collection device (SCD) is provided for detecting one or more target antigens or analytes that may be present in a sample. A sample collection device may be used in conjunction with a test device. In one aspect, a system for detecting one or more analytes is provided, the system comprising a SCD, a test device, and a reader, as further described herein.

In various embodiments, the SCD includes one or more upper sealed chambers, which may contain the same or different solutions. In one embodiment, the upper sealed chamber comprises at least two compartments or both comprise sub-chambers. In other embodiments, the upper chamber may comprise a pierceable, rupturable or destructible ampoule. In some embodiments, the SCD provides the necessary reagents to form a complex with one or more different target antigens that may be present in the sample, wherein the complex includes a capture moiety and a detectable label ; The test device provides the necessary tools to addressably capture one or more complexes formed thereby; the reader provides tools to detect one or more from the addressably captured complexes Signal.

In various embodiments, the upper sealed chamber includes extraction buffers and/or reagents, a sample collection device, a sample collection device holder, and a plurality of reagents, wherein the reagents include a plurality of specifically bound pairs, wherein , each pair comprising a label conjugated to a first specific binding agent and a capture moiety conjugated to a second specific binding agent, wherein the first specific binding agent and the second specific binding agent specifically bind the target antigen to form a complex . The capture moiety can be "captured" or immobilized, wherein a partner capture moiety disposed on a substrate binds to the capture moiety-specific binding agent conjugate.

In various embodiments, the plurality of specific binding agents includes sets of specific binding pairs, wherein each set includes specific binding agents that specifically bind one antigen of interest and a second set of specific binding pairs specifically binds a second, different antigen. Thus, multiple specific binding pairs contained in the SCD enable the detection of multiple different antigens.

In various embodiments, the capture moiety is an oligonucleotide, avidin, streptavidin, pyranosyl (pRNA), aptamer, or combinations thereof. In various embodiments, labels are metals, fluorophores, chromophores, or combinations thereof. In some embodiments, multiple specific binding pairs included in the SCD may include one type of capture moiety without a different capture moiety partner, e.g., each specific binding agent conjugated to pRNA, wherein, for a different Each group of antigens includes a different pRNA. In other embodiments, multiple specific binding pairs include one or more different capture moieties, for example, pRNA for one set of specific binding pairs and streptavidin for another set, or different types of capture moieties. combination of parts.

In some embodiments, the plurality of specific binding pairs contained in the SCD can include one type of label (e.g., specific binding pairs wherein each group is conjugated to a fluorophore with the same wavelength signal or a fluorophore with a different wavelength signal). group). In other embodiments, a particular binding pair may comprise a combination of different types of labels (eg, a combination of a metal and a fluorophore). In one embodiment, the capture moiety is pRNA and the label is europium.

In various embodiments, the specific binding agent is an antibody, so the specific binding pair comprises an antibody-labeled binding agent ("label probe") or an antibody-capture moiety ("capture probe"). In such embodiments, a "partner capture moiety" is contained on a test membrane disposed in a test device, the partner binds to a specific capture probe, e.g., a pRNA partner specifically binds to a capture probe. pRNA (i.e. capture moiety) on (antibody against target antigen).

In some embodiments, the different analytes detected are viruses or components of viruses (eg, polypeptides). In various embodiments, the different antigens are from influenza viruses and subtypes of influenza viruses. In one embodiment, the influenza viruses that can be detected are influenza A virus and influenza B virus and subtypes of influenza virus. One embodiment involves detection of influenza A strains and influenza B strains and subtypes of the form HxNy, where x can be 1 to 16 and y can be 1 to 9, or any combination of xy.

In yet another embodiment, the different analytes detected are one or more different infectious agents and/or different subtypes of one or more infectious agents. Such infectious agents include HIV, HCV and myobacterium, tuberculosis, bacteria, fungi, yeast, HSV, HPV or combinations thereof.

In various embodiments, the SCD includes a sampling implement that provides means for collecting a sample from a subject, wherein the included sampling implement is connected to the upper chamber via a sampling implement holder. The sampling implement is disposed at the distal end of a shaft, which may be solid, hollow or semi-permeable. In some embodiments, the sampling implement is a swab, comb, brush, scraper, stick, foam, wadding base, or rotating base.

In various embodiments, the SCD includes one or more sealed upper chambers, wherein the seal acts as a valve to control fluid communication between the upper and lower chambers of the SCD. In some embodiments, the valve may be a rupture valve, flapper valve, twist valve, screw valve, breakable valve, pierceable valve, or destructible valve. In other embodiments, the upper chamber may include one or more ampoules that prevent the solution contained therein from flowing into the lower chamber unless pressure is applied to rupture, break or break the ampoule thereby releasing any components contained therein.

One aspect of the present invention relates to an SCD comprising a reservoir upstream of the piston means, a plurality of sealable holes for delivering one or more solutions, for filtering one or more compounds from the sample to thereby A substrate facilitating storage of a sample and a reagent capable of specifically binding at least one analyte in said sample.

In another aspect of the present invention, there is provided a test device for detecting one or more analytes, wherein the device comprises a lateral flow membrane (lateral flow membrane) in the body, the upstream of the lateral flow membrane The chamber contains a fluid or solution, wherein a gap is provided between the chamber and the lateral flow membrane, thereby preventing fluid communication between the chamber and the lateral flow membrane. In one embodiment, said applied pressure pushes said gap tight, thereby establishing fluid communication between said chamber and a lateral flow membrane. In one embodiment, the opening to which the tip of the SCD fits is positioned directly over the pad of wicking material downstream of the gap and upstream of the lateral flow membrane.

In one embodiment, the test device chamber comprises one or more sub-chambers containing the same or different solutions. In other embodiments, the chambers of the subchambers comprise one or more ampoules that are rupturable, breakable or destructible. Thus, when pressure is applied to such ampoules, the ingredients are released in a controlled manner. As described herein, the test device may or may not include gap means for interrupting fluid communication between the chamber and the lateral flow membrane. The test gap may be formed to be zero to 3.0mm, 0.5mm to 3.5mm, 1.0mm to 2.5mm, 1.0mm to 3.0mm, or 2.0mm to 4.0mm.

In some embodiments, a test device can include a body housing a lateral flow membrane, wherein the body provides one or more windows through which the lateral flow membrane can be viewed. In various embodiments described herein, the test device includes a lateral flow membrane comprising a wicking substrate and an absorbent substrate disposed upstream or downstream of a test area on the lateral flow membrane. In some embodiments, a substrate for collecting small volume samples for archival purposes is provided in the SCD or test device. In one embodiment, the substrate providing such an archiving means is a filter, membrane or paper from which a small volume sample is collected, which is later removed from the device.

In various embodiments, the SCD and/or the test device include one or more identical identification tags that can be removed from one device and placed on the other device.

In some embodiments, the test device is formed to fit only (the shape of the specific adapter) the receiving port of the reader, if the upstream chamber is depressed, indicating that the wash buffer contained therein solution or tracking buffer has been released through the lateral flow membrane. In such an embodiment, the ligand-specific test device and reader provide a means to verify that the trace buffer or solution in the chamber upstream of the test device has been released so that any sample that has been washed passes through the lateral flow membrane. upstream of the lateral flow membrane. Thus, specific ligands provide a "safety tool" to prevent reading unprocessed samples.

In another aspect of the invention, the treated sample is run through the lateral flow membrane of the test device and can be left for 30 minutes to several hours. In various embodiments, multiple samples are run through the test device at 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, Samples were read after 11 or 12 hours to obtain a continuous and accurate signal.

In particular aspects of the invention, devices disclosed herein are used in methods of detecting one or more analytes that may be present in a sample. In some embodiments, the methods involve detecting one or more strains of an infectious agent. In one embodiment, a method involves detection of one or more influenza viruses and subtypes thereof using a device of the invention. For example, methods are provided for detecting influenza A and influenza B, as well as subtypes of influenza A that may be present in a single sample.

In one embodiment, a method is provided for determining whether a subject is infected with a pandemic, non-pandemic influenza, or a vaccine-available strain of influenza virus.

In some embodiments, the test device excludes any reagent or binding agent capable of specifically binding the target antigen.

In one aspect of the present invention, a reader is provided to detect a signal from a test device, wherein the reader is a UV LED reader. In various embodiments, the detected signal is a fluorescent signal from a detector molecule. In another embodiment, the detector molecules are lanthanides. In yet another embodiment, the lanthanide is europium.

In one embodiment, the reader includes a UV photodiode. In yet another embodiment, the reader includes a UV laser diode.

In another aspect of the invention, a reader is configured to include at least one hard standard. In another embodiment, the reader is configured to include at least two or more rigid standards. In various embodiments, the hard standard includes a label molecule that emits a detectable signal. In yet another embodiment, the label is a fluorescent label. In yet another embodiment, the fluorescent label is a lanthanide. In yet another embodiment, the lanthanide is europium.

In another aspect of the invention, the SCDs and test devices of the invention are used in methods of detecting one or more analytes associated with a disease, disease state, or other physiological condition. In various embodiments, the analyte is a biomarker associated with a condition involving the heart, liver, kidney, gut, brain, fetus or pancreas. In one embodiment, the analyte is associated with a cardiac condition (eg, myocardial infarction).

In various embodiments, the devices of the present invention can be used in any method for the detection of antigens or proteins (analytes) in a sample obtained from a subject by using immunoreactive specific plates or specific plates for the desired analyte. Specific binding reagents are used to detect any such analytes.

In aspects of the invention, the test device includes an upstream chamber including means for providing a wash/run buffer or liquid. In various embodiments, such buffers or liquids include additives such as signal/detector molecules that can be read by an optical reader or by the naked eye. In particular embodiments, the buffer or liquid is contained in a compartment consisting of a glass ampule or membrane bag, sac, or forms a filled sac. In yet another embodiment, such compartments are disrupted, broken up, or otherwise released by applying pressure to said compartments to release their constituents. In other embodiments, such a compartment is pierced or cut by an appendage or needle. In yet another embodiment, such compartments are protected by safety means against accidental or inadvertent release of their components.

Included by reference

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Description of drawings

The novel features of the invention are set forth with particularity in the claims. The features and advantages of the present invention will be better understood by referring to the following detailed description, which sets forth exemplary embodiments, in which the principles of the invention are utilized, in which:

Figure 1 shows the sample collection device.

Figure 2A shows a sample acquisition device with an enlarged view of the mixing chamber.

Figure 2B shows the sampling assembly.

Figure 3 shows the test setup.

Figure 4 shows the reader.

Figure 5 shows a sample collection device.

Figure 6 shows a sample collection device.

Figure 7 shows a test setup including a gap tool.

Figure 8 shows the sample collection device and testing device.

Figures 9A and 9B show test strips tested with different volumes of extraction buffer.

Figures 10A and 10B show the effect of background clean-up and different amounts of extraction buffer.

Figure 11 shows two different tests for detecting influenza A.

Figure 12 shows two different tests for detecting influenza A and influenza B.

Figure 13 shows the detection of influenza A using gold as a label.

Figure 14 shows the detection of influenza B using gold as a label.

Figure 15 shows the detection of influenza A using europium conjugates.

Figure 16 shows the detection of influenza B using europium conjugates.

Figure 17 shows the sensitivity of europium conjugates in the detection of influenza B.

Figure 18 shows bars tested with influenza A subtypes.

Figure 19 shows a buffer only control.

Figure 20 shows testing for influenza A and subtype H3N2.

Figure 21 shows testing for influenza B.

Figure 22 shows testing for Influenza A and subtype H5N1.

Figure 23 shows testing for Influenza A and subtype H1N1.

Figure 24 shows testing for influenza B and influenza A subtype H5N1.

Figure 25 shows the test for Epstein-Barr virus.

Figure 26 shows a test device comprising multiple test lines for different types and subtypes of analytes; 901 - filter or sample pad applied to the sample; 902 - reagent pad; 903 - test line for type 1 of analyte/infectious agent; 904 - type 2 of analyte/infectious agent; 905 - type 1 or subtype of type 2; 906 - subtype of type 1 or type 2; 907 - Control line.

Figures 27A to 27C illustrate various aspects of a test device; Figure 27A assembled test device 1001; Figure 27B unassembled test device with upper 1002, lower 1004 and test strips 1003 and wells/cabins for holding samples 1005 (aperture/port to receive a sample 1005); Figure 27C is a closer view of the spring/button chamber, the spring/button chamber can be pressed to rupture the buffer/liquid chamber arranged above the membrane/pad 1006 (rupture ), break (break) or puncture (puncture), the buffer/liquid chamber is in fluid communication with the test strip 1003.

Fig. 28A has shown the embodiment that SCD 1101 and test device 1105 components are connected, in this embodiment, provided ring lock (collar lock), when ring lock 1104 opens (turned in the lock position) ( That is, the ring lock 1104 provides a geometrically open channel/hole 1103 to the upstream chamber 1102 when allowing fluid flow from the upstream buffer/fluid chamber across the test device). Upstream chamber 1102 may be an ampoule or a shaped fill bladder.

Figure 28B shows an embodiment of a test device, wherein the device includes a safeguard cover 1107 over an upstream chamber 1106 containing a liquid (eg, wash/running buffer). Additionally, the device includes a reservoir 1109 in fluid communication with the sample well/chamber 1108, which may include a membrane or chamber for holding an archived sample.

Figure 28C shows a chamber (e.g., ampoule) 1111 with a break point 1110 disposed in a test device, wherein the test device is configured to provide a trough/channel 1112 and a support base 1113, the support base 1113 directs the liquid contents of the ampoule downstream to the test strip; FIG. 28D provides a top view of a chamber comprising a capsule/ampule 1114 downstream of the location 1115 where sample introduction occurs, the chamber configured to provide a liquid ramp (ramp) 1119 and carrier (support) 1118, the contents of the chamber pass through the carrier 1118 to the space 1117, and the space 1117 is in liquid communication with the absorbent pad 1116.

Figure 29 provides an example of a sampling device with an archive assembly; a sampling swab 1205 is attached to a hollow shaft 1201, a sleeve 1202 is positioned on the side of the hollow shaft 1201 in a hydrophobic frit ( 10+u) Upstream of 1206 and 1203, below is a filter paper that can take various shapes (Fig. 29B) with a region 1209 for holding archival samples and three three-dimensional regions 1210 that can eg hold reagent pellets/beads. In addition, reagent pellets/beads may also be provided in a chamber downstream of the filter paper 1208 which enables mixing of the sample prior to driving the sample through the tip 1204 .

Figure 30A shows a test strip using pRNA; Figure 30B shows a test strip using a monoclonal antibody.

Figure 31 shows a comparison of the sensitivity using pRNA and antibody, ie, Figure 31 shows comparing the sensitivity of influenza B detection using antibody and pRNA as capture.

Figure 32 provides a schematic representation of pRNA binding of multiple analytes on a test strip.

Figures 33A to 33H show hard standards; Figure 33A shows graphs of six different concentrations of europium read by a UV LED reader; Figure 33B shows six hard standards Figure 33C shows a graph of No. 1 concentration, wherein No. 1 europium concentration is about 2100 counts; Fig. 33D shows a graph of No. 5 concentration, wherein No. 5 europium concentration is about 2280 counts; Fig. 33E Figure 33F shows a graph for concentration No. 6, wherein Europium concentration No. 6 is about 3310 counts; Figure 33G shows concentration No. 2 33H shows a graph of concentration No. 4, wherein the concentration of Europium No. 4 is about 140,000 counts.

Figures 34A and 34B show the hard standards; Figure 34A provides graphs of seven different hard standards read by a UV LED reader; Figure 34B provides a test setup with multiple windows, when the test Signals are detected through the plurality of windows when the device is positioned in the reader.

Figure 35 shows an embodiment where a liquid-containing upstream chamber 1401 (e.g., an ampoule) is in fluid communication with a dry absorbent pad 1402 that expands when the chamber is manipulated to release its liquid contents , wherein the swelling increases the size of the absorbent pad 1403 such that the dry absorbent pad 1402 becomes in fluid communication with the sample 1405 disposed on the test strip 1406 . Thus, based on the density/type of absorbent material 1402, a predetermined time delay can be built into the system from the point of release of the sample 1405 through the test strip 1406 to the point of initiation of flow.

FIG. 36 shows test strips of subtype H5N1 virus, ie, fluorescence intensity of subtype H5N1 virus.

FIG. 37 shows a test strip of subtype H1N1 virus, ie, a graph of fluorescence intensity of subtype H1N1 virus.

FIG. 38 shows a test strip for influenza B virus, ie, a graph of fluorescence intensity of influenza B virus.

Figure 39 shows the sensitivity of the Nanogen Influenza A test using the MAB capture system.

Figure 40 shows the sensitivity of the Nanogen Influenza A test using the pRNA capture system.

Figure 41 shows the sensitivity of the Nanogen Influenza A test and membrane blocking using the pRNA capture system.

Figure 42 provides a comparison of the sensitivity of the Nanogen Influenza A test, ie, MAB, pRNA, and pRNA with blocking.

Detailed ways

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments have been provided by way of example only. Now, many changes, changes and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Various aspects of the invention relate to devices and binding-pair assays for the qualitative and/or quantitative analysis of selected analytes in a sample using specific binding and capture moieties. The invention is useful in a variety of assays for detecting one or more infectious agents present in a sample. Assays used in the present invention include, but are not limited to, competitive immunoassays, non-competitive immunoassays, sandwich immunoassays, and blocking assays.

However, the use of the present invention is not limited to immunoassays, such as other assays involving ligand-receptors, ligand-binding proteins, aptamer-binding agents, enzyme-substrates, by using the device or method of the present invention to detect chemical or biochemical reaction. According to one aspect of the invention, the methods and systems of the invention utilize sample collection devices, testing devices and readers. Various specific assay protocols, reagents, devices or analytes for practicing the present invention are known, see, for example, Nos. US Patent No. 6,448,001 or 5,415,994 and US Publication Nos. 2006/0040405, 2004/0014094, 2004/0048395 or 2005/0130120.

According to one aspect of the invention, a sample collection device is used to collect a sample, and to process the sample with an immunoreactive reagent providing detection means and capture means, the processed sample is then provided to a test device, which provides for reading /A tool to observe the signal provided by the detection tool. The signal can be read visually or with an instrument that relies on the combination of detection means (eg, conjugate label) employed. In addition, test devices can be configured to allow detection of multiple analytes. Such analytes may be from one or more infectious agents, including different strains and/or subtypes. Detection can include qualitative and/or quantitative measurement of one or more analytes.

Sample collection device. One aspect of the present invention relates to a sample collection device (SCD) that includes the necessary tools for collecting a biological sample and means for processing the sample and reacting binding reagents with one or more target analytes. Reagents and buffers required. As shown in FIG. 2B , an exemplary SCD includes an upper chamber 210, a sampling implement holder 209 attached to the upper chamber 210, a stem 208, and a sampling implement 207 assembled to form a sampling assembly ( sampling assembly).

The sampling assembly (FIG. 1) can be removed from a housing that includes a sample receiving tube 103, a lower chamber mixing or reagent area 104 that can accommodate specific binding to one or Reagents for various target antigens. Lower chamber 104 may include one or more chambers. For example, two chambers may be arranged in series in the lower chamber. The sampling assembly can be placed into the sample holding tube 103 of the housing to provide a unitary structure. In such a configuration, the sampling device 107 is located upstream of the lower chamber and is in fluid communication with the lower chamber. For sample collection (eg, throat-nasal sample collection), the length of the sampling assembly can be optimized. For example, the device (eg, unitary structure) can be about 1-9 inches in length, or about 3, 4, 5, 6, 7, 8, or 9 inches in length.

Rod 102 may be hollow, solid or semi-porous. Thus, in some embodiments, the rod actually provides a fluid communication path from the upper chamber 100 to the sampling implement 107 (eg, swab). The rod is held by a sample holder 101 fitted into a receiving end of the upper chamber 100 . For example, the rod may be hollow or semi-permeable, with the portion of the rod that extends into the upper chamber having a closed terminal end, thus providing a passage from the upper chamber down through the rod if the rod portion in the upper chamber breaks or ruptures. Fluid communication (ie, fluid flow) of the tip to a sampling implement (eg, a swab) containing a sample (eg, a biological sample).

The upper chamber may comprise one or more chambers. The upper chamber may be composed of a semi-rigid or compressible material and may be shaped as a bulb 99 . Such spheres can hold solutions.

The upper chamber can be sealed. Additionally, such seals may be pierced, ruptured or opened by a valve arrangement to provide fluid communication between the upper and lower chambers. In some embodiments, the solution in the upper sealed chamber is a buffered solution. In various embodiments, the volume of the solution in the upper chamber may be about 10 μL to about 500 μL, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 ,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380 , 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 μL. In one embodiment, the solution volume is 150 μL.

In some embodiments, a liquid solution containing a desired solvent (eg, a detection/capture specific binding agent, etc.) can be placed in the reagent region of the lower chamber in fluid communication with the upper chamber. As shown in FIG. 2A, liquid flows down from the upper chamber to the sampling device to extract a sample, which passes through a hole that can restrict/control the flow of liquid from the upper chamber to the lower chamber, e.g., the lower chamber Apertures may be included to control flow by size (eg, size of the perforation) or type of substrate or filter disposed on the proximal end 201 of the compartment in the lower chamber. The lower chamber may include a reagent area. In one embodiment, the reagent area (e.g., 203) comprises a solid substrate comprising the necessary reagents 202 (e.g., immunoassay reagents, such as detection and capture probes, etc.), the reagents 202 being formed as dry solid, separate Set solid or all-in-one solid.

Thus, if the sample is flushed down through a solution (eg, buffer) in the upper chamber, a mixture is created that carries the sample down to the reagent region of the lower chamber, which chamber includes the solid reagent 202 containing the substrate. The solid reagent is rapidly dissolved by the buffer, and the resulting solution is a mixture of the sample that may contain the analyte of interest and immunoassay reagents (eg, specific binding agents, label conjugates, capture probes, etc.). For example, solid reagents 202 may include labels and capture probes for use in assays that are capable of specifically binding target analytes. The integrated device 200 includes an upper chamber, a lower chamber, a sample collection device, and a luer lock 206 that locks into the side flow device for transfer of the reaction mixture for subsequent detection. Additionally, as noted above, the upper chamber can be designed with a depressible (eg, plastic) bulb 205, so that additional pressure is applied, if desired, to encourage fluid flow from the upper chamber into the lower chamber.

In one embodiment, the upper chamber includes a valve that controllably releases the solution contained in the upper chamber. For example, where the valve is a snap-valve, the user applies force to the valve stem to break the stem, so the breakaway feature allows buffer to pass through the stem into the lower chamber. Additionally, the sealed chamber may be a squeezable sphere that can be compressed (eg, a user applies pressure to the sphere) to control the flow rate of a solution (eg, buffer) to the sampling implement.

In some embodiments, the upper chamber consists of a spherical body assembly, which is a separate compartment containing a solution. Such solutions include extraction solutions, lysates, reagent solutions, buffers or preservative solutions. In one embodiment, the solution is a buffer used to transport the biological sample from the sampling implement down to the lower chamber.

Furthermore, such an upper chamber may comprise one or more compartments, such as shown in FIG. 5 . Each compartment can contain the same or different solutions 501 and 502 .

In addition, such solutions can include desired reagents, including extraction buffers, reducing agents, immunoreactive reagents (eg, anti-analyte-specific binding agents including detection labels (eg, conjugates) and capture moieties). Reagents for the reaction are shown at 503 and luer attachment to test strip 505 is shown at 504 .

In another embodiment, the sampling assembly is not integrally formed with the housing including the sample containment tube, see FIG. 6 . In such a configuration, sampling assembly 601 is used to collect a sample and transfer the sample to sample holding chamber 609 . The sample holding chamber can be open or closed to introduce the sample into the sample holding tube. It should be understood that any of the sample containment tubes disclosed herein may be of various geometric shapes including cylindrical, square, triangular, or any polygon, as desired. In some embodiments, the housing can include one or more sealable wells 603 that can be opened to add one or more selected reagents 607, buffers or washes.

For example, in one embodiment, whole blood is drawn into the sample holding chamber 609 . The sample then passes through a membrane 602 (eg, the membrane that separates red blood cells from plasma so that the plasma passes through) into the lower portion of the sample-holding tube to mix with various reagents, eg, required for an immunoassay. The immunological reagents required to target a particular analyte may be pre-selected and provided on a solid substrate 607 in the SCD, or added through holes 603 , or may be provided on a membrane 604 .

As the whole blood sample is released, the membrane 604 can act as a filter to prevent passage of blood components, allowing only plasma to pass through the end of the sample holding tube 605, which will be installed in the test device 606 . Other valves available include rotary valves, rupturable valves, stopcock valves, gate valves, ball valves, flapper valves, needle valves, butterfly valves, pinch valves, bellows valves, piston valves, slide valves, plug valves, diverter valves, or Electric valve.

As used herein, "capture probe" refers to a conjugate of a binding agent attached to a capture moiety, and "detection probe" refers to a conjugate of a binding agent attached to a label or signal generating moiety, wherein the capture probe Each of the and detection probes is capable of specifically binding to a target analyte. Furthermore, for clarity, a "capture moiety partner" (as described below) in the context of a test device refers to a complement, homologue or partner molecule that specifically binds to a capture moiety hosted on a capture probe.

Solid reagent components include powders, pellets, beads, lyophilized spheres, lyophilized powders, solid reagent components dried on a solid carrier (e.g., glass/plastic beads), or lyophilized on a solid carrier or combined with Solid supports are conjugated or dried directly in the mixing chamber or lower chamber. Such reagents are known in the art, for example as disclosed in CURRENT PROTOCOLS IN IMMUNOLOGY (Coligan, John E. et. al., eds. 1999).

In some embodiments, the extraction step of the sample occurs as the solution passes through the sample receptacle (eg, where the solution includes an extraction buffer). Additionally, the lower chamber may include a filter through which the extracted sample flows. For example, if a filter is placed at the proximal end of the lower chamber, the extracted sample flows through the filter (e.g., a mesh disk), thereby preventing certain components of the extraction mixture from entering the reagent containing solid reagent beads. In the regional compartment. Additionally, filter tools can also be used to block reagent beads during SCD transport and storage. As described herein, a reagent bead can include a detection and capture probe, or two separate beads can each include a detection or capture probe.

The filtration aspect allows the analyte of interest to migrate through the device in a controlled manner, carrying little, if any, interfering species. Aspects of filtration, if present, generally provide a higher probability of success depending on the type of sample being processed, which will be apparent to those skilled in the art (eg, whole blood samples versus throat swabs). In another embodiment, the SCD can also incorporate reagents to prevent cross-reaction with non-target analytes that may be present in the sample and/or to condition the sample; Examples of these reagents may include non-hCG blockers, anti-RBC reagents, Tris-based buffers, EDTA, etc., but are not limited thereto. Anti-RBC is usually used when whole blood is anticipated. In yet another embodiment, the SCD can incorporate other reagents, such as auxiliary specific binding members, liquid sample pretreatment reagents, and signal generating reagents (eg, substrates required for reaction with the label conjugate).

In another embodiment, the lower chamber may include some absorbent paper on which a predetermined percentage of the extracted sample is kept for archival purposes. After the extracted sample passes through the collection device and defines a portion of the extracted sample for archival purposes, the extracted sample contacts a reagent solution or solid (e.g., conjugate beads 202), while the sample liquid rapidly dissolves the conjugate beads and allows the reactants to mix And start the assay, proceed to the next assay step.

In other embodiments, the luer 105 provided on the end of the SCD may be replaced with or further attached to a valve structure that also serves to control the flow of liquid from the sample acquisition device. A valve may also be utilized in the practice of the invention, the valve being located in the upper seal chamber to provide a means of controlling the release of the solution contained in the upper seal chamber. Whether in the upper chamber, in the lower chamber, or between any compartments provided in the SCD, the valves can be of any type known in the art, such as rotary valves, snap action valves, rupturable valves, stopcock valves , gate valve, ball valve, needle valve, butterfly valve, pinch valve, bellows valve, piston valve, slide valve, plug valve, diverter valve or electric valve, but not limited thereto. In one embodiment, the valve may be a snap-action valve, opening the snap-action valve applies pressure in the stem of the valve extending into the upper seal chamber (100), thereby breaking the hollow stem 102 and allowing the sealed The contents of the upper chamber flow into the hollow tube at the end of which a sampling implement (eg swab 107) is placed.

In one embodiment, there is a valve at the end of the SCD. When the valve is in the closed position, the sample, or sample and reagents, can be held in the lower chamber 104 . When the valve is in the open position, the contents of the mixing subchamber are released, eg by gravity flow. Alternatively, the sample/reagents are released from the luer, luer valve or valve using the bulb assembly of the sample acquisition device. In a preferred embodiment of the invention, the contents from the end or outlet end of the sample collection device (SCD) are released from the SCD so that flow can be induced, regulated or adjusted.

In another embodiment, the ends of the SCD are opened to engage the SCD to the receiving end of the test device (eg, by friction fit) prior to releasing the solution from the upper sealed chamber. In such an embodiment, the flow of liquid from the end of the SCD into the test device is not regulated by the luer or valve structure. In another embodiment, the end of the SCD does not use a valve, but is left open. The SCD can be attached to the test device prior to releasing the buffer from the upper chamber. When the solution is released from the upper chamber, the sample is released and/or extracted from the collection implement by the solution, and the sample mixes with the reagents located in the lower chamber. The mixture then flows to a test device that is analyzed for the presence of one or more analytes. A water soluble membrane may be included in the upper chamber to slow the flow of the mixture from the SCD onto the test device. Such membranes are conventional and can be designed to permit the mixture to remain for varying periods of time sufficient for the reagents and sample analyte to mix and react. For example, such films can be prepared from proteins, polysaccharides or film formers.

In another embodiment, the end of the SCD includes a very narrow opening, allowing fluid to exit the end if no pressure is applied to the device or when pressure is applied to the device (e.g., through the spherical structure of the SCD, or if the housing is depressible (by applying pressure to the housing itself), the opening prevents fluid flow.

According to another aspect of the invention, the liquid conditioning means (eg, luer 105, luer valve combination, and valve) is closed so that the sample or the sample and one or more reagents remain in the lower chamber 104 for any length of time. The valve structure may then be mechanically opened in whole or in part to release the contents, optionally at an adjusted or adjusted rate, through the end or outlet port of the SCD into the test device.

Reagents used in the SCD of the invention may include one or more of salts, chelating agents, anticoagulants, detergents, stabilizers, diluents, buffers, enzymes, coenzyme factors, specific binding members, labels, solubilizing mucus etc. The one or more reagents may be compounds that facilitate analysis of the sample. Furthermore, such reagents can be readily adapted for use in the test devices of the present invention.

In one embodiment, the SCD is attached to a second device (e.g., a test device of the present invention), so that the opening of the valve structure releases the contents into the second device, or, the covering opening is removed (without the valve) cap 106, thereby releasing the contents into the second device. In another embodiment, engaging an SCD comprising a male luer and a test device comprising a female luer results in release of the contents of the reaction subchamber. In yet another embodiment, the contents from the tip can be released through the bulb assembly. If a valve structure is used in an SCD, it can be opened by various means to release the contents, for example, by opening a stopcock or by turning, rotating, twisting or sliding the valve structure (but not limited thereto), or by Removal of the cap opening the fluid flow path allows the valve to be opened allowing fluid to flow into the test platform.

Archive samples. According to an aspect of the invention, means for archiving a portion of a sample are provided. In some embodiments, the SCD or the test device, or both, include an archival means, which may include an absorbent or absorbent substrate (e.g., paper or film), short capillaries of defined length, or small reservoirs/compartments, to keep a portion of the sample in the lower chamber. In some embodiments, this archiving mechanism is located at a location in the device prior to the sample encountering the reagents.

In one embodiment shown in Figure 28, a small chamber 1109 capable of providing a small reservoir for archiving samples is provided in the test device adjacent to a bay/well 1108 for delivering samples to the test device. Such an archiving compartment may be configured to be removable, or the substrate on which the archiving sample is disposed is itself removable from said compartment. For example, a filter/membrane material sized to fit into a compartment will be used to collect a predetermined volume of sample (eg, cells, cellular components, proteins, nucleic acids, etc.). The test device may include a bag 1106 of wash buffer with a housing or lid 1107 .

In another embodiment, the SCD includes means for maintaining archived samples, see Figure 29A. For example, in the SCD lower compartment, filter paper and hydrophobic membranes can be provided to hold samples for archival purposes. Various combinations of materials are possible. In one embodiment, the tool for archiving includes three disks 1203, 1206, 1207, which may or may not be in contact with each other. In another embodiment, the SCD (FIG. 29A) can be configured with a case 1202 that provides a tool for moving the size of the tube/casing closer to the swab 1205 attached to the rod 1201, thus , as the liquid leaves the swab, the liquid will remain very close to the swab, thereby increasing the efficiency of extracting the liquid from the swab. Denote the reagent beads as 1208 and the outlet as 1204.

As shown in Figure 29B, the tray may include a grid portion 1210 and a pad portion 1209, wherein the pad portion is designed to hold archival samples. The pad portion may consist of any absorbent/absorbent material and may comprise 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the surface area of the disc. In addition, the mesh portion may include a three-dimensional (3D) base that is raised relative to the surface of the disc. Such 3D can provide a grid into which reagent beads can be set. Such beads may range in size from about 0.5, 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5.0, 5.5 mm to about 6.0 mm.

In one embodiment, the archived material is cells or cellular components, including proteins, peptides, protein fragments, or nucleic acid molecules. Thus, samples can be archived for further testing based on the type of molecule archived (eg, protein versus nucleic acid). In addition, an archive tray 1207 (such as shown in FIG. 29A ) provides a means of storing samples and keeps said samples from approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 21 days to 30 days stability. For example, RNA is stable for about 2, 3, 4 or 5 days.

In another embodiment, the archiving disc is placed in a preservative solution that prolongs the storage time of the archival sample by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks. Of course, depending on the field setting, samples may be stored indefinitely (eg, the samples are frozen once).

In another embodiment, the reaction compartment in the lower chamber can be removed from the sample-holding tube and the sample-holding tube placed in a housing (eg, a plastic tube). In one embodiment, a compartment (eg, cage) holds a small volume of sample mixture, and a preservative can be added to the sample mixture for storage. In another embodiment, the solution provided in the upper chamber or the reaction solution in the lower chamber may also contain preservatives necessary for archiving the liquid sample. Such preservatives are known in the art. See, eg, US Patent RE29061; Buccholz et al. Transfusion. 1999 Sep;39(9):998-1004; Quiagen specialty reagents, available at Quiagen.com.

In one embodiment, archived samples are kept for later testing (eg, by RT-PCR, see Example 8).

Sample identification. The SCD also includes 3 identical identification marks (eg, a barcode allowing at least 109 unique values) for the patient identification number located anywhere on the sampling implement or sample holding tube (eg, attached to the outer tube 103). Markers are peelable and self-adhesive. Hold a marker on SCD 103. The stripped copy can be placed on the test device (see Figure 3) and placed on any equipment paperwork or archive reservoir. The barcode format will be a common standard such as Codabar 303 or 305. In other embodiments, the identification mark can be a signal transponder known in the art, including a radio frequency transmitter, a light transmitter or an electromagnetic wave transmitter, but is not limited thereto.

A compartment in the upper or lower chamber. According to some aspects of the invention, in the lower chamber, the SCD includes one or more compartments that can house reagents, filters, membranes, or reservoirs. In one embodiment, the upper chamber of the SCD may comprise one, two or more compartments, each compartment may also contain a solution. In some embodiments, such compartments may contain the same or two different solutions, reagents, buffers, or combinations thereof. In addition, multiple compartments can be arranged in series in the lower chamber (eg, multiple cages in series). Additionally, in the disclosure herein, such chambers may be referred to as "sub-compartments".

In one embodiment, the compartment is relative to the end of the sampling implement and contains a liquid or solid reagent component including a binding agent that is specific for a particular analyte (or type of assay). For example, a liquid or solid reagent composition includes a specific binding agent (eg, an antibody) capable of specifically binding an analyte that may be present in the sample. In some embodiments, a single reaction or mixing compartment is utilized in the SCD that is at the end of, and in fluid communication with, the sampling implement. In other embodiments, one or more compartments may be used, wherein one compartment serves as a lysis or extraction compartment and a second compartment at the end of the first compartment serves as a reagent-sample mixing compartment. In other embodiments, the filtering means may be disposed proximally of one or more compartments disposed at the end relative to the sampling implement. Filtering tools can be utilized to remove specific components from a sample prior to extraction/lysing during processing, prior to sample-reagent mixing, or prior to release from the SCD. Furthermore, if multiple compartments are present in the sample holding tube, the same or different filter means may be provided on such multiple compartments.

sample

A sample is any material to be tested for the presence and/or concentration of an analyte. In general, a biological sample can be any sample taken from a subject (eg, a non-human animal or human) and can be used in a test device. For example, a biological sample can be any bodily fluid, cell or tissue sample from a biopsy. Bodily fluids may include, but are not limited to, blood, urine, saliva, semen, feces, saliva, bile, cerebral fluid, nasal fluid, urogenital fluid, nasal aspirate, spinal fluid, and the like. A biological sample can also include any sample derived from a sample taken directly from a subject (eg, a human). For example, a biological sample can be the plasma fraction of a blood sample, serum, protein, or nucleic acid extract from collected cells or tissues, or a biological sample can be derived from a sample that has been treated in a manner that enhances the quality of the sample. Detectability, e.g., the sample is a lysis buffer containing a mucolytic agent that breaks down the mucens in a nasal sample, thereby significantly reducing the viscosity of the sample, and a detergent that lyses the virus , to release the antigen and make the antigen available for assay detection. Samples may be from any host animal including, but not limited to, humans, birds, porcine, equines, bovines, murines, cats, dogs or sheep.

For example, samples may be derived from any source such as physiological fluids, including blood, serum, plasma, saliva or saliva, sputum, eye fluid, nasal fluid, nasopharyngeal fluid or nasopharyngeal swab or aspirate, sweat, urine, Milk, peritoneal fluid, mucus, synovial fluid, peritoneal fluid, transdermal fluid, pharyngeal exudate, bronchoalveolar lavage fluid, tracheal aspirate, cerebrospinal fluid, semen, cervical mucus, vaginal or urethral fluid , amniotic fluid, etc. Here, liquid homogeneous mixtures and gravy of cellular tissues (eg, hair, skin and nail fragments) are also considered biological fluids. Pretreatment may involve preparing plasma from blood, diluting or treating viscous fluids, etc. Treatment methods may involve filtration, distillation, separation, concentration, inactivation and addition of reagents of interfering components. For the performance of industrial, environmental or food production assays as well as diagnostic assays, other samples than physiological fluids can be used, such as water, food, soil extracts, etc. Additionally, solid materials suspected of containing an analyte may be used as test samples when the solid material is modified to form a liquid medium or release the analyte. The selection and pretreatment of biological, industrial and environmental samples prior to testing is well known in the art and requires no further description.

Other areas covered include: diagnosis of livestock disease; analysis of bacterial contamination of meat, poultry, fish; inspection of food factories, restaurants, hospitals or other public facilities; analysis of environmental samples including beach water, sea, lake or swimming pool contamination . Analytes detected by these tests include viral and bacterial antigens as well as chemicals (including, for example, lead, pesticides, hormones, drugs and their metabolites), hydrocarbons and a wide variety of organic or inorganic compounds.

test device

The present disclosure provides a test device, particularly an immunoassay device, for determining the presence or absence of a plurality of analytes in a liquid sample. Generally, test devices of the present disclosure (see FIG. 3 ) include a matrix defining an axial flow path. Typically, the matrix also includes a sample receiving zone, one or more test zones, and one or more control zones. In various embodiments, the test area includes test and control areas, which are overall addressable lines.

Here, the terms "axial flow membrane", "lateral flow membrane", "test membrane", "test strip" or "matrix" are used interchangeably in the context of the test device used, where capillary action is used to move or convey the test liquid, or, by air pressure, hydraulic accumulation (direct pumping of the test liquid using pistons or rotating bellows or other types of pumps, electrostatic movement by electric fields, gravity, etc.) to pump the liquid, thereby moving the liquid without capillary action.

According to an aspect of the invention, the test device as shown in Figure 3 comprises a hole/chamber into which the tip of the SCD of the invention is engaged by a friction fit, luer lock, adapter or valve . Aperture/chamber 302 provides an opening through which the sample flows from the SCD into the test device. A blood separation membrane may be provided in a chamber that provides unidirectional flow. In another embodiment, such a membrane may also be disposed in the SCD (eg, the exact end of the sample swab device).

Upstream of the holes is a compartment 307 which is in fluid communication with the holes which are in fluid communication with the absorbent substrate 309 . Additionally, a compartment may include one or more sub-compartments 308 containing one or more solutions. In the context of a test device, a sub-compartment may be made of a pierceable, perforable, rupturable (eg ampoule) or depressible bladder material. However, in one embodiment, the ring disengages, and when additional force is applied to the buffer/rinse compartment 308, only the buffer is released. As described herein, such compartments may be manipulated by applying sufficient pressure to puncture, rupture or depress the compartment to release its contents. Additionally, due to the application of force to such a compartment (eg, thumb pressing down), a spear, thorn or appendage penetrates into the compartment, thereby piercing the compartment.

In one embodiment, the test device comprises two parts, wherein one part includes a sample application part 302 and a sample application compartment upstream part 308 containing a wash or running buffer. In another embodiment, the upstream portion may comprise one or more (e.g., two) compartments, which may contain the same or different buffers, wherein each compartment may be operated independently Or operate each compartment simultaneously to expel its contents.

In another embodiment, the compartment 307 itself may be semi-rigid, pliable or compressible, or bladder-like, which provides a means for squeezing the compartment to expel any contents therein. Thus, in some embodiments, a user may apply pressure to compartment 307, thereby releasing the contents thereof (whether contained in the compartment or contained in a sub-compartment) into the Absorbent base 309 . Such a solution can be used as a wash or chase buffer to mobilize or enhance the movement of the treated sample mixture into the test strip 310 through the absorbent pad. Typically, such liquid solutions may include wash buffer, saline, or any desired container. Additionally, in some embodiments, such solutions may include reagents, enzymes, labels or compounds. The wash buffer mobilizes any unbound label to migrate across the detection zone along the strip, thereby reducing background.

In addition, the downstream of the test strip 310 is disposed on an adsorption substrate 311 . The test film substrate may overlap or be adjacent to the water-absorbent substrate and the absorbent substrate, respectively, or may overlap or be adjacent to one of the water-absorbent substrate and the absorbent substrate. Additionally, in some embodiments, the upper housing 306 or the lower housing 312 of the test device may include identification marks 303 and 305 that identify and correspond to the same identification marks on the SCD and may also identify the test device batch number (e.g. for quality assurance and tracking purposes). A window 304 through the upper housing permits visualization and reading of the results.

In a related embodiment, the matrix further comprises an absorbent zone disposed downstream of the lateral flow substrate, membrane or matrix. In one embodiment, an absorbent pad may be positioned upstream of the lateral flow membrane. In another embodiment, an absorbent pad is positioned directly below the sample access aperture. Furthermore, in a preferred embodiment, the test area, including the test and control areas, is visible (see Figure 8).

Suitable materials for making absorbent substrate 311 include, but are not limited to, hydrophilic polyethylene materials or pads, acrylic fibers, fiberglass, filter paper or pads, dry paper, pulp, fabric, and the like. For example, the lateral flow film absorbent region may comprise materials such as hydroentangled nonwoven acrylic fibers (i.e., New Merge (available from DuPont) or HDK materials (available from HDK Industries, Inc.)), nonwoven polyethylene , such materials are treated to improve the hydrophobic properties of the material.

security tools. In some embodiments, the safety means 301 is placed on the depressible chamber 307 so that the contents of the chamber cannot accidentally flow out into the channels in fluid communication with the lateral flow membrane. The safety means may be a lid or flange that lifts to expose the depressible chamber or a button provided on the depressible chamber.

Furthermore, such a safety tool can be used as an adapter for a specific homogeneous adapter, luer or valve present on the end of the SCD. Thus, for example, the safety tool may cover the hole into which the end of the SCD engages, before releasing the sample into the test device. In another embodiment, the reader is designed so that the test device can only be inserted into the containment compartment if the security cover is first removed. For example, a test device with the safety cap removed indicates that the sample has been introduced into the test device and the running buffer has been released from compartment 307 , upstream of the well (adapter/safety cap). In one embodiment, holes are provided above absorbent pad 309 .

Clearance tool. In some embodiments, the test device includes a gap disposed between the lateral flow membrane (eg, absorbent pad) and the channel in fluid communication with the buffer reservoir. The gap serves to keep any solution contained in the button reservoir separate from the assay sample until an appropriate time according to the progress of the assay (see Figure 7). In some embodiments, the gap may be approximately 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9 or 10 mm. In one embodiment, the gap is greater than 0 and less than 3 mm. Thus, in case the user applies pressure to the compartment, i.e. upstream of the sample well, the gap shown between 703 and 704 is forced closed and the solution contained in the compartment flows in the direction shown at 701 towards the absorbent pad and through the over the absorbent pad to allow the sample to pass through the test strip. As noted above, solutions may include any desired buffers, reagents, compounds, dyes, labels or beads. It should be understood that the gap embodiments disclosed herein may be adapted to any test device configuration disclosed herein.

In one embodiment, the test device is a lateral flow test strip, preferably (though not necessarily) housed in a housing, and designed to be read by a reader (see Figures 3, 27).

In one embodiment, in Figures 27A, 27B, and 27C, the SCD-treated sample is introduced into the test device 1001, whereupon the tracking or running buffer is released and follows the sample through the absorbent pad and into the test strip 1003 , in which specifically patterned capture agents bind their counterpart capture probes. Thus, if a specific analyte is present, that specific analyte will be bound by the detection probe and by the capture probe (as described above), which will bind its specific counterpart capture moiety, the specific paired The object capture moiety is affixed to a defined location or thread on the test strip 1003. FIG. 27B shows the assembly of the test device, including upper housing 1002 , lower housing 1004 and aperture 1005 .

In one embodiment, the wash/run buffer is contained in a foil, pouch or buster-type pack (e.g., similar to a ketchup/condiment pack) that is disposed in the test device, i.e., the sample entry bay 302 , 504, 605, 1005, 1108 upstream (see Figure 27C). The capsule or pack can be designed so that it is symmetrical about two orthogonal axes so that the capsule or pack can be easily loaded into the test device. Thus, in one embodiment, pressing down on the lid of the test device disposed on the bag causes the bag to rupture, thereby releasing the contents therein.

In another embodiment, the button portion may include a piercing appendage that, when the button is depressed, pierces the bag thereby releasing the contents therein. A leaf spring or cantilever spring (see Figure 27C) can be placed between the bag and the button and create pressure on the bag to ensure release of all contents. Additionally, the geometry of the test device is configured to direct wash buffer to the absorbent pads 904/905,1006. In addition, the geometry of the button, spring, and housing also reduces porosity in the pack area, allowing wash buffer to flow in any direction as desired, even against gravity (eg, upward), but not back into the pack storage area.

The number and size of the produced wells as well as the geometry of the produced wells can be adjusted relative to each other in order to allow the flushing buffer to flow out of the pack in a predetermined manner.

In one embodiment, piercing appendages (eg, needles) will provide a liquid resistance barrier at the top of the pack to allow liquid to exit the lower portion of the pack in the direction of the absorbent pad. The needle used for piercing can also be tapered to achieve or enhance this function.

In one embodiment, the spring (see FIG. 27C ) is an integral part of the button, upper housing or lower housing (see FIG. 27B ), or it can be a separate component. Overall, the spring is configured to be easily installed and Seal the wash/run buffer compartment.

In one embodiment, the sides of the button are designed to minimize pinch points when the button is depressed. The sides can also be designed to provide a baffle-like function, thereby minimizing the risk of liquid leaving the test device.

The geometry of the feature supporting the end of the absorbent strip is designed to allow the piercing feature (eg, a needle) to pass through the pack without allowing the pack to form a seal between the pack and the support feature. The function of the needle is to pierce the absorbent pan and bag. In another embodiment, only the bag with the absorbent disc positioned directly adjacent to the pierced hole is pierced.

In one embodiment, the wash/run buffer in the test device is contained within a rupturable/breakable substrate (eg, an ampoule). Pressure applied to the sealing membrane or button ruptures the ampoule, releasing its contents. In one embodiment, channels, trenches or grooves are designed to direct the buffer to the suction pan.

In one embodiment, the hole for receiving the end of the SCD includes a detachable ring ("lock ring") 1104 (see FIG. 28A ) that is attached to the SCD assembly and that breaks away when the SCD 1101 is removed. The device body 1105 is tested to release wash/run buffer from the compartment/reservoir 1102, upstream or just upstream of the well. In another embodiment, when the lock ring is twisted into the locked position, the sample is dispensed onto the test device while buffer or wash buffer is released from the upstream compartment. For example, the locking ring will include a geometrically shaped channel 1103, hole or opening that aligns with the opening, channel or hole of the rinse/buffer compartment only when the ring is in the locked position. Any compartment in the one or more upstream regions 1102, 1106 that may be used to deliver buffer/wash or any other liquid may utilize such a lock ring.

In an alternative embodiment, the SCD 1101 may include a lock ring 1103 that fits into the test device body 1103 and transitions from an unlocked position to a locked position 1104.

In one embodiment, as shown in Figure 28B, the upstream rinse/buffer compartment comprises a soft membrane (e.g., forming a fill-seal package) or an ampoule that, when minimal force is applied (e.g., a user presses with a finger) When used, the membrane or ampoule is prone to cracking/rupturing. Such an onion skin (onion skin) compartment 1106 can also be covered by a hard removable cover 1107, which prevents a sudden rupture of the onion skin. Samples enter the testing device through a chamber 1109, which may have a narrow channel 1109 to retrieve archived samples.

As shown in FIG. 8, the sample is delivered to the test strip through an SCD 802 including a rod and a swab 806. Upstream of the test strip is a compartment 801 with a buffer or other liquid. The test strip includes a test zone 803 and control zones A, B, C 804. The detection probe will provide a detectable signal through the conjugated label. The test device is then inserted into the reader 400, as shown in Figure 4, where the signal from the tag is measured and/or detected. In another embodiment, the test strip 401 can be inserted into the removable disc 402 within a very short read time period (-20 seconds) after the short assay processing time period has been completed, which is within one read time period. Allows for higher test throughput in the case of extractors. Also, in another embodiment, the test strip can be inserted into the reader prior to adding the sample.

In any of the embodiments herein involving the release of wash/run buffer from a chamber upstream of the sample (e.g., the sample entry compartment), a time delay feature can be built into the test device such that there is a delay between the introduction of the sample and the wash/run buffer. A period of time has passed. For example, when a dry absorbent pad base is in a wet state (ie, after release of wash buffer), the absorbent pad base swells and due to the swelling connects, otherwise the absorbent strips are disconnected (see Figure 34). For example, sample 1405 is applied. The ampoule or base 1401 containing the wash buffer is ruptured/disrupted, thereby releasing the liquid into the dry absorbent pad portion 1402 which expands 1403 and provides liquid to the absorbent pad portion containing the sample 1405 connected. The sample/buffer flows through the test strip via absorbent pad 1406 .

In another embodiment, a fibrous membrane of predetermined length/density is placed between the wash buffer region and the absorbent membrane, which is capable of delaying contact of the wash buffer with the absorbent membrane, thereby acting as a time delay mechanism. The buffer moves along the fibrous membrane by capillary action and accumulates at the ends of the membrane fibers until it reaches the absorbent membrane and flows through the sample placed on the absorbent membrane. In another embodiment, the buffer accumulates at the ends of the membrane fibers until there is sufficient volume to bridge the gap separating the fiber membrane from the water-absorbent membrane.

In other embodiments, a plunger or spring mechanism is built into the test device that acts to reduce the volume of the compartment/ampule to ensure that its contents are dispersed onto the absorbent pad. The plunger may be moved forward by the user applying pressure to a button, or a spring-loaded plunger may be driven forward in an automated fashion (eg, provided in the reader). When the plunger is driven forward, the plunger forms a seal so liquid only passes through the outlet to the absorbent pad.

In one embodiment, the transport of liquid along the test strip is based on capillary action. In another embodiment, the liquid transport along the matrix is based on non-absorbent lateral flow, wherein all dissolved or dispersed liquid sample components are carried at substantially equal rates and flow transversely through the matrix with relatively unimpaired flow , as the preferred retention of one or more constituents would occur, for example, in materials with one or more constituents interacting chemically, physically, ionically, or otherwise. See, eg, US Patent No. 4,943,522, which is hereby incorporated by reference in its entirety.

Any suitable material can be used to fabricate the devices disclosed herein, such materials include rigid or semi-rigid materials, water-impermeable materials such as glass, ceramics, metals, plastics, polymers or copolymers, or combinations thereof combination. In some embodiments, the SCD or test device comprises a plastic, polymer, or copolymer, such as a fracture-resistant polymer or copolymer, such as polypropylene, an isomorphic polymer, polycarbonate, or cycloalkene or cycloalkene copolymer thing. Furthermore, the device of the present invention can be manufactured by suitable manufacturing methods such as injection molding, blow molding, machining or compression molding.

A test strip substrate as used herein refers to a material attached to a capture moiety using conventional methods in the art. A variety of materials can be used as a substrate, including any material that can serve as a carrier for the attachment of molecules of interest. Such materials are well known to those skilled in the art and include, but are not limited to: organic or inorganic polymers; natural and synthetic polymers, which include, but are not limited to: agarose, cellulose, nitrocellulose, cellulose acetate Dextran, other cellulose derivatives, dextran, dextran derivatives and dextran copolymers, other polysaccharides, glass, silica gel, gelatin, polyvinylpyrrolidone (PVP), rayon, nylon, polyethylene, polypropylene , polybutene, polycarbonate, polyester, polyamide, vinyl polymer, polyvinyl alcohol, polystyrene and polystyrene copolymer, polystyrene cross-linked with divinylbenzene, etc., acrylic resin , acrylates and acrylic acid, acrylamide, polyacrylamide, polyacrylamide blends, copolymers of ethylene and acrylamide, methacrylates, methacrylate derivatives and copolymers, other polymers with various functional groups and Copolymers, latex, butyl rubber, and other synthetic rubbers; silicon, glass, paper, natural sponge, insoluble proteins, surfactants, red blood cells, metals, nonmetals, magnetic materials, or other commercially available media or made of solid or Composite materials consisting of semi-solid substrates coated with a material improving the hydrophilic properties of the strip substrate, such as polystyrene, Mylar, polyethylene, polycarbonate , polypropylene, polybutene, metals such as aluminum, copper, tin or mixtures of metals coated with dextran, detergents, salts, PVP and/or treated with electrostatic or plasma discharges A mixture of metals to add charge to a surface, thereby imparting hydrophilic properties to the surface.

In one embodiment, the lateral flow membrane comprises a porous material such as a high density polyethylene sheet material manufactured by Porex Technologies Corp. of Fairburn, Ga., USA. The sheet material has an open pore structure with a typical density of 0.57 gm/cc at 40% void volume and an average pore diameter of 1-250 microns, with an average of typically 3-100 Microns. In another embodiment, the marking zone comprises a porous material, such as a non-woven acrylic fiber (similar to a sample holding tube), such as New Merge or HDK material. Typically, the porous material can be supported by a general waterproof layer (eg, Mylar), or can be laminated on top of a waterproof layer. When employed, the backing is secured to the substrate by an adhesive (e.g., 3M 444 double-sided tape). Typically, a waterproof backing is used for membranes of small thickness. A wide variety of polymers can be used, provided they do not bind non-specifically to the assay components and do not interfere with the flow of the liquid sample. Exemplary polymers include polyethylene, polypropylene, polystyrene, and the like. The matrix can be self-supporting if desired. Other membranes that are subject to non-absorbent flow, such as polyvinyl chloride, polyvinyl acetate, copolymers of vinyl acetate and vinyl chloride, polyamides, polycarbonates, polystyrene, and the like, may also be used. In another embodiment, the lateral flow membrane comprises materials such as untreated paper, cellulose blends, nitrocellulose, polyesters, acrylonitrile copolymers, and the like. The marker region can be configured to provide a hygroscopic flow or a non-absorbent flow, typically of a type similar or identical to that provided in at least a portion of the sample-receiving region. In another embodiment, the marking zone comprises a non-woven fabric such as Rayon or fiberglass. Other marker zone materials suitable for use in the present invention include the chromatographic materials disclosed in US Patent No. 5,075,078, which is incorporated herein by reference.

In another embodiment, the test strip substrate is treated with a solution including a material blocking agent and a marking stabilizer. Blocking agents include bovine serum albumin (BSA), methylated BSA, casein, acid or base hydrolyzed casein, skim milk powder, fish gelatin, and the like. Stabilizers are readily available and well known in the art, for example, stabilizers can be used to stabilize labeled reagents. In some embodiments, the upstream compartment containing solution 307 may comprise a plurality of ampoules which may be selectively pierced or ruptured to release their contents. Thus, in one embodiment, the The blocking agent is contained in one ampoule for preconditioning (eg, "blocking") the test strip (ie, lateral flow membrane), while another ampoule is reserved for flushing the sample through the test strip.

In various disclosures herein, the test strip/lateral flow membrane includes a plurality of test zones, one test zone being designated 803 in FIG. 8 . The test zone typically includes a preselected capture moiety, wherein the preselected region includes a capture moiety that is a capture molecule partner for an analyte-specific binding agent (eg, a monoclonal antibody). In many of the embodiments presently contemplated, multiple labeled reagents are incorporated into the SCD so that they are permeable to liquid samples that come into contact with the device. These multiple labeled reagents can be analyte-specific or control reagents and can have different detectable characteristics (e.g., different colors), so that when these reagents are used in the same device, one of the labeled A labeled reagent can be distinguished from another labeled reagent, or, in preferred embodiments, the multiple labeled reagents have different capture moieties. When the labeled reagent frequently binds to a specific analyte of interest and is subsequently processed by a test device including a test strip, the concentration of labeled reagents with different specificities (including analyte-specific reagents and control-labeled reagents) Discriminative detection would be a desired property. Typically, however, the ability to differentially detect labeled reagents with different specificities based on the labeling component alone is not necessary due to the presence of defined test and control zones in the device whereby labeled reagents accumulate in designated zones. of.

In some embodiments, each detection probe is conjugated to a fluorescent label that emits at a different wavelength. Where multiple specific binding agents are included in the SCD, if the multiple specific binding agents comprise several different sets of specific binding pairs, each binding pair of a given set includes a different label than any other set, where multiple Each group complements the plurality of specific binding agents. For example, a set of influenza A-specific binding antibodies can be conjugated to one fluorescent label (i.e., conjugated to a first fluorescently-labeled detection probe), while a set of specific binding antibodies (i.e., conjugated to a second Fluorescently labeled detection probes), the third group, the fourth group or the fifth group may each comprise a detection probe conjugated to a different fluorescent label. Of course, it should be apparent that the detection probes may also comprise different types of labels (eg, a first set comprising a fluorescent label, a second set comprising a metal, while the control may comprise a chromophore).

In one embodiment, the fluorescent markers emit at sufficiently different wavelengths that several test lines can be distinguished.

This description provides the development and use of single or multiple control zones in a single immunoassay device, which are arranged in a predetermined manner relative to each test zone, thereby allowing easy identification of the test zone in the device. Each of the one or more analytes of interest. This description also provides control areas for making various shapes, physical or chemical characteristics and colors. In part, the use of such a control zone allows for easy use of the immunoassay device and allows for the identification of multiple analytes during a single assay. (See Example 11).

In one embodiment, the test device does not include any reagent contained therein that is capable of specifically binding to the analyte (eg, an antibody specific to H5N1).

Typically, the test area includes one or more control areas, which are used to verify that sample flow is as expected. Each control zone includes a spatially distinct region that includes a fixed member of a particular binding pair that interacts with a labeled control reagent. In a special case embodiment, a procedural control zone accommodates an authentic sample of the analyte of interest or a fragment thereof. In this embodiment, a labeled reagent can be utilized, wherein the liquid sample delivers the labeled reagent to the test and control zones; the labeled reagent that is not bound to the analyte of interest then binds to the Authentic samples of the analytes of interest. In another embodiment, the control line includes antibodies that are specific for, or provide for, immobilization of the labeled reagent. In operation, the labeled reagent is maintained in each of the one or more control zones even in the absence of any or all of the analyte of interest in the test sample.

In some embodiments, a labeled control reagent is introduced into the flow of liquid sample in the SCD or test device. For example, in a test device, a control reagent may be contained in an upstream solution/buffer reservoir, as described herein (see Figure 3). In another example, a labeled control reagent can be added to the liquid sample prior to application of the sample to the test device (eg, present in a mixing subchamber in an SCD).

For example, exemplary functions of labeled control reagents and zones include confirming that the fluid flow of the sample effectively solubilizes and mobilizes labeled reagents from the SCD that are captured in one or more defined test zones. In addition, the control can confirm that a sufficient amount of fluid is currently passing through the test and control regions of the test strip so that a sufficient amount of capture moiety can complex with the corresponding specific capture probe to a specific analyte (i.e., by an antigen-specific binding agent). reaction. In addition, the control reagent confirms that the immune complex (e.g., analyte-analyte-specific binding agent) moves through the test zone in an amount onto the test zone comprising the test and control zones, whereby the accumulation of labeled analyte will produce a visible signal, or, in the case of a positive test result in the test zone, a readable signal. Furthermore, another function of the control area may be to serve as a reference area that allows the user to identify the test results displayed as readable areas.

Because the devices of the present invention may include one or more control zones, each control zone will become visualized by developing a labeled control reagent and its corresponding control zone, wherein, regardless of the one or more analyte(s) of interest presence or absence, each control zone has the desired intensity for all control zones after the liquid sample is in contact with the device.

In one embodiment, a single labeled control reagent will be captured by each control zone on the test strip. Typically, if multiple control zones are present, such labeled control reagent will be deposited on or in the labeling zone in an amount exceeding the total binding capacity of the combined control zones. Thus, the amount of capture reagent specific to the control label can be deposited in an amount that allows the desired signal intensity to be generated in one or more control zones and allows each control zone to retain the desired amount of labeled contrast reagents. Each control zone preferably provides a desired and/or pre-designed signal (in terms of intensity and form) after completion of the assay. Examples of expected pre-designed signals include: equal intensity signal in each control zone; or, other signal intensities in the control zone following a desired pattern of increasing, decreasing.

In another embodiment, each control zone will be specific to a unique control reagent. In this embodiment, the labeling zone may include multiple and different labeled control reagents to equalize the number of control zones in the assay or related alteration. Therein, each labeled control reagent can become restricted in one or more predetermined and specific control zones. These labeled control reagents can provide the same detectable signal, or provide a distinguishable detectable signal based on accumulation in the control zone (eg, with a different color label or other detection system).

In another embodiment, the control zone may comprise a combination of the two control zones described in the two previous examples, specifically, one or more control zones are capable of inhibiting or binding a single labeled control reagent, the same Other control areas on the test strip will be capable of binding one or several other specifically labeled control reagents.

In one embodiment, the labeled control reagent includes a detectable moiety that binds to one member of a particular binding pair. Typically, the labeled control reagent is selected to be different from that recognized by means capable of inhibiting the analyte of interest in the test zone. reagent. Furthermore, labeled control reagents are generally not specific for the analyte. In typical embodiments, the labeled control reagent is capable of binding the corresponding member of a specific binding pair or control capture partner immobilized on or in the control zone. Thus, the labeled control reagent is confined directly in the control zone.

In another embodiment, the detectable moiety forming the labeling component of the labeled control reagent is the same detectable moiety as the labeling component of the labeled test reagent used as the analyte of interest. In typical embodiments, the labeling component of the labeled control reagent is different from the labeling component of the labeled test reagent so that assay results are readily ascertainable. In another general embodiment, the control reagents and test markers comprise colored beads, such as colored latex. Also, typically, control and test beads comprise different colors.

In another embodiment, the labeled control reagent includes streptavidin, avidin, or biotin, and the control capture moiety includes a corresponding member of such a specific binding pair that readily and specifically combined with each other. In one example, the labeled control reagent includes biotin and the control capture partner includes streptavidin. Those skilled in the art will appreciate that other members of a particular binding pair can alternatively be used, including, for example, antigen/antibody reactions that are not associated with the analyte. In another embodiment, a capture partner may comprise any binding moiety disclosed herein.

The purpose of the control zone is that the appearance of a signal in the control zone indicates when the test result can be read (even for negative results). Thus, when the desired signal appears in the control line, it can indicate the presence or absence of a signal in the test zone.

In another embodiment, a control zone comprising a marker that becomes visible in the test area when the test area is wet is utilized. This type of control zone is disclosed in the following patent application, namely: currently pending US patent application serial number 09/950,366 filed September 10, 2001, published as US patent application publication number 20030049167 ; currently pending US Patent Application Serial No. 10/241,822 filed September 10, 2002, published as US Patent Application Publication No. 20030157699.

In a special case embodiment, one or more control zones of this type are utilized. In another embodiment, a combination of the following types of control zones can be used, including: types that utilize labeled control reagents and control zones; types that display control zones when wet. This allows for an easy method to elucidate the control zone while allowing the use of reagent-based control zones to determine that redissolution and migration of reagents in SCD-treated samples has been effective and to identify specific responses along the test device, The path defined by the absorbent pad, test strip and absorbent pad occurred as expected. This embodiment includes the use of one or more control zones, for each control zone of the assay other than the control zone on the end or downstream end of the test strip, the one or more control zones when the test zone is wet One or more control zones become visible.

This specification also provides tools for setting up rapid multi-analyte assays, which are needed in many fields of environmental monitoring, medicine, and especially in the field of infectious diseases. For example, contemplated devices include means for differential diagnosis of influenza A or B and their subtypes (e.g., influenza A, H5N1) that may result in different treatments or for differential diagnosis of influenza A in one step , Influenza B and/or RSV. Such devices permit a single sample to be assayed for multiple analytes at once, and advantageously, such devices allow for a considerable reduction in hands-on time and duration of diagnostic procedures, generally to the benefit of the physician or user. Thus, multiple immunological reagents can be utilized in the SCD of the invention, wherein the multiple immune reagents comprise a population of specific binding agents comprising pairs conjugated to label and capture moieties, respectively, Thus, the plurality of immunological reagents includes a plurality of populations, each population being specific for a different analyte than any other population. For example, the plurality of immunizing agents may be specific to several types of one pathogen or different pathogens.

Various analytes are assayed using the devices and methods of the present disclosure. In a particular device for assaying a sample for one or more analytes of interest, a collection of analytes of interest may be referred to as a panel. For example, a test series may include influenza A, influenza B, influenza A subtype, respiratory syncytial virus (RSV), adenovirus, and different types of parainfluenza virus (e.g., type 1, type 2, type 3 type, etc.) in any combination (or all combinations). Another test series may include tests for selecting one or more upper respiratory tract infections including, for example, Streptococcus pneumoniae, Mycoplasma pneumoniae and/or Chlamydia pneumoniae. A further test series may be designed for the diagnosis of sexually transmitted diseases including, for example, Chlamydia, Trichomonas and/or Gonorrhea. In each case, a specific assay series designed to provide a signal for a range of specific analytes on a test device is readily obtained by combining different sets of detection and capture probes in the SCD described herein. Thus, a particular SCD will employ a test strip with detection reagents (not specific for the analyte of interest), providing all the reagents necessary to detect the analytes of the particular test series detected when using the test device. In other embodiments, a broad range test device may include non-specific capture moieties for several series of analytes from related or different pathogens, for example, for HIV and HCV antigens, HIV and tuberculosis, A, B and Detection of subtype influenza A, bacterial and viral infections. Thus, a single test device can be used with an SCD comprising immunoreactants for analytes of different test series, as long as efficiency and cost-effectiveness are enhanced.

For example, optionally, the test series can include various other target analytes including SARS-associated coronavirus (coronavirus), influenza A; the hepatitis test series includes hepatitis B surface antigen or antibody, hepatitis B core antibody ( hepatitis B core Ab), hepatitis A virus antibody and hepatitis C virus screening (selection); phospholipid test series include anticardiolipin (Anticardiolipin) antibody (IgG, IgA and IgM isotypes) screening; arthritis test series include class Rheumatic factor, antinuclear antibody and uric acid; Epstein Barr test series include Epstein Barr nuclear antibody, Epstein Barr virus capsid antigen and Epstein Barr virus, early antigen screening; other test series include HIV test series, lupus test series, H.Pylori Test series, toxoplasma test series, herpes test series, Borrelia test series, rubella test series, cytomegalovirus test series, tests using analytes including isotroponin and myoglobin and/or CKMB and others Trial series of recent myocardial infarction. Those skilled in the art will appreciate that various assay series can be assayed by means of immunoassays utilizing the devices disclosed herein. Immunoassay methods are well known in the art. See, eg, CURRENT PROTOCOLS IN IMMUNOLOGY (A Laboratory Guide to Immunology, Coligan, John E. et al., eds. 1999).

Numerous analytical devices known to those skilled in the art can be adapted to detect multiple analytes in accordance with the present invention. By way of example, dipstick devices, lateral flow devices, and flow-through devices, in particular those immunoassays, may be modified according to the invention for the detection and identification of multiple analytes.示例性的横向流动装置包括那些在第4,818,677、4,943,522、5,096,837(RE35,306)、5,096,837、5,118,428、5,118,630、5,221,616、5,223,220、5,225,328、5,415,994、5,434,057、5,521,102、5,536,646、5,541,069、5,686,315、5,763,262、5,766,961、5,770,460 , 5,773,234, 5,786,220, 5,804,452, 5,814,455, 5939,331, 6,306,642 US Patent Nos. Other lateral flow devices that can be modified for differential detection of multiple analytes in fluid samples include US Pat. Exemplary dipstick devices include those described in US Patent Nos. 4,235,601, 5,559,041, 5,712,172, and 6,790,611. It will be appreciated by those skilled in the art that the aforementioned patents may, and do frequently, disclose more than one assay configuration, and that additional disclosures for these are also incorporated herein. Advantageously, the improvements described are applicable to various assays (especially immunoassays), configurations.

The SCD or test device of the present invention can be configured to utilize existing analyte detection systems. For example, the SCD of the present invention can be configured for use with an existing test setup, or for a test setup, an existing test setup can be constructed/modified as disclosed herein. Some exemplary devices that can be modified in this manner include dipsticks, lateral flow, cartridges, multiplexed, microtiter plates, microfluidics, plates or arrays or high throughput platforms such as those在以下的专利中公开的装置：第4,235,601、4,632,901、5,559,041、5,712,172、6,790,611、6,448,001、4,943,522、6,485,982、6,656,744、6,811,971、5,073,484、5,716,778、5,798,273、6,565,808、5,078,968、5,415,994、6,235,539、6,267,722、6,297,060、7,098,040、 6,375,896、4,818,677、4,943,522、5,096,837(RE 35,306)、5,096,837、5,118,428、5,118,630、5,221,616、5,223,220、5,225,328、5,415,994、5,434,057、5,521,102、5,536,646、5,541,069、5,686,315、5,763,262、5,766,961、5,770,460、5,773,234、5,786,220、5,804,452、5,814,455、 US Patent Nos. 5,939,331 and 6,306,642. Other lateral flow devices that can be modified for the differential detection of multiple analytes in fluid samples include those described in the following patents: , US Patent Nos. 6,306,642, 7,109,042, 5,952,173 and 5,914,241. Exemplary microfluidic devices include those disclosed in US Patent Nos. 5,707,799, 5,837,115 and WO2004/029221. Each of the above patent publications is hereby incorporated by reference in its entirety.

reader

The systems and methods include: an immunoassay device, in combination with a reader 400, specifically a reader 400 with a built-in computer, e.g., a reflectance and/or fluorescence based reader; data processing software , using data reduction and curve fitting algorithms, optionally in combination with trained neural networks, for precise determination of the presence or concentration of an analyte in a biological sample. As used herein, a reader refers to an instrument used to detect and/or quantify data, eg, a test strip included in the test device 401 . Data can be visible to the naked eye, but need not be. The method comprises the steps of: performing an immunoassay on a patient sample; reading the data with a reflectance and/or fluorescence based reader; and processing the resulting data with processing software employing data reduction. Preferred software includes curve fitting algorithms (optionally combined with trained neural networks) for determining the presence or amount of an analyte in a given sample. The data obtained from the reader can then be further processed by a medical diagnostic system, providing a risk assessment or a diagnosis of a medical condition as output. In an alternative embodiment, the output may be used as input into a subsequent decision support system such as a neural network trained to evaluate such data.

In various embodiments, depending on the signal to be detected from the test device (e.g., LRE Medical, USA), the reader may be a reflectance reader, a transmission reader, a fluorescence reader, a biochemical fluorescence reader , magnetic reader or current reader (or two or more test series). In one embodiment, the reader includes a receiving port designed to receive a test device such that a test device can only be inserted into the receiving port if the entry aperture has been depressed as described below Upstream of the depressible (eg, button) device, the test device can be fitted into the receiving port 402 . Thus, in such embodiments, the test device is placed in the reader only when the contents of the reservoir (e.g., wash buffer) are released, ensuring that the sample has "run-through" The lateral flow membrane included in the test setup.

In one embodiment, the reader is a UV LED reader that detects a fluorescent signal. The fluorescent signal is excited by a light emitting diode emitting in the UV region of the spectrum and within the absorption peak of the fluorescent signal (eg, lanthanide labels). The emitted fluorescent signal is detected by a photodiode, and the wavelength of the detected signal can be limited by the use of a long-pass filter, where the long-pass filter blocks light that deviates from emission and absorbs wavelengths at and around the peak emission wavelength of the emitting fluorescent label of light. In other embodiments, a band pass filter may be used instead of the long pass filter. Additionally, the excitation light can be limited by a bandpass filter.

In another embodiment, the diodes are UV laser diodes. Any conventional UV, LED or photodiode can be used.

In any such embodiment, the excitation source and detector are mounted in a single machine or modular block. To simplify the reading of the fluorescent signal generated on the test strip. In yet another embodiment, such a machine further includes a rigid standard as described herein (eg, FIGS. 33A and 33B ).

In one embodiment, the axis of the excitation light is at 90 degrees to the test device or a test strip included in the test device. In addition, the axis of the emitted light is at an angle other than 90 degrees to the test strip.

In one embodiment, the wavelength of the excitation light is limited by a short pass filter. In yet another embodiment, the wavelength of the excitation light is limited by an experimental series of bandpass filters and shortpass filters. In yet another embodiment, the wavelength of the detected light is limited by an experimental series of bandpass filters and longpass filters. The reader may be configured to detect any of the signal emitters/markers described herein. In one embodiment, the label can be any of the lanthanides described herein. In yet another embodiment, the lanthanide used is europium.

As noted herein, in one embodiment, a reader is configured to contain one or more hard standards. Accordingly, the reader can be designed to provide a standard (for example, acrylic (acrylic) as described herein ) (encase)) tools (eg, clamps), the standard set on the center of about 3mm, 4mm, 5mm or 6mm (eg see Figure 33A, Figure 33B, Figure 34).

In one embodiment, the reader is adapted to the receiving port of the test device of Figures 27A and 27B, which itself is configured with the security device of Figure 27C. In one embodiment, the reader will accept the test device 401, but not process the test device 401, if the button is not pressed; or, if the wash buffer control does not generate a positive signal, etc., the reader will The test set is accepted and read, but the result will be rejected. In this latter embodiment, the wash/run buffer provided in the compartment/capsule provided upstream of the sample in FIG. 27C may contain a control signal (e.g., a label emitting at a different wavelength ).

Signals acquired by the reader are processed using data processing software employing data reduction and curve fitting algorithms, optionally in combination with trained neural networks, to give positive or negative results for each test line, or to quantify samples The concentration of each analyte in the test is associated with an outcome indicative of the risk or presence of a disease or condition. Optionally, the results can be input into a decision support system and processed to provide as output a risk-improving assessment of the medical condition. In one embodiment, the entire procedure can be automated and/or computer controlled.

Multi-analyte point of care systems

Rapid flu tests have been on the market for many years. Most of these tests are lateral flow immunoassay tests utilizing gold or latex as the chromogenic reagent. While most new rapid immunoassays are able to distinguish influenza A from influenza B, only a few have lines for both types A and B on a single test strip. However, none of these tests were designed to distinguish subtypes of influenza A. Thus, these tests may be able to detect avian influenza, but none of them can prove whether a patient has been infected with a seasonal influenza A virus or a more serious type of avian influenza named H5N1 (or the current potential pandemic influenza A virus). influenza) infection. The present invention was designed based on the idea of being able to detect A, B and differentiate subtype H5N1 from seasonal influenza (subtypes H1 and H3) when applied to a hypersensitive assay with improved reproducibility , and the present invention is easy to use. Efforts have been made to apply a variety of new techniques to new device designs, such as premixing the sample with the conjugate, using chasing or wash buffers to reduce background, and employing the unique generic capture agent pRNA ( allow multiple analyte detection with high sensitivity), highly sensitive fluorescent labeling, etc. The combination of these approaches has enabled a novel and highly effective influenza rapid test that is more sensitive, provides a low-cost product, is easy to perform, and has the ability to distinguish seasonal influenza from epidemic avian influenza H5N1.

In one embodiment, the combination of features described herein is reliable for the superior sensitivity and reproducibility of assays constructed in accordance with the present invention to use the novel system for concentrating the ligand from the test sample at the test site of the test strip ( concentrated ligand), the use of metal sols or other colored particles as marker systems allows for continuous visual observation of fluorescence over a period of one or several hours after the minimum time required to complete the assay. Eliminate background noise while maintaining excellent sensitivity by including a controlled release buffer in the test instrument to wash away excess/unbound label. In addition, compare the color of one or more control sites to the test sites. In some embodiments, a filtering means is included in the sampling instrument or testing instrument, in which filtering means limits the introduction of contaminants from natural or raw biological samples to the test site. In yet another embodiment, the filter means is a membrane and includes detection and capture probes 604 disposed thereon.

assay method

In one embodiment, the assay method comprises the steps of: applying a sampling instrument to a subject or a biological sample of a subject to collect a sample (e.g., secretions from inside the nose, mouth, throat, ear); making the collection element Acts on a biological sample obtained from a subject; inserts the collection instrument into the sample collection device housing chamber, squeezes the upper chamber to break the snap-action valve, and causes the buffer to flow down to the sampling instrument, thereby immersing the The upper biological sample and the buffer and sample mixture flow into a reaction chamber (eg, lower chamber) where multiple capture and detection probes bind to their specific target analytes. Subsequently or simultaneously, the mixture is expelled from the end of the SCD by means of a complementary capture moiety attached to the capture probe into a test device comprising an immobilized capture moiety designed to capture the complex of the analyte and the detection/capture probe . Thus, for a specific analyte, specific capture probes are designed to be complementary to the immobilized capture moiety. Furthermore, as disclosed herein, capture moieties are positioned in different sites/patterns on the lateral flow membrane, enabling the identification and/or quantification of a specific analyte if a signal or spot is detected via a signal emitting label detection. Thus, by patterning specific capture probes on lateral flow membranes, as disclosed herein, assays can detect test series of the same or related infectious agents, or even test series of unrelated infectious agents.

In some embodiments, a sandwich immunoassay format is used, however, any conventional format including competition assays may be used. Typically, indirect capture of formed immune complexes is used in a sandwich format. One or more analytes in the sample are contacted with one or more pairs of detection probes and capture probes. Each pair comprises a detection probe and a capture probe, wherein the detection probe is a conjugate comprising a label and a specific binding agent (SBA) capable of specifically binding to the analyte, and the capture probe is a conjugate comprising a detection moiety and another capable of binding to the analyte. Conjugates of SBAs that specifically bind the same analyte. Examples of specific binding agents include antibodies and aptamers. In one example, since each of the two SBAs is the same target antigen or analyte, each is a specific binding partner. Examples of SBAs are well known in the art and include, but are not limited to, antibodies, aptamers, or oligonucleotides. In a sandwich assay, the analyte is bound by both the detection probe and the capture probe. The detection moiety is part of a specific binding pair, the other partner of the pair is immobilized on the test device so as to capture the immune complex as it flows through the test device. The use of different capture moieties for each different analyte allows detection of multiple analytes from a single sample and reaction sequence on the test device. In most cases, the labels used for each analyte are different. However, with specific positioning for each analyte as a capture zone with a different capture moiety for each analyte, it is possible to use the same label for all analytes.

Examples of sandwich immunoassays performed on test strips are described in US Patent Nos. 4,168,146 and 4,366,241, both of which are incorporated herein by reference. Examples of competitive immunoassays are described in US Patent Nos. 4,235,601, 4,442,204, and 5,208,535, all of which are incorporated herein by reference. Some additional exemplary devices that may be adapted to incorporate one or more components of the invention include dipsticks, lateral flow, cassettes, multiplex microplates, microtiter plates, microfluidics, plates or arrays, or high throughput platforms ，例如那些在以下专利中公开的装置：第6,448,001、4,943,522、6,485,982、6,656,744、6,811,971、5,073,484、5,716,778、5,798,273、6,565,808、5,078,968、5,415,994、6,235,539、6,267,722、6,297,060、7,098,040、6,375,896、7,083,912、5,225,322、6,780,582、 US Patent Nos. 5,763,262, 6,306,642, 7,109,042, 5,952,173, and 5,914,241. Exemplary microfluidic devices include those in US Patent Nos. 5,707,799 and WO2004/029221.

In one such approach, the test strip comprises an enzyme-labeled binding partner for the analyte in a region downstream from the sample application region. If an analyte is present in the test sample, the analyte will combine with its labeled binding partner to form a complex, which will flow along the test strip to the detection zone, which contains The substrate is also capable of providing a detectable enzymatic reaction, eg, a color reaction in the presence of the enzyme. The test strip also contains a region where a predetermined amount of analyte is immobilized such that any labeled binding partner that does not combine with the analyte will be captured due to the absence of the analyte in the sample and thus be inhibited from reaching the detection zone.

Alternatively, the test strip may comprise a binding partner comprising a moiety having a detectable physical property. For example, a labeled binding partner may include one or more moieties, e.g., chemical groups detectable based on specific physical properties including, but not limited to, colored species of fluorescent agents, phosphorescent molecules, radioisotopes, Electroactive parts, particles such as gold, etc.

In another approach, detection is based on the competition between a specific analyte (e.g., a ligand, antigen, or antibody) whose amount is determined in the sample and a known amount of a tracer, using the analyte and marker The tracer competes for a limited number of available binding sites on the binding agent that is specific towards the analyte and the tracer, where the tracer is usually a labeled form of the analyte or a suitable analogue thereof. For example, if the concentrations of tracer and binding agent are fixed, and the only variable is the level of analyte, then unknown levels of analyte can be measured by determining the bound and/or free tracer body weight in the system. . By comparing the value determined in the assay with the value given by a range of known amounts of the analyte processed in the same manner, the amount of the analyte in the sample can be determined by this comparison.

Typically, tracers used in such assays require instrumentation and/or processing of the tracer in order to determine the tracer in the bound and/or free fraction of the analyte as a measure of the analyte . For example, in an assay using an enzyme as a label or label to the tracer, the enzyme must be visualized with a suitable developer. When the label or label is a fluorescent material, the bound and/or free portion of the tracer is determined by using a suitable instrument for determining fluorescence.

Alternatively, the tracer used in the assay is a ligand labeled with a particulate label that is visible when bound to a support or to an analyte that has bound to a binding agent on the support, whereas No further processing is required wherein the ligand is bound with a binding agent or analyte. See also US Patent No. 4,703,017, which is hereby incorporated by reference.

In another specific aspect, the non-nucleic acid based screening assays of the invention include any solid phase assay, lateral flow assay or perfusion assay. Typically, a solid phase immunoassay device comprises a solid support to which a member of a ligand-receptor pair, usually an antibody, antigen or hapten, is bound. Common early forms of solid supports were plates, tubes or beads made of polystyrene, which are well known in the art of radioimmunoassays and enzyme immunoassays. Recently, many porous materials such as nylon, nitrocellulose, cellulose acetate and other porous polymers have been used as solid supports.

In one embodiment, a sample is collected from the subject by means of the sampling instrument 102 and returned to the cylinder housing of the SCD device 200 . First, the SCD can be inserted into the test device or, after insertion into the test device, release the solution contained in the upper sealed chamber of the SCD, effectively washing the sample and solution into the mixture below towards the reaction chamber. Liquid or solid reagents containing detection and capture probes that target one or more of the different analytes disclosed herein are disposed in the reaction chamber, thereby forming a reaction chamber bound to the detection and capture probes. complex of analytes. The sample is then released from the SCD into the test device through an aperture that seals contact between the SCD and the test device from the external environment (eg, prevents spillage, suspension, or contamination). The sample mixture can flow as a result of the force of weight or atmospheric pressure introduced by pressing the SCD (upper sealed chamber) into the test device. The sample is pushed by capillary forces and/or by wash buffer included in the test device 307, thereby forcing any analyte-probe complexes across the lateral flow membrane contained in the test device. The capture probe and the complementary immobilized capture moiety bind or hybridize to each other at predetermined lines or points on the lateral flow membrane 803 or 804, whereby the detection probe (by means of a conjugated label contained in the detection probe) will provide a detectable The detectable signal can then be read to determine which analytes are present in the processed sample.

In one embodiment, the test device with the processed sample thereon may be set aside for a period of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours or 8 hours before the result is read, The test device can still provide just as accurate results if the results are read 15 or 20 minutes after treatment. As a result, the generated signal is stable for a long time, allowing the results to be read at a later time after the actual execution of the test. This is a major improvement over point-of-care diagnostics, where manpower and time resources are limited in field conditions, and test settings can be performed at remote distances that are not easily accessible quickly.

binding reagent

One aspect of the invention resides in a binding reagent disposed in an SCD. For example, in some immunoassays, pairs of antibodies are used, each member of which can specifically bind the same target analyte, where one antibody is a capture antibody and the other is a detection antibody. The capture antibody is directly or indirectly linked to a capture moiety that is "captured" by a homologously immobilized capture moiety disposed on a solid support (eg, a nitrocellulose membrane). In addition, a detection antibody (ie, detection probe) is linked to a detectable label. Preferably, the detection antibody is labeled by conjugation to a physically detectable label and forms a complex upon contact with a sample containing the analyte of interest. The antibody-analyte complex can then be immobilized on a solid support by means of a capture moiety. The resulting complex immobilized on the solid support can be detected by means of a label.

In one embodiment, the SCD reagent solution or solid phase substrate includes a plurality of different detection probes, each detection probe capable of binding to a different target, and each detection probe capable of being labeled with or capable of obtaining a detection signal , thereby indicating the presence of each target by information on the signal in the test zone for that target (i.e., in the test device); wherein the targets for at least two capture moieties are infectious agents, pathogenic microorganisms Or a marker indicative of a disease, disorder or condition of the host from which the sample solution is taken, wherein at least two capture moieties are capable of binding to different components or markers of the same infectious agent or pathogenic microorganism, or to Different markers for the same disease, disorder or condition caused by a pathogen or pathogenic microorganism are targeted for those capture moieties. In addition, the SCD will also include multiple distinct capture probes, each paired with a detection probe, with these pairs defined by their ability to bind a specific target analyte.

As used herein, the term "specifically binds" refers to the binding specificity of a specific binding pair. "Specific binding pair member" means a member of a specific binding pair (sbp), and refers to two distinct molecules wherein one molecule specifically binds to the second molecule either chemically or physically. For example, a pair of pRNA or aptamer/target antigen pair, or streptavidin-biotin provides exemplary specific binding pair members or sbp. These two molecules are related in the sense that their binding to each other enables them to distinguish their binding partners from other assay components with similar characteristics. The members of a specific binding pair are called ligand and receptor (anti-ligand), sbp member and sbp member (partner), and the like. The molecule may be an sbp member for agglutination of the molecule; for example, an antibody against an immune complex of a second antibody and its corresponding antigen may be considered as an sbp member for the immune complex.

In addition to antigen and antibody specific binding pair members, other specific binding pairs include, by way of example and without limitation, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences , effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, peptide sequences or chemical moieties (such as digoxin/anti-digoxin) and antibodies specific to sequences, chemical moieties or whole Proteins, polymer acids and bases, dyes and protein-binding agents, peptides and specific protein-binding agents (e.g., ribonucleases, S-peptides, and ribonucleic acid S-peptides), metals and their chelators ,etc. In addition, specific binding pairs may include members that are analogs of the original specific binding members, eg, analyte-analogues or specific binding members produced by recombinant techniques or molecular engineering.

An sbp member is an analog of another sbp member if both the sbp member and the other sbp member are capable of binding to another identical complementary sbp member. For example, such sbp members may be ligands or receptors modified by substituting a group for at least one hydrogen atom, such as a labeled ligand or a labeled receptor. The sbp member can be similar or complementary to the analyte, or an sbp member that is complementary to the analyte. If the specific binding member is an immunoreactant, the specific binding member may be a monoclonal or polyclonal antibody, a recombinant protein or antibody, a chimeric antibody, mixtures or fragments thereof, and mixtures of antibodies and other specific binding members. Other examples of binding pairs can be incorporated into the detection molecules disclosed in the following patents: US Pat.

"Antibody" refers to a polypeptide substantially encoded by one or more immunoglobulin genes or a fragment thereof, and includes any antibody comprising monoclonal antibody, polyclonal antibody, multispecific or bispecific antibody that binds to a specific antigen Immunoglobulins. A complete antibody includes two heavy chains and two light chains. Each heavy chain is composed of a variable region and a first, second and third constant region, while each light chain is composed of a variable region and a constant region. Antibodies have a "Y" shape in which the stem of the Y consists of the second and third constant regions of two sets of heavy chains bonded together by disulfide bonds. Each arm of Y consists of the variable and first constant regions of a single heavy chain bonded to the variable and constant regions of a single light chain. The variable region of the light chain and the variable region of the heavy chain are responsible for the combination of antigens. The variable regions of these two chains usually contain three hypervariable loops called complementarity determining regions (CDRs) (light (L) chain CDRs include LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs include HCDR1, HCDR2, and HCDR3 ) (as defined by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition (1991), vols. 1-3, NIH Publication 91-3242, Bethesda Md.). These three CDRs are placed between flanking frames of known framework regions (FRs), which are more highly conserved than the CDRs and form the backbone to support the hypervariable loops. The constant regions of the heavy and light chains are not contained in the antigen-binding agent, but instead exhibit various effector functions. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as a number of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes and subclasses, which include IgG, IgG1, IgG2, IgG3, IgG4, IgM, respectively , IgA, IgA1 or IgA2, IgD and IgE. Generally, an antibody is an immunoglobulin with an area on its surface or in a cavity that specifically binds to a specific spatial and polar organization of another molecule and thus refuses to complement this composition. Antibodies can be polyclonal or monoclonal. Antibodies can comprise complete immunoglobulins or fragments of complete immunoglobulins. Fragments of complete immunoglobulins may include Fab, Fv and F(ab') ₂ , Fab', and the like. Antibodies may also include chimeric antibodies or fragments of chimeric antibodies produced by recombinant methods. The term "antibody" as used herein is classified based on the amino acid sequence of the constant region of the heavy chain of the antibody. The main classes of antibodies are IgA, IgD, IgE, IgG, and IgM, and some of these classes are, for example, divided into subclasses.

In addition to intact immunoglobulins, the term "antibody" as used herein refers to fragments of intact immunoglobulins (i.e., at least one immunologically active portion of an immunoglobulin molecule), e.g., Fab, Fab', F(ab' ) _2. Fv fragments, single chain antibody molecules, multispecific antibodies formed from any fragment of an immunoglobulin molecule including one or more CDRs. Furthermore, the antibodies used herein may comprise one or more CDRs from a particular human immunoglobulin grafted into the framework regions of one or more different human immunoglobulins.

"Fab" with respect to an antibody refers to that portion of an antibody that consists of a single light chain (variable and constant regions) disulfide-bonded to a single heavy chain variable region and a first constant region.

"Fab" refers to a Fab fragment that includes a portion of the hinge region.

"F(ab') ₂ " means a dimer of Fab'.

"Fc" with respect to an antibody refers to that portion of an antibody that consists of the second and third constant regions of the first heavy chain that are disulfide bonded to the second and third constant regions of the second heavy chain . The Fc portion of an antibody is responsible for various effector functions, but is inactive for antigen binding.

"Fv" in reference to an antibody refers to the smallest fragment of the antibody that bears the entire antigen combining site. The Fv fragment consists of the variable region of a single light chain linked to the variable region of a single heavy chain.

"Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked directly to each other or via a peptide linker sequence (Huston 1998).

"Single-chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of scFv linked to the Fc region of the antibody.

The term "epitope" as used herein refers to a group of atoms and/or amino acids on an antigen molecule to which an antibody binds.

As used herein, the term "monoclonal antibody" as used herein refers to an antibody or fragment of an antibody obtained from a population of substantially homogeneous antibodies, i.e., including individual individuals of the population, except for those capable of naturally occurring mutations that may be present in small amounts. Antibodies are the same. Monoclonal antibodies directed against a single epitope on an antigen are highly specific. Monoclonal antibodies are relative to polyclonal antibodies, which usually include different antibodies against different epitopes on the antigen. Although traditionally, monoclonal antibodies are derived from hybridization, the monoclonal antibodies of the present invention are not limited by the method by which they are produced. For example, monoclonal antibodies of the invention can be made by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or by recombinant DNA methods (see, eg, U.S. Patent No. 4,816,567).

The term "chimeric antibody" as used herein refers to antibodies, as well as fragments of such antibodies, in which a portion of the heavy and/or light chains is the same as that obtained from a specific species or belonging to a specific antibody class or subclass The corresponding sequences in the antibody are identical or have homology, and the rest of the heavy and/or light chains are identical or have homology to the corresponding sequences in antibodies obtained from other species or belonging to another antibody class or subclass, As for the antibody fragment, it is sufficient that the antibody fragment exhibits desired antigen-binding activity (Cabilly et al., US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:68516855 (1984)).

The term "humanized antibody" as used herein refers to an antibody or antibody fragment that is a human immunoglobulin (recipient antibody) in which some or all of the residues from the CDRs of the recipient are derived from Substitution of residues in CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit of , avidity and capacity. In some instances, FR residues of the human immunoglobulin are substituted by corresponding non-human residues. In addition, humanized antibodies may comprise residues which are found neither in the recipient antibody nor in the grafted CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable regions, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and of the FR regions. All or substantially all of the FR regions are human immunoglobulin sequences. The humanized antibody optionally will also comprise at least a portion of an Fc region of an immunoglobulin, typically that of a human immunoglobulin. For a more detailed description, see Jones et al., Nature, 321:522525 (1986); Reichmann et al., Nature, 332:323329 (1988); Presta, Curr. Op. Struct. Biol., 2:593596 (1992); and Clark, Immunol. Today 21:397402 (2000).

The present invention provides anti-H5 monoclonal antibodies produced by mouse hybridoma cell lines 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2. These monoclonal antibodies are named for the hybridoma cell lines that produce them. Therefore, the anti-H5 mAbs produced by the mouse hybridoma cell lines 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2 were named mAbs 8H5, 3C8, 10F7, 4D1, 3G4, and 2F2, respectively. The monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2 specifically bind to the hemagglutinin of subtype H5 avian influenza virus. The mouse hybridoma cell lines 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2 were deposited in the China Center for Type Culture Collection (CCTCC, Wuhan, China, Wuhan University) on January 17, 2006, with the preservation number CCTCC-C200607 (hybridoma cell line 8H5), CCTCC-C200605 (hybridoma cell line 3C8), CCTCC-C200608 (hybridoma cell line 10F7), CCTCC-C200606 (hybridoma cell line 4D 1), CCTCC-C200604 (hybridoma cell line 3G4) and CCTCC-C200424 (hybridoma cell line 2F2).

In various embodiments, monoclonal antibodies are provided that block the binding of unit monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 or 2F2 to the hemagglutinin of subtype H5 avian influenza virus. Such blocking mAbs may bind to the same epitope on hemagglutinin recognized by mAbs 8H5, 3C8, 10F7, 4D1, 3G4 or 2F2. Alternatively, those blocking monoclonal antibodies may bind to epitopes that spatially overlap with those recognized by monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 or 2F2. These blocking monoclonal antibodies can reduce the binding of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4, or 2F2 to subtype H5 avian influenza virus hemagglutinin by at least about 50%. Alternatively, the blocking monoclonal antibodies may reduce binding by at least about 60%, preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80%, more preferably at least About 85%, even more preferably at least about 90%, even more preferably at least about 95%, most preferably at least about 99%.

Testing the ability of monoclonal antibodies to reduce the binding of known monoclonal antibodies to H5 hemagglutinin can be determined by routine competition assays, for example, as in Antibodies: A Laboratory Manual ("Laboratory Manual"), Cold Spring Harbor Laboratory (Cold Spring Hong Kong Laboratory), the method described in Ed Harlow and David Lane (1988). For example, such an assay can be performed by pre-coating a microtiter plate with antigen; incubating the pre-coated plate with serial dilutions of an unlabeled test antibody mixed with a selected concentration of a labeled known antibody; The incubation mixture was washed; each dilution of the test antibody was tested and the amount of known antibody bound to the plate was determined. The more the test antibody competes with known antibodies for binding to the antigen, the more the combination of antibody and antigen will be reduced. Typically, 96-well plates are precoated with antigen and radioactive or enzymatically labeled to determine the ability of unlabeled antibodies to block binding to labeled antibodies.

First, monoclonal antibodies can be produced by the hybridoma method described by Kohler et al., Nature, 256:495 (1975). In the hybridoma method, a mouse or other suitable host animal is immunized with one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected into the host animal by multiple subcutaneous or intraperitoneal injections. It may be useful to conjugate the immunizing agent to a protein known to confer immunity to the host animal being immunized (eg, serum albumin or soybean trypsin inhibitor). Examples of adjuvants that may be employed include Freund's competition adjuvant and MPL-TDM. Following immunization, the host animal's lymphocytes that produce or are capable of producing antibodies will specifically bind to the antigen used for immunization. Alternatively, lymphocytes can be immunized in vitro. The desired lymphocytes are collected, and the lymphocytes are fused with myeloma cells using a suitable fusion agent (for example, polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59- 103, Academic Press, 1996).

The hybridoma cells thus prepared are bred and grown in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of the unfused, parental hybridoma cells. For example, if the parental hybridoma cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridoma will typically include hypoxanthine, aminopterin, and thymine (HAT medium), These substances prevent the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently with selected antibody-producing cells, support stable high-level growth of antibody, and are sensitive to a medium such as HAT medium. Among these cells, preferred myeloma cell lines are murine myeloma lines such as those obtainable from the Salk Institute Cell Distribution Center in San Diego, California (San Diego, Calif. USA) MOP-21 and Cell lines obtained from MC-11 mouse tumor and cell lines obtained from SP-2 or X63-Ag8-653 cells obtained from American Type Culture Collection in Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

The medium in which the hybridoma cells are grown is assayed for the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or in vitro binding assays, eg, by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of monoclonal antibodies can be determined by Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

After identification of hybridoma cells producing the desired specificity, avidity, and/or activity, these cells can be subcloned by limiting dilution procedures and grown according to standard methods (Goding, Monoclonal Antibodies: Principles and Practice , pp.59-103, Academic Press, 1996). Suitable media for this purpose include, for example, DMEM or RPMI-1640 bases. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

Secreted by subclones, suitably isolated from culture medium, ascitic fluid, or serum by conventional immunoglobulin purification procedures, such as by protein A-sepharose, hydroxyapatite column chromatography, gel electrophoresis, dialysis, or affinity chromatography of monoclonal antibodies.

The monoclonal antibody of the present invention can also be produced by traditional genetic engineering methods. DNA molecules encoding the heavy and light chains of the monoclonal antibody are isolated from hybridoma cells, eg, by PCR, using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibody. Then, the DNA molecule is inserted into the expression vector. The expression vector is transfected into a host cell that does not otherwise produce immunoglobulin, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells. Host cells are cultured under conditions suitable for antibody expression.

The antibody of the present invention can bind to H5 hemagglutinin with high specificity and affinity. Antibodies should have low, preferably no, cross-reactivity with other subtypes of hemagglutinin. In one aspect, the invention provides antibodies that bind to H5 hemagglutinin with a _KD value of less than 1 x 10 ^-5 M. Preferably, the K _D value is less than 1×10 ^-6 M. More preferably, the K _D value is less than 1×10 ⁻⁷ M. More preferably, the K _D value is less than 1×10 ⁻⁸ M.

Antibodies of the invention may comprise a "Y"-shaped structure traditionally composed of two heavy chains and two light chains. In addition, antibodies can also be Fab fragments, Fab' fragments, F(ab) ₂ fragments, or Fv fragments, or other partial fragments of the traditional "Y"-shaped structure that retain binding affinity for hemagglutinin. Fragments may bind hemagglutinin with higher or lower affinity than conventional "Y" shaped structures.

Antibody fragments can be generated by proteolysis of intact antibodies (see, eg, Morimoto et al., J. Biochem. Biophys. Methods, 24:107-117, (1992) and Brennan et al., Science, 229:81 (1985)). These fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol., 11:548-557 (1999); Little et al., Immunol, Today, 21:364-370 (2000)). For example, Fab' fragments can be directly recovered from E. coli and chemically combined to form F(ab)' ₂ fragments (Carter et al., Bio/Technology, 10:163-167 (1992)). In another embodiment, the leucine zipper GCN4 is used to form F(ab)' ₂ fragments to facilitate the assembly of F(ab)' ₂ molecules. According to another approach, Fv fragments, Fab fragments, F(ab)' ₂ fragments can be isolated directly from the recombinant host cell culture medium. Other techniques for generating antibody fragments will be apparent to those of ordinary skill in the art.

The invention provides isolated nucleotide molecules encoding antibodies or fragments thereof that specifically bind to H5 hemagglutinin. Nucleotide molecules encoding antibodies can be isolated from hybridoma cells. The nucleotide sequence of a molecule can be determined using conventional techniques known to those of ordinary skill in the art. The nucleotide molecules of the present invention can also be prepared using traditional genetic engineering techniques and chemical synthesis. In one aspect, the invention provides an isolated nucleotide molecule encoding the variable region of the heavy chain of an anti-H5(HA) antibody, or a portion of such a nucleotide molecule. In another aspect, the invention provides isolated nucleotide molecules encoding the CDRs of the antibody heavy or light chain variable regions.

In one aspect, the invention provides isolated nucleotide molecules encoding the variable regions of the heavy and light chains of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2. The encoded monoclonal antibodies 8H5, 3C8, 10F7, Nucleotide sequences of the heavy chain variable regions of 4D1, 3G4 and 2F2. The light chains encoding monoclonal antibodies 8H5, 3C8, 10F7, 4D1 and 2F2 are mentioned in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 18 and SEQ ID NO: 24, respectively. The nucleotide sequence of the variable region. The present invention also includes degenerate analogs of the nucleotide sequences encoding the variable regions of the heavy and light chains of the monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2.

In another aspect, the present invention provides a combination of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 16 An isolated nucleotide variant having sequence identity to the nucleotide sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24 or SEQ ID NO: 26. In one embodiment, for SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: The sequence of ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 24 or SEQ ID NO: 26, the nucleotide variants have at least 70% sequence identity, preferably at least 75% sequence identity, more preferably Preferably at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, more preferably at least 95% sequence identity.

The present invention also provides isolated nucleotide molecules encoding antibody fragments that are still capable of specifically binding to subtype H5 avian influenza virus.

The present invention also provides an isolated nucleotide molecule encoding an antibody heavy chain variable region comprised in SEQ ID NO: 28-30, SEQ ID NO: 34-36, SEQ ID NO: 40-42, the amino acid sequences mentioned in SEQ ID NO: 46-48, SEQ ID NO: 52-54 and SEQ ID NO: 58-60. The invention provides isolated nucleotide molecules encoding antibody light chain variable regions comprised in SEQ ID NO: 31-33, SEQ ID NO: 37-39, SEQ ID NO: 43 -45, the amino acid sequences mentioned in SEQ ID NO: 49-51, SEQ ID NO: 55-57 and SEQ ID NO: 61-63.

The invention provides recombinant expression vectors comprising the isolated nucleotide molecules of the invention. The invention also provides host cells transformed with the nucleotide molecule. In addition, the present invention provides a method of producing an antibody of the present invention, the method comprising culturing a host cell under conditions in which the nucleotide molecule is expressed to produce the antibody, and isolating the antibody from the host cell.

Antibody peptide sequence

From the nucleotide sequences of the variable regions of the heavy and light chains of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2 their respective amino acid sequences have been deduced. Monoclonal antibodies 8H5, 3C8, 10F7, 4D1 are mentioned in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 25, respectively Amino acid sequences of heavy chain variable regions of , 3G4 and 2F2. It is mentioned in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 19 and SEQ ID NO: 27 that the light chains encoding monoclonal antibodies 8H5, 3C8, 10F7, 4D1 and 2F2 can be The nucleotide sequence of the variable region. In one aspect, the invention provides an anti-H5 antibody comprising a heavy chain variable region comprising as in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 17. The amino acid sequence mentioned in SEQ ID NO: 21 and SEQ ID NO: 25. In another aspect, the present invention provides an anti-H5 antibody comprising a light chain variable region comprising as described in SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID Amino acid sequences of NO: 19 and SEQ ID NO: 27.

In another aspect, the present invention provides the amino acid sequences mentioned in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 17, SEQ ID NO: 21 and SEQ ID NO: 25 Antibody heavy chains having variable regions of at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity , more preferably have at least 90% sequence identity, most preferably have at least 95% sequence identity.

In another aspect, the invention provides an antibody light chain comprising a variable region that is identical to SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 19 and SEQ ID NO: 27 The amino acid sequences mentioned in have at least 70% sequence identity, preferably at least 75% sequence identity, more preferably at least 80% sequence identity, more preferably at least 85% sequence identity, More preferably at least 90% sequence identity, most preferably at least 95% sequence identity.

The amino acid sequences of the CDRs of the variable regions of the heavy and light chains of monoclonal antibodies 8H5, 3C8, 10F7, 4D1, 3G4 and 2F2 have been identified as follows:

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 8H5 are mentioned in SEQ ID NO: 28-30, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of monoclonal antibody 8H5 are mentioned in SEQ ID NO: 31-33, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 3C8 are mentioned in SEQ ID NO: 34-36, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of monoclonal antibody 3C8 are mentioned in SEQ ID NO: 37-39, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 10F7 are mentioned in SEQ ID NO: 40-42, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of monoclonal antibody 8H5 are mentioned in SEQ ID NO: 43-45, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 4D1 are mentioned in SEQ ID NO: 46-48, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of monoclonal antibody 4D1 are mentioned in SEQ ID NO: 49-51, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 3G4 are mentioned in SEQ ID NO: 52-54, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of monoclonal antibody 3G4 are mentioned in SEQ ID NO: 55-57, respectively.

The amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain of monoclonal antibody 2F2 are mentioned in SEQ ID NO: 58-60, respectively. The amino acid sequences of CDR1, CDR2 and CDR3 of the light chain of monoclonal antibody 2F2 are mentioned in SEQ ID NO: 61-63, respectively.

Table 1 Six strains of monoclonal antibody CDR amino acid sequences

In another aspect, the invention provides an anti-H5 monoclonal antibody heavy chain or fragment thereof comprising the following CDRs: (i) one or more CDRs selected from SEQ ID NO: 28-30; (ii) selected from SEQ ID NO: 28-30; (iii) one or more CDRs selected from among SEQ ID NO:40-42; (iv) one or more selected from SEQ ID NO:46-48 (v) one or more CDRs selected from SEQ ID NOs: 52-54; or (vi) one or more CDRs selected from SEQ ID NOs: 58-60. In one embodiment, the anti-H5 monoclonal antibody heavy chain or a fragment thereof includes three CDRs having the amino acid sequences mentioned in SEQ ID NO: 28-30, respectively. In another embodiment, the anti-H5 monoclonal antibody heavy chain or a fragment thereof includes three CDRs having the amino acid sequences mentioned in SEQ ID NO: 34-36, respectively. In another embodiment, the anti-H5 monoclonal antibody heavy chain or fragment thereof comprises three CDRs having the amino acid sequences set forth in SEQ ID NO: 40-42. In another embodiment, the anti-H5 monoclonal antibody heavy chain or fragment thereof comprises three CDRs having the amino acid sequences set forth in SEQ ID NO: 46-48. In another embodiment, the anti-H5 monoclonal antibody heavy chain or a fragment thereof comprises three CDRs having the amino acid sequences set forth in SEQ ID NO: 52-54. In another embodiment, the anti-H5 monoclonal antibody heavy chain or a fragment thereof includes three CDRs having the amino acid sequences mentioned in SEQ ID NO: 58-60, respectively.

In another aspect, the CDRs contained in the anti-H5 monoclonal antibody heavy chain or fragment thereof of the present invention may be included in SEQ ID NO: 28-30, 34-36, 40-42, 46-48 and 58-60 Substitution, addition and/or deletion of one or more amino acids in the mentioned amino acid sequence. Preferably, amino acid substitutions, additions and/or deletions may only occur at three amino acid positions. More preferably, amino acid substitutions, additions and/or deletions may only occur at two amino acid positions. More preferably, amino acid substitutions, additions and/or deletions may only occur at one amino acid position.

In another aspect, the invention provides an anti-H5 monoclonal antibody light chain or fragment thereof comprising the following CDRs: (i) one or more CDRs selected from SEQ ID NO: 31-33; (ii) selected from SEQ ID NO: 31-33; (iii) one or more CDRs selected from among SEQ ID NO:43-45; (iv) one or more selected from SEQ ID NO:49-51 (v) one or more CDRs selected from SEQ ID NOs: 55-57; or (vi) one or more CDRs selected from SEQ ID NOs: 61-63. In one embodiment, the anti-H5 monoclonal antibody light chain or a fragment thereof includes three CDRs having the amino acid sequences mentioned in SEQ ID NO: 31-33, respectively. In another embodiment, the anti-H5 monoclonal antibody light chain or a fragment thereof includes three CDRs having the amino acid sequences mentioned in SEQ ID NO: 37-39, respectively. In another embodiment, the anti-H5 monoclonal antibody light chain or fragment thereof comprises three CDRs having the amino acid sequences set forth in SEQ ID NO: 43-45. In another embodiment, the anti-H5 monoclonal antibody light chain or fragment thereof comprises three CDRs having the amino acid sequences set forth in SEQ ID NO: 49-51. In another embodiment, the anti-H5 monoclonal antibody light chain or fragment thereof comprises three CDRs having the amino acid sequences set forth in SEQ ID NO: 55-57. In another embodiment, the anti-H5 monoclonal antibody light chain or a fragment thereof includes three CDRs having the amino acid sequences mentioned in SEQ ID NO: 61-63, respectively.

In another aspect, the CDRs contained in the light chain of the anti-H5 monoclonal antibody of the present invention or a fragment thereof may include sequences from SEQ ID NO: 31-33, 37-39, 43-45, 49-51, 55-57 and One or more amino acids substituted, added and/or deleted from the amino acid sequences mentioned in 61-63. Preferably, amino acid substitutions, additions and/or deletions may only occur at three amino acid positions. More preferably, amino acid substitutions, additions and/or deletions may only occur at two amino acid positions. More preferably, amino acid substitutions, additions and/or deletions may only occur at one amino acid position.

Table 2 is bound to the amino acid sequence of the 7aa peptide of 8H5mAb or 3C8mAb

Variants generated by substitution, addition and/or deletion of amino acids in the variable regions of the antibodies described above or the CDRs described above retain the ability to specifically bind to subtype H5 avian influenza virus. The invention also includes antigen-binding fragments of such variants.

The monoclonal antibody variants of the present invention can be prepared by conventional fundamental engineering methods. Nucleotide mutations can be introduced into DNA molecules using methods known to those of ordinary skill in the art. Alternatively, the nucleotide molecules encoding the heavy and light chains can be prepared by chemical synthesis.

In another aspect, the screening method of the present invention comprises the following steps: (i) cultivating a peptide display library under conditions suitable for peptide expression; (ii) contacting the culture solution with the monoclonal antibody of the present invention; (iii) selecting phage clones that specifically bind to the monoclonal antibody. The monoclonal antibodies used for screening may include, but are not limited to, monoclonal antibodies 8H5, 3C8, 10F7, 4D1 and 3G4. Example 12 included herein details an assay using a peptide phage display library for the successful screening of short peptides that bind to the monoclonal antibodies of the invention.

Table 3 is bound to the sequence of the 12aa peptide of 8H5mAb

The term "analyte" as used herein refers to a compound or composition to be detected or assayed, and has at least one epitope or binding site. The analyte can be any substance for which a naturally occurring analyte-specific binding member exists or for which an analyte-specific binding member can be prepared, eg, carbohydrates and lectins, hormones and receptors, complementary nucleic acids, and the like. Furthermore, possible analytes include virtually any compound, composition, aggregate or other substance that can be immunodetected. That is, the analyte or portion thereof should be an antigen or hapten having at least one determinant site, or should be a member of a naturally occurring binding pair.

Analytes include, but are not limited to, toxins, organic compounds, proteins, peptides, microorganisms, bacteria, viruses, amino acids, nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs (including those used for therapeutic Drugs used for legitimate purposes), pollutants, pesticides and metabolites, or antibodies to any of the above substances. The term "analyte" also includes any antigenic substance, hapten, antibody, macromolecule and combinations thereof. A non-exhaustive list of exemplary analytes is set forth at column 19, line 7 through line 26 through line 42 of US Patent No. 4,366,241, the disclosure of which is incorporated herein by reference. Further descriptions and enumerations of representative analytes are found in US Patent Nos. 4,299,916, 4,275,149 and 4,806,311, all of which are incorporated herein by reference. In some embodiments, the SCD or test device is configured to detect multiple different analytes (eg, Figure 26).

labeling reagent

"Labeling reagent" is meant to include a detectable label (eg, a detection probe) attached to a specific binding member. The attachment can be covalent or non-covalent, but the method of attachment is not critical to the invention. The label enables the labeling reagent to produce a detectable signal that correlates with the presence of the analyte in the fluid sample. The specific binding member of the labeling reagent is selected to bind directly to the analyte or indirectly to the analyte by assisting the specific binding member, as described in more detail below. Labeling reagents may be incorporated into the test device upstream of the site from the capture zone, may be combined with a fluid test train to form a fluid solution, may be added to the test device separately from the test sample, or may be deposited or otherwise immobilized at the capture zone. Furthermore, specific binding members may be labeled before or during assay performance by suitable attachment methods.

"Mark" means any substance capable of generating a signal detectable by visual or instrumental means. Various labels suitable for use in the present invention include labels that generate signals by chemical means or physical means. Such labels may include enzymes and substrates, chromogens, catalysts, fluorophores or fluorophore-like compounds and/or particles, magnetic compounds and/or particles, chemiluminescent compounds and/or particles, and radioactive labels. Other suitable labels include particulate labels, for example, colloidal metal particles such as gold, colloidal non-metallic particles such as selenium or tellurium, dyes or colored particles such as dyed plastics or dyed microorganisms, organic polymer latex particles and Liposomes, colored beads, polymer microcapsules, sacs, erythrocytes, erythrocyte ghosts, or other vesicles that directly contain visible substances, etc. Typically, a visually detectable label is used as a label for the labeling reagent, thereby providing a direct visual or instrumental readout of the presence or amount of the analyte in the test sample without generating an additional signal of the component at the detection site.

Additional labels that may be utilized in the practice of the present invention include chromophores, electrochemical moieties, enzymes, radioactive moieties, phosphorescent groups, fluorescent moieties, chemiluminescent moieties, or dots, or more specifically, radiolabels, fluorophores labeling, quantum dot labeling, chromophore labeling, enzyme labeling, ligand affinity labeling, electromagnetic spin labeling, heavy atom labeling, probes labeled with nanoparticle light scattering or other nanoparticles, fluorescein isothiocyanate ( FITC), TRITC, rhodamine, tetramethylrhodamine, R-phycoerythrin, Cy-3, Cy-5, Cy-7, Texas Red, Phar-red, other Allophycocyanin (APC), epitope tags such as FLAG or HA epitopes and enzyme tags such as alkaline phosphatase, horseradish peroxidase, ^I2 -galactosidase, alkaline Phosphatases, β-galactosidases, or acetylcholinesterases such as digoxigenin or dinitrophenyl and hapten conjugates, or capable of forming such as streptavidin/biotin, avidin Members of binding pairs of complexes of protein/biotin or antigen/antibody complexes including, for example, rabbit IgG and anti-rabbit IgG; fluorophores such as umbelliferone, fluorescein, isothiocyanate Fluorescein, rhodamine, tetramethylrhodamine, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green green), stilbene, lucifer yellow, Cascade Blue, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, such as those including Phosphor lanthanide complexes of europium and terbium, Cy3, Cy5, molecular beacons and their fluorescent derivatives, luminescent materials such as luminol; astigmatic or plasmon resonant materials such as gold or silver particles or quantum dots; or radioactive materials including ¹⁴ C, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, Tc99m, ³⁵ S, or ³ H; alternatively, spherical shells and generated labels using any other method known to those skilled in the art signal-labeled probes. For example, detectable molecules include, but are not limited to, fluorescein and others known in the art as described in the following publications: e.g., Principles of Fluorescence Spectroscopy, Joseph R. Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the ^6th Edition of the Molecular Probes Handbook by Richard P. Hoagland.

A number of signal generating systems can be employed to achieve the objects of the present invention. A signal generating system generates a signal related to the presence of an analyte (ie, target molecule) in the sample. A signal generating system may include all reagents required to generate a measurable signal. Other components of the signal generating system may be included in the developing solution and may include substrates, enhancers, activators, chemiluminescent compounds, cofactors, inhibitors, scavengers, metal ions, binding specific binding substances requiring signal generation, etc. . Other components of the signal generating system may be coenzymes, reacting with enzyme products, other enzymes and catalysts, and the like. In some embodiments, where visual inspection is desired, the signal generating system provides a signal detectable by an external tool through the use of electromagnetic radiation. An exemplary signal generating system is described in US Patent No. 5,508,178.

In some embodiments, a nucleic acid molecule can be linked to a detection probe (eg, an antibody-linked oligonucleotide), such that the nucleic acid molecule functions as a label utilizing the nucleic acid label. For example, a reagent solution or substrate included in an SCD may include a detection reagent—a plurality of detection and capture specific binding agents (SBAs)—comprising a plurality of oligonucleotides that act to provide a detectable signal such that for a given subpopulation of SBAs (specific for a particular analyte), the conjugated oligonucleotides were prestained with a different stain, while antibodies against another subpopulation (specific for a different analyte) were serially separated Nucleic acid stains that bind nucleic acid molecules in a specific manner. Examples include intercalating stains such as phenanthridine and acridinium (e.g., ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1, and ethidium homodimer monoazide-2, ethidium monoazide, and ACMA); some microgrove binders, for example, indole and imidazole (for example, Hoechst 33258, Hoechst 33342, Hoechst 34580, and DAPI); and promiscuous nucleic acid stains, for example, acridine Orange (can also be embedded), 7-AAD, actinomycin D, LDS751 and hydroxystilbamidine. All of the above-mentioned nucleotide stains are commercially available from suppliers (eg, Molecular Probes, Inc). Other examples of nucleotide stains include the following stains from Molecular Probes: Cyanine dyes such as SYTOX Blue, SYTOX Green, SYTOX Orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1 , TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO-3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1 , TO-PRO-3, TO-PRO-5, JO-PRO-1, LO-PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR Gold, SYBR Green I, SYBR Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11 , -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), YTO-64, -17, -59 , -61, -62, -60, -63 (red). Other detectable labels include chemiluminescent and chromogenic molecules, optical or electron density labels, and the like.

As described in certain embodiments, the markers comprise semiconductor nanocrystals, eg, quantum dots (ie, Qdots) described in US Patent No. 6,207,392. Qdot is commercially available from Quantum Dot Corporation. Semiconductor nanocrystals useful in the practice of the present invention include: nanocrystals of Group II-VI semiconductors, for example, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, ZnS , ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, and HgTe, and their mixed compositions; nanocrystals of III-V semiconductors, for example, GaAs, InGaAs, InP, and InAs, and their mixed compositions. Group IV semiconductors such as germanium or silicon, or organic semiconductors are also feasible under certain conditions. Semiconductor nanocrystals may also include alloys comprising two or more semiconductors selected from the group consisting of the above Group III-V compounds, Group II-V compounds, Group IV elements, and combinations thereof .

In some embodiments, a fluorescent receptor is attached as a label to a detection probe (ie, a binding moiety conjugated to a detection molecule). In one embodiment, the fluorescent energy acceptor molecule can be formed by reacting the compound with pure oxygen to form a fluorescent compound, or the compound can be reacted with an auxiliary compound converted therefrom to form a fluorescent compound. Said auxiliary compounds may be included in the buffer contained in the SCD and/or test device. In other embodiments, a fluorescent energy receptor can be included as part of a compound that also includes a chemiluminescent agent. For example, fluorescent energy acceptors may include metal chelates of rare earth elements such as europium, samarium, tellurium, and the like. These materials are particularly attractive due to their sharp luminescent bands. In addition, lanthanide labels such as europium(III) provide efficient, long-term signal emission and are resistant to photobleaching, allowing test devices containing treated/reacted samples to be set aside for extended periods of time if desired.

Long-lived fluorescent europium(III) chelate nanoparticles have been shown to serve as labels in various heterogeneous and homogeneous immunoassays. See, eg, Huhtinen et al. Clin. Chem. 2004 Oct;50(10):1935-6. Assay performance can be enhanced when these inherently labeled nanoparticles are used in conjunction with time-resolved fluorescence detection. In heterogeneous assays, the dynamic range of the assay at low concentrations can be extended. In addition, the dynamic properties of the assay can be improved by using highly specific activity nanoparticle labels coated with antibodies instead of conventionally labeled detection antibodies. In homogeneous assays, europium(III) nanoparticles have been shown to be efficient donors in fluorescence resonance energy transfer, enabling simple and rapid high-throughput screening. Heterogeneous and homogeneous nanoparticle label-based assays can be used with various sample matrices such as serum, heparin, plasma, and mucus.

In some embodiments, a label disclosed herein (eg, a fluorescent label) is included as a nanoparticle label conjugated to a biomolecule. In other words, nanoparticles can be utilized using detection probes or capture probes. For example, in the practice of the invention (e.g., nanoparticle-based immunoassays), one can utilize europium(III)-labeled nanoparticles that are linked to monoclonal antibodies or streptavidin to detect sample analytes in . The nanoparticles serve as a substrate to which a specific binding agent is attached and which binds the analyte to either the detection (ie label) moiety or the capture moiety.

In various embodiments of the invention, the labels utilized are lanthanide metals. Lanthanides include, but are not limited to, europium, samarium, terbium, or dysprosium. Non-specific background fluorescence has a decay time of only about 10 ns, so such background disappears before the sample fluorescence is measured. In addition, lanthanide chelates have large Stokes shifts. For example, the Stokes shift of europium is almost 300nm. Such a large difference between excitation and emission peaks means that fluorescence measurements are made at wavelengths where background influence is minimal. In addition, the emission peaks are very sharp, which means that the detector can be set to very fine limits and the emission signals of different lanthanide chelates can be easily distinguished from each other. Thus, in one embodiment, one lanthanide or multiple different lanthanides may be used in the same assay.

Hard standard. In one embodiment, the fluorescence reader is configured to include an integral or permanent standard ("hard standard"). As used herein, the term "hard standard" means a device, including internal, integrated or permanent standards, for reading a test sample in such a way as to detect/quantify one or more The same marker used in the piece to tag the sample is read in terms of. In one embodiment, the hard standard and test markers include lanthanides (eg, europium III). Figure 33A to Figure 33H.

In one embodiment, the reader is an LED comprising a lamp emitting in the UVA (400nm-315nm) portion of the spectrum. Emission is in the visible part of the spectrum. Some exemplary or conventional LEDs or photodiodes are disclosed in US Patent Nos. 7,175,086, 7,135,342, and 7,106,442, the contents of which are fully incorporated herein.

In another embodiment, the reader includes at least two hard standards of differing quantities (eg, high concentration markers and low concentration markers), thereby providing a two-point calibration of the reader. For example, two (2) lanthanide hard standards (such as europium) are permanently mounted on the reader slide and can be read during each test read. In this way, two hard standards can be used in order to determine lower detection limits (ie, during analyte quantitative determination or for determining the lowest detection threshold in qualitative determination). Here, fluorescence is read and plotted as percent fluorescence (y-axis) versus concentration (x-axis). The straight line between the two readings for each hard standard on this graph allows the intercept of the noise (no markers) to be measured, giving the lowest detection limit measurement.

In some embodiments, the test device includes a chamber (compartment or fluid sac) containing a wash or running buffer for removal of unbound label thereby reducing or eliminating background noise. In various embodiments, a device including a hard standard provides accurate qualitative and quantitative determination of an analyte present in a sample and labeled with a label, wherein the label is the same as that used in the hard standard.

In some embodiments, rigid standards are embedded or cast in glass, polymeric materials including plastic, vinyl, or acrylic (FIG. 33B). The embedded markers can be molded to a suitable shape/size. Alternatively, such rigid standards may be cut to size to be integrated into the reader. In one embodiment, the rigid standard is cut into rectangles, squares, rectangles, circles or arbitrary polygons. In one embodiment, the rigid standard is cut into a rectangular shape comprising a height of about 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.10, 0.11, 0.12, 0.125, 0.126, 0.127, 0.128, 0.129, 0.130, 0.135, 0.140, 0.150 inches, about 0.01, 0.02, 0.03, 0.035, 0.039, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0. , 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95 or 1.0 inches, about 0.01, 0.02, 0.03, 0.035, 0.039, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1.0 inches in size.

In one embodiment, readers using a hard standard as a reference are used to normalize relative to a population, e.g., to plot subsequent reader performance relative to a predetermined "gold standard," as shown in the table below :

Table 4

Gold standard test S0 1000 900 S1 5400 5000 S2 10200 11000 S3 19000 20000 S4 22000 23000 S5 50000 50000

Thus, with test reader and reference standard measurements on the y- and x-axes, respectively, the lower detection limit is the pitch at which the plotted line intersects the noise level (read without markers).

In one embodiment, the test device includes different pRNAs, each of which is patterned based on a specific analyte, and the complementary SCD includes a plurality of capture antibodies linked to a pRNA homologous to the immobilized pRNA on the test device. pRNA, and the plurality of capture antibodies include different subpopulations of antibodies specific for different analytes. In addition, the SCD reagent solution or substrate (eg, lyophilized solid substrate) includes multiple europium(III)-labeled antibodies or detection probes composed of the same subpopulation of antibodies specific for different analytes. Additional lanthanide labels that may be implemented in the present invention are known in the art, for example, as disclosed in US Patent No. 7,101,667. See also, for example, Richardson F.S., "Terbium(III) and Europium(III) Ions as Luminescent probes and Stains for Biomolecular Systems" (Terbium(III) and Europium(III) Ions as Probes and Stains for Biomolecular Systems ) Chem. Rev., 82:541-552 (1982).

Thus, depending on the marker chosen, in some embodiments the signal will be visible to the naked eye, while in other embodiments a reading instrument is utilized in the practice of the invention.

capture part

In some embodiments, one member of a pair of complementary capture moieties will bind to the analyte-specific binding agent and the other member will be immobilized on a thread or spot, respectively (Figure 8A). As noted herein, the term "capture moiety" refers to a binding moiety specific for a partner or a complementary capture moiety (e.g., a pRNA specific for a complementary pRNA, or an avidin/streptavidin avidin/streptavidin-biotin). In one embodiment, the test device comprises a combination of different capture moieties, wherein the capture moieties comprise different amounts and/or types of substances. For example, a test strip disposed in a test device may include one or more addressable test lines comprising pRNA, antibody, and specific binding member (eg, avidin/biotin). Thus, in various embodiments where two capture moieties of the same type are disposed on a test strip, each capture moiety is specific for a different complementary partner molecule. For example, for two pRNA addressable lines, each line would include a pRNA with a different sequence that would specifically bind to a complementary sequence pRNA that itself binds to a target for a different analyte. (eg, antibodies to influenza A as opposed to influenza B). Furthermore, in such configurations, additional test lines may comprise different capture moieties (such as antibodies or avidin) that themselves bind their cognate counterpart molecules. Thus, in one embodiment, the test strip comprises 2, 3, 4, 5, 6, 7 or 8 addressable test lines which may comprise 1, 2, 3 or 4 different types of capture moieties .

Where multiplex (i.e., multiple analyte) detection is desired, utilizing multiple capture moieties, an antibody specific for an analyte will include a member of a specific pair of complementary capture moieties to specifically bind Antibodies to the second and different analyte will include members of the second and different specificity pair of complementary capture moieties, and so on. Thus, in one embodiment, a plurality of different analytes can be detected, wherein cognate members of a pair of capture moieties are immobilized at discrete locations on a test membrane included in a test apparatus. For example, the plurality of antibodies in the SCD includes antibodies against multiple influenza virus strains and/or subtypes, wherein the antibodies include a detection antibody-capture antibody pair, the capture antibody having a specific capture moiety. Furthermore, each family of antibodies in the plurality of antibodies is defined by the specific target analyte to which the antibody binds. Thus, all capture antibodies directed against a specific target analyte will have the same capture moiety, for which reason homologous/complementary capture moieties are provided in the test device.

In various embodiments, capture moieties include oligonucleotides, avidin, streptavidin, pyranosyl RNA (pRNA), antigen-antibody binding pairs selected for high affinity, Aptamers or their combination. In additional embodiments, the oligonucleotide is DNA or RNA. Furthermore, in some embodiments, different combinations of capture moieties are utilized in the same detection system of the invention. For example, capture moieties for one particular analyte include oligonucleotide pairs, while capture pairs for a different analyte include capture moieties comprising pRNA, or avidin or streptavidin, among others. In other embodiments, combinations of different types of capture moieties are utilized in the devices and assays of the invention to detect multiple analytes (e.g., multiple capture antibodies, whereby each family specific for a single analyte The capture antibody is linked to one capture moiety and other antibodies specific for the analyte are linked to other capture moieties such as aptamers, pRNA or streptavidin).

In one embodiment, all capture moieties are pRNA, and there are multiple pairs of pRNA capture moieties and pRNA partner capture moieties (eg, one conjugated to a specific binding agent, cognate pRNA immobilized on a lateral flow membrane).

pRNA

In one aspect of the invention, a combination of complementary pyran RNA (pRNA) sequences is included in an SCD/test device of the invention capable of specific detection of multiple analytes simultaneously ( FIG. 32 ). In various embodiments, one of a pair of homologous pRNA sequences is immobilized in a test area or a specific strip in a test device, and the other of the pair of homologous pRNA sequences is conjugated to a binding moiety that is specific for binding to target analytes. Thus, in one embodiment, the analyte of interest is captured by the immobilized pRNA by interacting with the immobilized pRNA sequence with its binding pair pRNA. The analyte is detected by a detection molecule conjugated to a second binding moiety 1301, which is also specific for the same target analyte.

In some embodiments, pRNA binding partners are selected from, but are not limited to, the following pRNAs.

table 5:

All oligonucleotides have a 4'-C12 amino group and a 2'-hexanol group

Thus, in the case of multiple capture probes (eg antibodies linked to pRNA), each capture probe is linked to a capture moiety, for which purpose the cognate capture probes are immobilized on a test strip 310 comprised in the test device, 903, 904, 905, 906 in predetermined positions (Fig. 3, Fig. 26, Fig. 27A to Fig. 27C). See also Figures 39-42, which illustrate the actual sensitivity of the pRNA trap.

Thus, a sequence immobilized on a specifically addressable thread on a test strip will specifically bind to a complementary pRNA conjugated to an anti-analyte binding moiety (eg, an antiviral antibody). The efficacy of such specificity is illustrated in Example 6 (eg, Figure 30A and Figure 30B).

In some embodiments, a test device comprising one or more pRNA binding pairs provides about 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 ,0.9,1.0,1.2,1.5,1.7,2.0,2.5,3.0,3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5,8.0,8.5,9.0,9.5,10.0,15,20,30 , 40 or 50 ng/mL sensitivity.

In some embodiments, the pRNA is attached to the membrane (ie, test strip) using a protein linker. For example, pRNA can be conjugated to a hydrophilic protein. In one embodiment, the adapter protein has at least from about 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7500, 8000, 9000、10000、20000、30000、40000、50000、60000、70000、80000、90000、100000、110000、120000、130000、140000、150000、160000、170000、180000、190000、200000、225000、250000、300000、350000至Molecular weight of about 450,000. Such adapters may be in sizes from about 5 to 10, 6 to 11, 7 to 12, 8 to 13, 9 to 14, 10 to 15, 11 to 16, 12 to 17, 13 to 18, 14 to 19, 15 to 20, 16 to 21, 17 to 22, 18 to 23, 19 to 24, 20 to 25, 21 to 26, 22 to 27, 23 to 28, 24 to 29, 25 to 30, 35, 40, 45 or 50AA long . Linkers can be peptides or polypeptides. In one embodiment, the linker is BSA or IgG.

In one embodiment, the pRNA is bound to the hydrophilic protein/peptide through a covalent bond between the pRNA molecule and the hydrophilic protein. A solution containing the pRNA-protein complex is applied to a defined area (eg nitrocellulose) on the test membrane, whereby the protein anchor is irreversibly bound to the membrane. Subsequently, this pRNA can be used in an assay. In one embodiment, the ankyrin/connexin is a hydrophilic protein and the test membrane is nitrocellulose.

As noted previously, test devices may include addressable test lines utilizing different types of capture moieties (eg, antibody, nucleic acid, pRNA, avidin/streptavidin/biotin in combination). In one embodiment (eg, FIG. 32 ), at least one addressable line or specific capture region 1305, 1312 comprises a pRNA 1304, 1311 sequence bound to a solid support 1308 (eg, nitrocellulose, poly styrene, glass, plastic, metal, etc.) and is specific in binding to a cognate pRNA sequence conjugated to a second binding moiety 1302, 1310 specific for a particular target analyte 1306 . Furthermore, the second binding molecule is the counterpart of the first binding molecule, said second binding molecule being conjugated to a detection molecule 1301, 1309 (eg a fluorescent label). As demonstrated here in Example 6, pRNA is efficiently immobilized and can specifically bind complementary sequences, allowing detection of analytes. Thus, pRNA provides a new tool for the detection of multiple analytes based on the large specificity and affinity of the pRNA molecule.

In one embodiment, pRNA molecules are disposed on 1, 2, 3, 4, 5, 6 or 7 different addressable lines (ie capture zones) in the test device. In other embodiments, pRNA is used in combination with antibodies, nucleic acid binding pairs, avidin/streptavidin, digoxin/anti-digoxigenin. For example, the test device includes a test strip with 5 addressable test lines/capture zones, where each test zone is sensitive to a different analyte (eg, influenza A or influenza B) or subtype (eg, influenza A's pandemic and non-pandemic subtypes) are specific. Thus, for example, two test areas use pRNA binding, while one uses streptavidin/avidin-biotin, another uses immobilized antibodies, and yet another uses DNA/RNA. In this example, for each type of binding system there is a complementary binding partner specific for the target analyte "captured" on a particular test zone. In addition, the analyte is also bound by a conjugate conjugated to a detection molecule/label (eg, a fluorescent label), as described herein. Thus, once the Analyte-detector-binding partner complex (Analyte-detector-binding partner complex) flows through the test device, the complex is captured in a different test zone with an immobilized binding partner.

Thus, a core aspect of these SCD/test devices of the invention is that they can be configured to detect a variety of analytes, including cells, cellular components (eg, cell markers, cell surface markers), proteins (eg, enzymes), and the like.

In one embodiment, the SCD/test device of the present invention may be used in a method of assaying any pathogenic condition for which the specific corresponding analyte is known or will be determined in the future. SCDs and test devices can be configured to provide any combination of detection reagents disclosed herein (eg, pRNA, nucleic acid, antibody, specific binding partner (eg, avidin/biotin)). For example, during the detection/diagnosis of myocardial infarction (MI), a plurality of analytes corresponding to MI can be determined. Markers for a variety of situations are known in the art, such as cardiac markers disclosed in US Pat.

In one embodiment, a device of the invention (see Example 10) will be designed that provides a three-in-one assay for proteinaceous cardiac markers of myocardial infarction.

In one embodiment, the sample is applied in such a way that it can be mixed with a mixture (serial or parallel) of first conjugates conjugated to a detection reagent (e.g. a combination of gold particles and a fluorescent label), and capable of being mixed with a second binder conjugated to a first member of a pRNA cognate binding pair, wherein the second binder may or may not comprise an immunological binding reaction and/or a pRNA cognate binding pair specific binding material.

If the system is designed with the desired pre-incubation (pre-incubation) such that the first and second binders are able to attach to the analyte and bridge the analyte to form a complex, then such a step can be at the reagent to the test zone or During capture zone flow. Optionally, flow can be stopped to bring the reaction closer to completion. In yet another embodiment, the reagents are mixed in the SCD of the present invention prior to being applied to the test device.

The mixture flows along the surface of or within the solid material/substrate (e.g., a test strip) by capillary action or hydrostatic pressure by any of several mechanisms, or by other non-capillary action, The first and second cognate binding pair are brought into contact as the reaction mixture passes/passes through the test/capture zone. If the first and second binders have bridged analytes (Figure 32), the detection reagent (e.g. europium) will accumulate in the test/capture zone, creating parsed signal.

In embodiments where detection of multiple different analytes is attempted, different detection reagents are used for each analyte, each different analyte being captured in a single detection/capture zone. A device reader was used to differentiate the signal for each analyte. In one embodiment, a device reader can detect the signal, providing both qualitative and quantitative measurements.

In another embodiment, for each target analyte, a unique pRNA cognate binding pair is utilized.

In another embodiment, a wash buffer or reagent buffer is released through a flow path (as described herein), wherein the buffer may include energy release that causes any of several forms, available The device, reader, or visually detects any of the several forms of energy release described. For example, the reaction may result in the release of light, electrons, or may require the input of electrons to the substrate to release light. Such reactions are well known to those skilled in the art.

After contacting the fluorescent compound with the running/wash buffer, such a reaction can cause the fluorescent compound to be primed to fluoresce, resulting in the release of electrons, protons, neutrons, which can be detected by a device or reader as described here , The release of neutrons.

Aptamer

In some embodiments, the capture moiety is an aptamer molecule that can be utilized interchangeably with a capture probe, or can be used interchangeably as an immobilized capture moiety included in the axial flow membrane of the test device. Aptamers include nucleic acids identified from a candidate mixture of nucleic acids. In preferred embodiments, the aptamer comprises a nucleic acid sequence substantially homologous to the nucleic acid ligand isolated by the SELEX method. Substantial homology means that the degree of major sequence identity exceeds 70%, most preferably exceeds 80%. The "SELEX" method used herein involves the combination of selected nucleic acid ligands that interact with the target analyte in a desired effect, such as binding to a protein, such that those selected nucleic acids are amplified. An optional iterative selection/amplification step may select for one or a small number of nucleic acids that most strongly interact with the target antigen/biomarker from a pool containing a very large amount of nucleic acid. Cycles of selection/amplification steps are performed continuously until the selected target is achieved. The SELEX method is described in the following US patents and patent applications: US Patent Application Serial No. 07/536,428 and US Patent Publication Nos. 5,475,096, 5,270,163.

infectious agent

In various embodiments of these compositions and methods, the infectious agent can be any pathogen including, but not limited to, bacteria, yeast, fungi, viruses, eukaryotic parasites, and the like. In some embodiments, the infectious agent is influenza virus, parainfluenza virus, adenovirus, rhinovirus, coronavirus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, HIV, enterovirus, pap papillomavirus, coxsackievirus, herpes simplex virus, or Epstein-Barr virus. In other embodiments, the infectious agent is Mycobacterium, Streptococcus, Salmonella, Shigella, staphylcococcus, Neisseria, Clostridium, or coli. It will be apparent to those skilled in the art that the compositions and methods of the invention are readily adaptable to different assays by utilizing different assay series of binding agents (e.g., antibodies) specific for the type or subtype of infectious agent. infectious agent.

Generally, the common type of an infectious agent may be a generic type of an infectious agent, or any initial or first typing or identification of an infectious agent. The classification of the infectious agent may be the type or species of the infectious agent, or any secondary or subsequent classification of the infectious agent. According to the present invention, the identification of common types or subtypes of infectious agents can be performed by various suitable test setups. For example, identification of a common type of an infectious agent may include one or more screening tests for 1) a specific common type of an infectious agent, 2) a specific desired or selected common type of an infectious agent, Or 3) all or substantially all relevant common types of infectious agents, or combinations thereof. Similarly, identification of subtypes of infectious agents may include one or more screening tests for: 1) one or more specific subtypes of infectious agents; 2) specific subtypes of infectious agents; 3) One or more specific subtypes of the infectious agent selected based on additional information related to the subject, for example, a specific population or geographic location One or more suspected or expected subtypes; 4), one or more potentially pandemic or epidemic subtypes of an infectious agent that is identical or related to the infectious agent tested for the common type, or a combination thereof.

According to another aspect of the present invention, the method provided by the present invention may optionally or additionally comprise identification of common types and/or subtypes of a second infectious agent closely related to the first infectious agent, or alternatively, the second infectious agent Infection by the second infectious agent is associated with, or may be combined with, infection by the first infectious agent. In one embodiment, the methods provided herein include the identification of common types and subtypes of viruses and bacteria. For example, HIV infection may be associated with a specific bacterial infection, so it would be useful to identify common and subtypes of HIV as well as mycobacteria and/or Pneumocystis carina. In particular, the present invention provides methods involving the identification of HIV and one or more types of mycobacteria and/or Pneumocystis carinii.

In another embodiment, the methods provided herein include identification of common types and subtypes of the first virus and the second virus. For example, the methods provided herein may include identification of common types and subtypes of HIV and hepatitis viruses, or alternatively, may include identification of common types and subtypes of HIV and common types of hepatitis viruses or specific strains of hepatitis viruses . In one embodiment, the methods and compositions of the invention can be utilized in assays for the detection of E. coli 0157, a very dangerous and often fatal infectious species, in the presence of other enteric or infectious species. Another example would be to test patients for influenza infection, where it is known that mutations or variations of strains within subtypes will occur and that some forms of influenza are much more pathogenic than others. Yet another example is testing for different types of HIV, such as testing for HIV-1 and HIV-2. In one aspect, identification of common types of human immunodeficiency virus (HIV) may include screening for the presence of HIV, while identification of subtypes of HIV may include screening for HIV-1, HIV-2, and/or other HIV subtypes. type to filter. Similarly, identification of common types of herpesviruses such as herpes simplex virus (HSV) may involve screening for the presence of HSV, whereas identification of subtypes of HSV may include screening for HSV type 1 and/or HSV type 2, or screening for the presence of HSV Epstein-Barr virus and/or EBV subtypes were screened.

In yet another specific aspect, identification of common types of enteroviruses may include identification of one or more enteroviruses (e.g., poliovirus, coxsackievirus, echovirus , specified enteroviruses, etc.), while identification of subtypes of enteroviruses may include screening for polioviruses (e.g., serotypes 1-3), Cuxackievirus A (e.g., serotypes 1-22 and 24 type), Cuxackievirus B (eg, serotypes 1-6), echoviruses (eg, serotypes 1-9, 11-27, 29-31), and specified enteroviruses (eg, enterovirus 68-71) etc. to filter.

Typically, for bacterial infectious agents, identification of common types and subtypes of bacterial infectious agents includes the identification of the genus and one or more classes or strains of bacterial infectious agents associated with infection and/or resistance to the infectious agent. filter. In one embodiment, identification of common types and subtypes of bacterial infectious agents comprises screening for mycobacteria and one or more classes of mycobacteria, including but not limited to tuberculosis, avium, bovis , chelonei, fortuitum, intracellulare, kansasii, leprae, etc. In another embodiment, identification of common types and subtypes of a bacterial infectious agent comprises screening for Salmonella and one or more classes of Salmonella, including but not limited to typhi, enteritis, and the like. In yet another embodiment, identification of common types and subtypes of a bacterial infectious agent comprises screening for Shigella and one or more classes of Shigella, including but not limited to dysenteriae . In yet another embodiment, identification of common types and subtypes of a bacterial infectious agent comprises screening for Streptococcus and one or more classes of Streptococcus including, but not limited to, pneumoniae , Pyogenes (group A) and so on. In yet another embodiment, identification of common types and subtypes of bacterial infectious agents includes screening for E. coli and one or more strains of E. coli, including but not limited to enterotoxigenic strain.

According to the present invention, the screening test used to identify common types and subtypes of infectious agents may be any suitable test known or later discovered in the art. For example, the screening test of the present invention can be a non-nucleic acid test or a chemical test, and the non-nucleic acid test includes but not limited to protein, peptide, amino acid, ligand. In one embodiment, the screening test of the invention is a test based on the presence or absence of one or more structural proteins (eg, glycoproteins, envelope proteins, polysaccharides, etc.) of an infectious agent. In another embodiment, the screening test of the invention is a test based on the presence or absence of one or more antigens or epitopes or antibodies to an infectious agent. In yet another embodiment, the screening test of the invention is a test based on the presence or absence of one or more substances released or metabolized by an infectious agent. In yet another embodiment, the screening test of the invention is a test based on the presence or absence of one or more substances derived from host cells, wherein said one or more substances are associated with the infectious agent Related to or arising from the infection of an infectious agent.

In a specific aspect, the non-nucleic acid screening assays of the invention include commonly used ligand binding or immunoassays, such as ligand or immunochromatographic assays. Many of these assays are based on highly specific interactions between specific binding pairs. Examples of such binding pairs include antigen/antibody, hapten/antibody, lectin/saccharide, apoprotein/cofactor, biotin/avidin (streptavidin). Additionally, many of these assays involve devices (e.g., solid phase, lateral flow test strips, flow-through tests) that have one or more of the binding pairs attached to a movable or immovable solid phase material. Members, the solid phase material is, for example, latex beads, glass fibers, glass beads, cellulose strips, or nitrocellulose membranes (US Pat. Nos. 4,703,017, 4,743,560, 5,073,484).

In one embodiment, the methods and devices of the present invention are used to detect or identify influenza A subtypes and/or influenza B and/or influenza C.

Influenza viruses belong to the genus orthomyxovirus in the family Orthomyxoviridae. ssRNA envelopes the virus with helical symmetry. The diameter of the coated particles is 80nm-120nm. RNA is tightly bound to nucleoprotein (NP), thereby forming a helical structure. The genome was segmented with 8 RNA fragments (7 for influenza C). Four basic antigens appeared, namely hemagglutinin (H), neuraminidase (N), nucleoprotein (NP), matrix (M) protein.

NP is a type-specific antigen occurring in three forms, A, B and C, which provides the basis for the classification of human and non-human influenza viruses. The matrix protein (M protein) surrounds the nucleocapsid and constitutes 35%-45% of the particle mass. In addition, two surface glycoproteins were seen as rod-shaped protrusions on the surface. Hemagglutinin (H) is composed of two subunits, H1 and H2. Hemagglutinin mediates viral attachment to cellular receptors. Neuraminidase molecules are present in lesser amounts in the envelope. Antigenic differences in the hemagglutinin and neuraminidase of influenza A viruses provide the basis for their classification into subtypes, for example, A/Hong Kong/1/68(H3N2) indicates isolation from a single patient in 1968 Influenza A virus of the H3N2 subtype.

In various embodiments, the methods and devices of the present invention are directed to the detection or identification of influenza A viruses defined by HxNy, where x is 1-16, y is 1-9, or any combination of xy. For example, in one embodiment, the methods and devices of the present invention are used to detect influenza A virus of H1N5 subtype. Therefore, multiple detection probes and capture probes for different subtypes of influenza virus are provided in the SCD of the present invention. In one embodiment, the assay is used to detect influenza A (with H1/H3 subtypes and the pandemic H5 subtype) and influenza B.

Specifically, the common type of influenza virus can be any type assigned based on the antigenic properties of the nucleoprotein and matrix protein antigens, such as influenza A, B, C, while the subtype can be based on One or more types of antigenically subdivided influenza viruses, such as one or more of influenza A or B viruses characterized by surface antigens such as hemagglutinin (H) and neuraminidase (N) Subtype.

In one embodiment, identifying common types of influenza viruses includes screening for influenza A viruses, influenza B viruses, and/or influenza viruses, and identifying subtypes of influenza viruses (e.g., influenza A viruses) includes screening for One or more predicted subtypes of A type A are screened, e.g., subtypes expected to be present in the population at the time of testing, optionally one or more suspected subtypes, e.g., in an outbreak (e.g., epidemic subtypes under surveillance for sexual and pandemic outbreaks). In another embodiment, identification of the common type of influenza virus includes screening for influenza A virus and influenza B virus, and identification of subtypes of influenza virus (eg, A virus) includes screening for One or more subtypes are screened for, for example, current vaccine subtypes or strains for the testing season, including subtypes and/or strains that are expected to circulate in the next influenza season. In yet another embodiment, identification of common types of influenza viruses includes screening for influenza A viruses and influenza B viruses, and identification of subtypes of influenza viruses (e.g., influenza A viruses) includes screening for influenza viruses used in the production of influenza vaccines One or more subtypes or strains, one or more subtypes or strains suspected of causing a pandemic outbreak (eg, one or more avian subtypes or strains such as H5N1 or derivatives thereof) are screened.

In yet another embodiment, identification of common types of influenza viruses includes screening for influenza A viruses and influenza B viruses, and identification of subtypes of influenza viruses (such as influenza A viruses) includes screening for one or more of the circulating viruses. Multiple common or predicted subtypes, including but not limited to a) H ₁ and H ₃ , b) H ₁ , H ₃ and H 3 ₂ , c) H ₁ , H ₂ , H ₃ and H ₉ , d) H ₁ , H ₃ and N ₁ , e) H ₁ , H ₃ , N ₁ and N ₂ , f) H ₁ , H ₃ and N 2 ₂ , g) N ₂ , h) N ₁ and N ₂ . For example, a screening test for identifying subtypes of influenza A virus may be for any of the subtype groups listed in a), b), c), d), e), f), g) or h) The presence of a subtype is identified without necessarily identifying the presence of a specific subtype within a group of subtypes. Alternatively, a screening test for identifying subtypes of influenza A virus may be performed on each and all of the listed in a), b), c), d), e), f), g) or h) The presence or absence of a subtype is identified, eg, the presence of a particular subtype within a group of subtypes is identified.

In yet another embodiment, identification of common types of influenza viruses includes screening for influenza A viruses and influenza B viruses, and identification of subtypes of influenza viruses (such as influenza A viruses) includes screening for one or Multiple pandemic or unexpected subtypes are screened for one or more pandemic or unexpected subtypes in circulation including but not limited to a) _H5 , b) _H5 and _H7 , c) H ₅ , H ₇ and H ₉ , d) N ₂ , N ₇ and N ₈ , e) H ₅ and N ₂ , f) H ₅ and N ₁ , g) H ₅ and N ₈ , h) H ₅ , N ₈ , H ₇ and N ₇ , i) H ₅ , H ₇ , H ₉ , N ₇ and N ₈ . For example, a screening test for identifying subtypes of influenza A virus may be performed on the subtypes listed in subtype groups a), b), c), d), e), f), g), h) or i). The presence of any one of the subtypes is identified without necessarily identifying the presence of a specific subtype within a group of subtypes. Alternatively, a screening test for identifying subtypes of influenza A virus may be performed for each of the subtypes listed in a), b), c), d), e), f), g), h) or i). The presence or absence of a species and all subtypes is identified, for example, the presence of a particular subtype within a group of subtypes is identified.

In another specific aspect, the common type of hepatitis virus may be a type A virus, a type B virus and a type C virus, each of which may have several subtypes including mutants. In one embodiment, identification of common types of hepatitis viruses includes screening for hepatitis A, B, and/or C, and identification of subtypes of hepatitis viruses includes screening for hepatitis A, B, and C, respectively. Virus subtypes or mutants were screened. In another embodiment, the identification of the common type of hepatitis virus includes screening for hepatitis B virus, and the identification of the subtype of hepatitis virus includes screening for one or more subtypes or mutant strains of hepatitis B virus . In yet another embodiment, the identification of the common type of hepatitis virus includes screening for hepatitis C virus, and the identification of the subtype of hepatitis virus includes one or more of the 1-9 subtypes of hepatitis C virus to filter.

In various embodiments, the methods and devices of the present invention can detect one or more different infectious agents. For example, a sampler may contain a plurality of different antibodies, wherein there are multiple subpopulations of antibodies whereby each subpopulation of antibodies specifically binds a different infectious agent. For example, the plurality of antibodies can include 2, 3, 4, 5, 6, 7, 8, 9, or 10 subpopulations, wherein the antibodies of each subpopulation of the plurality of antibodies specifically bind a different infectious agent. In some embodiments, the methods and devices of the present invention detect pandemic and non-pandemic infectious agents. In one embodiment, the pandemic and non-pandemic infectious agents are influenza viruses.

The explosive nature of influenza and the specific clinical features of the disease have given a reliable epidemiological record of this infectious disease since the early nineteenth century. Several epidemics have been recorded during the nineteenth century, but the first pandemic was not accurately recorded until 1889-1892. A second pandemic, probably of European origin, occurred in 1918-19, known as the Spanish flu, which killed 20-25 million people, mostly young adults.

Following the Spanish flu, there were regular successive pandemics in 1932–1933, 1947–1948, 1957, and 1968. The next pandemic is considered delayed. These later pandemics, similar to the one of 1890, affected millions of people with mild upper respiratory infections and resulted in a small number of deaths. The H1N1 (pig) virus probably appeared in 1918 and continued to circulate until 1957, when it was replaced by the H2N2 (Asian) virus. The H2N2 virus was prevalent until 1968, when the H3N2 (Hong Kong) strain emerged in 1968. The H1N1 virus reemerged in 1977 and did not replace the H3N2 subtype, and the two continued to circulate together. Therefore, there is an urgent need to screen subjects in an accurate and efficient manner to determine which virus strain and/or subtype an individual is infected with. Furthermore, in some cases, such sample collection and processing must be done in a point-of-care setting (e.g., on the battlefield, where there are large numbers of subjects to sample and process, and performing Human resources for this sampling are limited).

Thus, in one embodiment, the methods and devices of the present invention are utilized in an immediate fashion during the processing of a large number of samples, wherein the test results are visible (ie, read) some time after the test is completed. For example, the period of time may be 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 4 hours or 5 hours. In some embodiments, methods and devices in combination with the reagents disclosed herein provide high sensitivity and specificity, wherein fluorescence reads can be very similar results over an extended period of time. Thus, in some embodiments, a biological sample can be collected and processed, and then set aside for a substantial period of time before being read, which is advantageous in a point-of-care fashion, or when collected with limited manpower or time. Very advantageous in the case of a large number of samples and further processing of the samples.

In yet another aspect of the invention, the compositions and methods of the invention are used to detect any one analyte or analytes present in a sample. As noted above, for example, one or more analytes associated with MI can be detected by using different binding moieties that specifically bind to a marker associated with a condition. Thus, SCDs and test devices may contain reagents needed for diagnosing diseases or pathological conditions other than infectious diseases.

In certain embodiments, the one or more analytes are markers associated with a pathological condition or disease. In another embodiment, the one or more analytes are polypeptides associated with a nutritional state or condition. In yet another embodiment, the one or more analytes are cellular markers related to cell cycle and cell growth. In another embodiment, the one or more analytes are associated with cell proliferation and cell differentiation. In one embodiment, the cell marker is associated with cancer.

example

Example 1 - Determination of Volume and Tracking Buffer

To explore the optimal assay sample volume that would give the best sensitivity, and to investigate whether the tracking buffer would also increase the sensitivity and performance of the test strip.

Materials: Nitrocellulose Membrane: Millipore HiFlow 135 membrane, 2.5 cm wide, Cat. No. SHF1350425, Lot No. R68N46849, Code No. RK04414, Roll No. 04OLI. Use 1.0 mg/mL monoclonal anti-H5N1 antibody clone 3C8 to form bands on the membrane. A band was formed on the control line with 1.5 mg/mL rabbit anti-mouse antibody. Wick Material Pad: 0.05% Tween-20 treated polyester pad, Alhstrom 6613 grade. Absorbent pad prepared: Ahlstrom grade 222 paper, 3.5 cm wide, purchased from Fisher, Cat. No. 2228-1212, Lot No. 6150502. The anti-H53G4 gold conjugate was prepared by Nanogen POC in Toronto, Canada, and the OD of the 3G4 gold conjugate under the maximum absorption peak was 112.54. Extraction buffer: 50 mM Tris-Cl, pH 8.15, 0.75 M NaCl, 1% BSA, 0.1% pluronic F68, 0.05% digested casein, 2 mM TCEP and 0.02% NaN ₃ . Recombinant H5 hemagglutinin (0.2 mg/mL) was supplied by Nanogen POC, Toronto, Canada.

Protocol: Preparation of Lateral Flow Test Strips. The nitrocellulose membrane on which the antibody bands were formed was superimposed on the plastic card and overlapped with the wicking material pad and the absorbent pad by 1-2 mm. There is no cover tape applied to any components. Strips were cut to 5mm width.

Test: Mix 50-100 µL of extraction buffer with 1.1 µL of 3G4 gold conjugate and 2 µL of recombinant H5 hemagglutinin, or mix 50-100 µL of extraction buffer with 1.1 µL of 3G4 gold conjugate. The test was started by inserting the strips into the extraction buffer and the results were scanned at 15 minutes, 20 minutes, 25 minutes and 30 minutes. For experiments with tracking buffer, see Results for details.

Results: Figure 9A and Figure 9B are the bands tested with different volumes of extraction buffer at 15 minutes, 20 minutes, 25 minutes and 30 minutes. Figure 9A: Test strips were tested with different volumes of extraction buffer. At 15 minutes, 20 minutes, 25 minutes and 30 minutes, bands were removed and scanned with a Bio-Rad GS-800 Calibrated Densitometer. Rows 1 and 2: 50 µL extraction buffer; Rows 3 and 4: 60 µL extraction buffer; Rows 5 and 6: 70 µL extraction buffer; Rows 7 and 8: 80 µL extraction buffer solution; rows 9 and 10: 100 μL extraction buffer. Figure 9B: Comparison of the test line intensity of the test strip with different volumes of extraction buffer at different read times.

Table 6. Data analysis of Figure 9A and Figure 9B using Quantity One

Figure 10A: Effect of test strip 3 with different amounts of extraction buffer and background elimination on their negative control results.

Table 7. Data analysis of Figure 10A and Figure 10B using Quantity One software

Add 1.1 µL of 3G4 gold conjugate to different volumes of extraction buffer. The test was initiated by inserting the strip into the assay solution and scanning was performed at 15 minutes. (first lap). All bands were then transferred to tubes containing only extraction buffer and another 5 min run was performed. Scan the strips again. (Second lap).

Figure 10B: Test strip 3 with different amounts of extraction buffer and the effect of background subtraction on their sensitivity.

Add 1.1 µL of 3G4 gold conjugate and 2 µL of recombinant H5 hemagglutinin to different amounts of extraction buffer. The test was initiated by inserting the strip into the assay solution and scanning was performed at 15 minutes. (first lap). All bands were then transferred to tubes containing only extraction buffer and another 5 min run was performed. Scan the strips again. (Second lap).

Table 8. Data Analysis of Figure 4 Using Quantity One Software

discuss. In most lateral flow assay designs for point-of-care, the antibody conjugate is typically dried on a conjugate pad interfacing with nitrocellulose. The disadvantage of this design is that the dry conjugate sometimes does not release evenly and consistently from strip to strip due to the conjugate pad material. Conversely, in the case of premixing the antibody conjugate with the assay solution, it is more likely that the liquid flow will be consistent since once the wick material pad is wet, the flow rate is actually controlled by the membrane rather than by the wick material pad. Yes; as long as the liquid flow is consistent, the flow of the antibody conjugate should be more consistent and uniform. Therefore, in the above examples, the antibody gold conjugate was directly mixed with the assay solution.

Initially, 100 μL of extraction buffer was used for each test and the assay read time was set at 15 minutes. It was found that at the end of 15 minutes, not all of the assay solution had been consumed. There is waste of analyte and gold conjugate. Experiments were designed to investigate what the optimal assay volume would be in the relatively short read time range. Figure 9B and Table 2 show that when the volume of the assay solution is large, for example 100 μL, it takes a long time to obtain better sensitivity. However, with 50 μL of assay buffer, the color intensity of the test line did not increase much after 15 minutes at different reading times, indicating that most of the analyte and gold conjugate had passed through the capture line at 15 minutes . Although at 30 minutes, the color intensity of the bands tested with 100 μL of the assay solution reached almost the same level as that of the bands tested with a small volume of the assay solution at 15 minutes, it was clear that the 15-minute Tests are more competitive in the market than 30 minute tests.

However, because of the high concentration of gold conjugate in the relatively small volume of the assay solution, the background of the test strip shows a prominent red background, which reduces the contrast between the test line and the background and is not visually correct. Expected. Figure 10B shows that running the tracking buffer after 15 minutes can significantly clear the background, increasing the sensitivity of the test by 43%-63%. The data also show again that a small assay volume (50 [mu]L) is a better choice for testing.

Example 2 - Europium beads compared to prior art systems

Materials: Inactivated Influenza A Virus: Texas 1/77(H3N2) from Microbix Biosystems Ltd. Cat. No. EL-13-02 Lot No. 13037A3; Inactivated Influenza B Virus: Hong Kong 5 from Microbix Biosystems Ltd. /72, Cat. No. EL-14-03, Lot No. 14057A2; Quidel QuickVue A+B Test, Lot No. 702391.

Protocol: Virus Dilution - Prior to use, the influenza A and influenza B formulations were diluted with saline to 4096 HA/mL and 409.6 HA/mL, respectively. Assay procedure: The procedure described in the package insert of the Quidel test kit was carried out and briefly reviewed here. Dispense the entire extraction reagent solution from the reagent tube. Swirl the tube gently to dissolve its contents. Spike the virus into the tube. Place the swab into the extraction tube. Twist the swab at least three times while pressing the bottom and sides of the extraction tube with the swab tip. Leave the swab in the tube for 1 minute. Rotate the swab head against the inside of the tube while removing it. Place the test strip into the extraction tube. Read results at 10 minutes.

Results: Figure 11: QuickVue FluA+B was tested with different amounts of influenza A, Texas 1/77 virus. The test line is shown above the control line.

Table 9. Visualized Read Results and Data Analysis of Figure 4 Using Quantity One Software

Peak intensities were analyzed with Quantity One software. The software gives the values. Reading it by eye, any value below 4 will be negative.

Figure 12: Testing of QuickVue FluA+B with different amounts of influenza B, Hong Kong 5/72 virus.

The test line for type B is below the control line.

Table 10. Visualized Read Results and Data Analysis of Figure 4 Using Quantity One Software

Peak intensities were analyzed with Quantity One software. The software gives the values. In the case of a visual read, any value below 4 will be a negative result.

Table 11. Quidel QuickVue Flu A+B was tested with different amounts of B, Hong Kong 5/72 influenza virus.

Example 3 - Testing Limits of Detection of Influenza A and B Tests Using Gold Labels.

Materials: Anti-influenza A M4090913 gold conjugate prepared by Nanogen POC, Toronto, Canada. The OD at the maximum absorption peak is 102.69. The anti-influenza B antibody 2/3 gold conjugate was prepared by Nanogen POC, Toronto, Canada. The OD at the maximum absorption peak is 94.78. For nitrocellulose membranes, wicking pads, and absorbent pads, see above. Band the membrane with 1.5 mg/mL of anti-influenza B M2110171 and prepare a 1.5 mg/mL rabbit anti-mouse antibody from Nanogen POC in Toronto, Canada. Band the membrane with 1.5 mg/mL anti-influenza A 7304, and prepare 1.5 mg/mL rabbit anti-mouse antibody from Nanogen POC, Toronto, Canada. Inactivated Influenza A H3N2, Texas 1/77 was purchased from Microbix Biosystems Ltd., lot number 13037A3, 40960HA/mL.

Inactivated influenza B Hong Kong 5/72 was purchased from Microbix Biosystems Co., Ltd., batch number 14057A2, 40960HA/mL; molecular biology grade water, batch number 318105; 3 times extraction buffer, containing 0.15M Tris-Cl, pH 8.0, 2.25M NaCl, 0.3% Pluronic F68, 3% BSA, 6mM TCEP, 1.5% endocasein and 0.06% NaN ₃ ; 2 times extraction buffer containing 0.1M Tris- Cl, pH is 8.0, NaCl of 1.5M, 0.2% of Pluronic F68, 2% of BSA, 4mM of TCEP, 1% of endocasein and 0.04% of NaN ₃ ; 1 times extraction buffer, lot number ( lot#) 2232-003.

Protocol: Prepare lateral flow test membranes (see above); Virus Dilution: Influenza A Texas 1/77 was diluted to 409.6 HA/mL with 10 mM PBS with 1% BSA. Dilute inactivated influenza B Hong Kong 5/72 to 40.96 HA/mL with the same buffer before use. Influenza A and B tests using gold conjugates: For influenza A tests, add 16.7 µL of 3x extraction buffer, 1.96 µL of M4090913 gold conjugate, and varying amounts of inactivated influenza A virus to the tube . Add water to a final volume of 50 µL. For the influenza B test, add 25 µL of 2x extraction buffer, 2.12 µL of 2/3 gold conjugate, and varying amounts of virus to the tube. Add water to a final volume of 50 µL. The test is started by inserting the test strip into the assay solution. At 15 min, add 50 µL of 1x extraction buffer to the same tube and perform an additional 5 min test. After scanning with Bio-Rad GS-800Calibrated Densitometer, the results on the strips were read by eye and also analyzed with Quantity One software.

Results: Figure 13: Detection limit of the Influenza A and B tests using gold as a label.

Table 12. Visualized Read Results and Data Analysis of Figure 7 Using Quantity One Software

The peak intensity values are the results of Quantity One analysis.

Figure 14. Detection limit of influenza B test using gold as a label.

Table 13. Visualized Read Results and Data Analysis of Figure 9A and Figure 9B Using Quantity One Software

The peak intensity values are the results of Quantity One analysis.

Example 4 - Testing Limits of Detection of Influenza A and B Assays Using Fluorescent Europium Conjugates.

Materials: Wick material Pad: 0.05% Tween-20 treated polyester pad, 1.4 cm wide, supplied by Nanogen POC, Toronto, Canada; Absorbent pad: Ahlstrom 222 grade paper, 3.5 cm wide, purchased from Fisher, Cat. No. 2228- 1212, Lot No. 6150502; Nitrocellulose Membrane: Millipore HiFlow135 Membrane, 2.5cm wide, Cat. No. SHF1350425, Lot No. R68N46849, Code No. RK04414, Reel No. 04OLI; Influenza A Membrane: Anti-Influenza A Medix 7304 antibody at 1.5 mg/mL in test On the line, 1.5 mg/mL rabbit anti-mouse IgG is on the control line, banded by Nanogen POC, Toronto, Canada; anti-influenza A Fitzgerald M4090913 - Europium conjugate at 1% bead concentration prepared by Nanogen POC, Toronto, Canada ; Influenza B membrane: Anti-Influenza B Medix9901 antibody at 1.5mg/mL on the test line, rabbit anti-mouse IgG at 1.5mg/mL on the control line, banded by Nanogen POC in Toronto, Canada; Influenza B membrane : Anti-Influenza B M2110171 antibody at 1.5mg/mL on the test line, rabbit anti-mouse IgG at 1.5mg/mL on the control line, banded by Nanogen POC in Toronto, Canada; Anti-Influenza B HyTest 2/3- Europium conjugates at 1% bead concentration were prepared by Nanogen POC, Toronto, Canada; inactivated influenza A virus: Texas 1/77 (H3N2) from Microbix Biosystems Ltd., Cat. No. EL-13-02, Lot No. 13037A3; inactivated Live Influenza B virus: Hong Kong 5/72 from Microbix Biosystems Ltd., Cat. No. EL-14-03, Lot No. 14057A2; 3x extraction buffer prepared at Nanogen, San Diego, containing 0.15 M Tris-Cl, pH 8.0, 2.25M NaCl, 3% BSA, 0.3% Pluronic, 0.06% NaN ₃ , 1.5% endocasein and 6mM TCEP, batch number 2232-001; virus dilution buffer: with 1 % BSA in 10 mM PBS; antibody-europium conjugate dilution buffer: 10 mM PBS, 1% BSA and 0.2% Tween-20.

plan. Preparation of lateral flow test strips: Lay the nitrocellulose membrane with antibody strips formed on it on a plastic card, and overlap with the wick material pad by 1-2 mm, and stack one end of the nitrocellulose strip with the absorbent pad for 1-2 mm. 2mm. There is no cover tape applied to any components. Strips were cut to 5mm width. Virus Dilution: Inactivated Influenza A Positive Control and Influenza B Positive Control were diluted to 409.6 HA Units/mL and 40.96 HA Units/mL, respectively, to be determined using the Influenza Test.

Pretreatment of antibody-europium conjugates: 1% antibody-europium conjugates were sonicated for 4 minutes in an ultrasonic bath. The amount of conjugated beads was then diluted to 0.04% with antibody-europium conjugate, followed by an additional 4 minutes of sonication.

Influenza A and B testing procedure with antibody-europium conjugate: To 16.67 μL of 3x extraction buffer in a test tube, add 5 μL of 0.04% antibody-europium conjugate and various amounts of virus. Add water to a final volume of 50 µL. Insert the lateral flow test membrane into the assay solution. Wait 15 minutes at room temperature. Add 50 µL of 1x extraction buffer to the same tube. Wait 5 minutes at room temperature. Place the lateral flow test membrane in a dish and measure the fluorescence intensity. Each test condition was repeated 5 times.

Results: Figure 15: Analytical sensitivity of the influenza A test using europium conjugates.

Table 14. Statistical analysis of raw data for the analytical sensitivity of the influenza A test with europium conjugates.

Figure 16: Analytical Sensitivity of Influenza B Test Using Medix 9901 and Europium Conjugate on Membrane.

Table 15. Statistical analysis of raw data for the analytical sensitivity of the influenza B test with europium conjugates.

Figure 17: Testing the analytical sensitivity of the Influenza B test using M2110171 and europium conjugates on the membrane.

Table 16. Statistical analysis of raw data for the analytical sensitivity of the influenza B test with europium conjugates.

Results: Tables 5-7 show that the leading rapid influenza immunoassay (Quidel QuickVue Flu A+B test) currently on the market has a detection limit of 4.1 HA units for Influenza A Texas 1/77 and a detection limit for Influenza B Hong Kong 5/72 Influenza has a detection limit of 1.6 HA units. The influenza rapid test of the present invention can detect at least 0.41HA of Influenza A Texas 1/77 and Influenza B Hong Kong 5/72 of 0.082HA when using a gold label and when using a limited assay volume and tracking buffer . In detecting Influenza A Texas 1/77 and Influenza B Hong Kong 5/72, the rapid influenza test of the present invention is 10 times and 20 times more sensitive than the Quidel QuickVue Flu A+B test, respectively. By using fluorescent europium conjugates, the rapid influenza test of the present invention can detect influenza A Texas 1/77 down to 0.021 HA units, the signal/noise (S/N) ratio is 2.03, and the rapid influenza test of the present invention can detect Detected Hong Kong 5/72 Influenza B as low as 0.0021 HA unit with an S/N ratio of 4.96. If a S/N ratio of 2 is considered the detection limit of the europium-based assay, detection of influenza B will be possible down to 0.001 HA units/test. Or about a 1600-fold increase in sensitivity compared to the Quidel assay for type B. This study clearly demonstrates that improvements to the nitrocellulose assay technique have indeed significantly improved POC assay performance.

Example 5

Laboratory test results were used to initially demonstrate the principle that H5N1 influenza can be differentiated from seasonal human influenza viruses using the proposed product design and instrumentation.

Materials: 3x extraction buffer containing 0.15M Tris-Cl, pH 8.0, 2.25M NaCl, 0.3% Pluronic F68, 3% BSA, 1.5% casein and 0.06% NaN ₃ , Lot 2232-001; 1x Extraction Buffer, Lot 2232-003; 0.2M Tris(carboxyethyl)phosphine Hydrochloride (TCEP), Lot 2232-078; Molecular Biology Grade Water, Lot 318105; Europium Dilution Buffer solution, Lot RC 6-8-06; 10 mM PBS, pH 7.2, containing 1% BSA, Lot HR 7-19-06; 1% HyTest 2/3 Europium conjugate with 0.025 mg antibody, Lot No. E2/3-270706-2; 1% M4090913 Europium Conjugate with 0.025 mg Antibody, Lot No. E13-270706-2; 1% 3C8 Europium Conjugate with 0.1 mg Antibody, Lot No. E3C8-180806; from Canada Anti-influenza A60010044P, anti-influenza B 9901, anti-H53G4 and rabbit anti-mouse antibodies were used to form bands on nitrocellulose membranes prepared by Nanogen POC in Toronto. For all three test lines, the antibody coating concentration was 2.0 mg/mL. The antibody coating concentration of the control line was 1.5 mg/mL. The distance between each test line and the distance between the test line and the control line is 5mm ± 0.5mm; wick material pad: 0.05% Tween-20 treated polyester pad, 1.4cm wide, by Nanogen POC in Toronto, Canada Provide; absorbent pad: Ahlstrom 222 grade paper, 3.5cm wide, purchased from Fisher, item number 2228-1212, lot number 6150502; inactivated influenza A H1N1, Beijing/262/95 influenza was purchased from NIBSC, 40 μg HA activity/ampule; Inactivated Influenza A H3N2, Texas 1/77 was purchased from Microbix Biosystems Co., Ltd., Lot No. 13037A3, 40960HA/mL; Inactivated H5N1, Ck/HK/Yu22/02 Influenza from Wantai, China; Inactivated Influenza B Hong Kong 5/72 influenza was purchased from Microbix Biosystems Co., Ltd., lot number 14057A2, 40960HA/mL; Epstein-Barr virus was purchased from ATCC, VR-1491, strain B98-8, lot number 14057A2.

Procedure: Influenza test strips with multiple test lines were prepared: the nitrocellulose membrane on which the antibody bands were formed was superimposed on a plastic card and superimposed 1-2 mm with the wick material pad and the absorbent pad. There is no cover tape applied to any components. Strips were cut to 5mm width. Dilute inactivated influenza A H3N2, Texas 1/77 influenza and Hong Kong 5/72 influenza B 10-fold and 100-fold with _10mM PBS/1%BSA; reconstitute inactivated H1N1 Beijing 262/ 95; 1% antibody europium conjugate was sonicated for 4 minutes in a Branson 1210 ultrasonic bath. The individual conjugates were then diluted to 0.04% with europium conjugate dilution buffer, followed by sonication 3 times for 4 min each in an ice bath. In some embodiments, all 3 europium conjugates are co-diluted in the same tube. In this case, the concentration of europium conjugate per antibody was still 0.04% beads. Assay preparation:

For each assay, add the following components to the tube: 16.67 μL 3x extraction buffer; 0.5 μL 0.2M TCEP; 5 μL diluted europium conjugate (if all europium conjugates are diluted in the same tube medium) or 15 μL of europium conjugates (5 μL each if diluting each europium conjugate individually); virus as indicated in the figure; adjust final volume to 50 μL with _H2O .

Test: Start the test by inserting the test strip into the assay solution. After 15 minutes, 50 μL of 1x extraction buffer was added to the tube and the test was performed for an additional 5 minutes. At 20 minutes, read the test strip results with a fluorescent reader. Each test was repeated three times.

Results: Figure 18: Images of Influenza Rapid Test Strips under UV light after testing the strips with buffer, Influenza A H3N2, H1N1 and H5N1 subtypes, Influenza B and Epstein-Barr virus.

Figure 19: Influenza rapid test with buffer only.

Figure 20: Influenza rapid test using influenza A H3N2, Texas 1/77 subtype.

Figure 21: Rapid Influenza Test with Influenza B.

Figure 22: Influenza rapid test using influenza A H5N1, Ck/HK/Yu22/02 subtype influenza.

Figure 23: Influenza rapid test using H1N1Beijing/262/95 subtype influenza.

Figure 24: Influenza rapid test with H5N1 and Influenza B.

Figure 25: Rapid influenza test using Epstein-Barr virus.

Results: Based on initial test results, antibodies have been selected for the detection of influenza A, B and H5 subtypes. Strips were prepared with three different test lines detecting A, B and H5 subtypes, respectively. The results in Figures 1 to 7 show that when influenza B was tested, only the influenza B peak was observed. When testing for H1N1 or H3N2, only influenza A test peaks were seen and no peak corresponding to the H5 subtype was observed. According to one embodiment herein, once the anti-H1 and H3 antibodies are selected, another test line corresponding to H1 and H3 (together) is used to band the membrane. Subsequently, in addition to the influenza A peak, another peak corresponding to H1/H3 will also be observed. When testing the H5N1 subtype, both the Influenza A peak and the H5 peak were observed, with the Influenza A peak showing a stronger signal than the H5 peak. This result is considered an additional optimization of MAb selection and antibody loading, other factors within the assay were optimized. No cross-reactivity of influenza tests with Epstein-Barr virus was observed. These results clearly demonstrate that the method discovered here can obtain multi-analyte test results without interference between test lines.

The results shown above have clearly demonstrated that a novel fully integrated POC technology has been discovered that is highly sensitive and can independently detect different virus types and virus subtypes on the same test strip.

Example 6. Rapid Influenza B Assay Using pRNA as a Capture

The pRNA was immobilized on the membrane to determine the efficacy of a complementary pRNA capture conjugated with an anti-Influenza B nucleoprotein antibody to generate a signal and form antibody-antigen-antibody complexes when B virus is present in the sample.

Materials: Wick material Pad: 0.05% Tween-20 treated polyester pad, supplied by Nanogen POC, Toronto, Canada, 1.4 cm wide; Absorbent pad: Grade 222 paper from Ahlstrom, 3.5 cm wide, purchased from Fisher, Cat. No. 2228- 1212, Lot No. 6150502; Nitrocellulose Membrane: Millipore HiFlow 135 Membrane, 2.5 cm wide, Cat. No. SHF1350425, Lot No. R68N46849, Code No. RK04414, Reel No. 04OLI; Nitrocellulose Membrane, M2110171 at 1.5 mg/mL striped on Nitrocellulose , lot number M083106 for M2110171; nitrocellulose membrane, 1.35 mg/mL of 102a4-5G4 banded on nitrocellulose membrane, lot number MZ-42-74247-pRNA135 for 102a4-5G4; anti-influenza B HyTest2/3 - Toronto, Canada Nanogen POC prepared with beads conjugated to 1% europium, lot number E2/3-270706-3; inactivated influenza B virus: Hong Kong 5/72 from Microbix Biosystems Ltd, cat. no. EL-14-03 , lot number 14057A2; 3x extraction buffer prepared at Nanogen in San Diego, containing 0.15M Tris-Cl, pH 8.0, 2.25M NaCl, 3% BSA, 0.3% pluronic, 0.06% NaN ₃ , 1.5% endocasein, and 6 mM TCEP, Lot No. 2232-001; 0.2M TCEP, Lot No. 2232-078; 1X Extraction Buffer, Lot No. 2232-003; Europium Conjugate Dilution Buffer, Lot No. RC 6-8-06; Virus dilution buffer: 10 mM PBS with 1% BSA; 10 mM PBS, Lot SP09-22-06; Europium-conjugated antibody dilution buffer: 10 mM PBS, 1% BSA and 0.2% Tween-20; protein 5G4 for conjugation to pRNA to be immobilized on membrane; anti-influenza B nucleoprotein M2110171 purchased from Fitzgerald, Lot #578; pRNA 102a4-amn synthesized and activated by PDITC and 102b4-amn (a total of 13mer).

plan:

Preparation of pRNA antibody-conjugate

Table 17. Calculate the amount of oligonucleotide and antibody required for conjugation

In two separate tubes, dissolve the activated pRNAs 102a4amn (229 nmol in total) and 102b4amn (164 nmol in total) in 150 μL of water.

a. For each pRNA, aliquot 50 μL into 3 microcentrifuge tubes and dry them using a spin vac.

b. Place 1.5 mg of 5G4 monoclonal antibody into one spin filter and 2.0 mg of M2110171 monoclonal antibody into another spin filter, thereby concentrating both antibodies. Antibody was washed with 0.1M sodium borate buffer. The final concentrations of 5G4 and M2110171, determined by absorbance at 280 nm, were 33.35 mg/mL and 28.6 mg/mL, respectively.

c. Take 34 μL of concentrated 5G4 (1.15 mg) and 57 μL of concentrated M2110171. Adjust the above two volumes to 65 µL using 0.1 M borate buffer.

d. Add each antibody to the corresponding pRNA and allow the reaction to proceed for 15-20 hours at room temperature.

e. The pRNA antibody conjugate was then purified with a Sephadex G-50 column equilibrated with 0.01 M PBS with a bed volume of 7 mL.

1. Preparation of nitrocellulose membrane banded with 102a4-5G4.

2. NPOC in Toronto forms bands on membranes, see Materials.

3. Preparation of Lateral Flow Test Strips:

Lay the nitrocellulose membrane on which the antibody or 102a4-5G4 band is formed on the plastic card, one end overlaps the wick material pad by 1-2mm, and the other end overlaps the absorbent pad by 1-2mm. Strips were cut to 5mm width.

4. Virus dilution:

The inactivated influenza B virus was diluted to 4.096 HA units/mL with 0.01M PBS and 1% BSA buffer.

Antibody europium conjugate pretreatment

1% anti-influenza B 2/3-europium conjugate was sonicated for 4 minutes in a bath sonicator. A 10 μL amount of conjugated beads was then diluted to 0.02% with antibody-europium dilution buffer and sonicated for 20 seconds using a probe proximity sonicator (Fisher, model 550) at a power adjustment of 2 ( 4 pulses) with 10 s incubation in ice between each pulse (every 5 s).

102b4-M2110171 (0.54 mg/mL after purification) was diluted to 0.1 mg/mL with 10 mM PBS.

assay process

Take 50 µL of the assay mix and add the assay mix to the tube. Insert the test strip into the assay mix. Wait 15 minutes at room temperature. Add 50 µL of 1x extraction buffer to the same tube and keep for another 5 min. Read the test strips with an LRE fluorescence reader.

a. Preparation of assay mix for formation of strips with 102a4-5G4 band

Table 18.

b. Preparation of Assay Mixture for Formation of Bands with Anti-Influenza B Antibody M2110171

Table 19.

result

Figure 30A: Raw signal of a test strip with 102a4-5G4 conjugate on it.

Table 20. Net peak heights of the signals shown in Figure 30A

Table 20.

HA unit/test 1 2 AVG signal/noise 0 360.0 331.9 345.9 1.0 0.002 521.1 447.1 484.1 1.4 0.004 682.0 783.3 732.7 2.1 0.008 1048.5 916.2 982.4 2.8 0.016 1799.0 1594.3 1696.7 4.9 0.032 2745.4 3257.2 3001.3 8.7

Figure 30B: Raw signal of a test strip with antibody M2110171 on it.

Table 21. Net peak heights of the signals shown in Figure 30B

Figure 31 compares the sensitivity of two influenza B test strips.

The data clearly show that: 1) pRNA can be immobilized on nitrocellulose membranes when conjugated to proteins such as 5G4 antibody; 2) pRNA can bind complementary oligonucleotides conjugated to antibodies on nitrocellulose membranes. Acid hybridization; 3) After conjugation with pRNA, the specific antibody in the analyte still works. The results indicate that specific pairs of pRNAs can be used as capture systems in lateral flow rapid assays.

Example 7. Reader europium detection

Readers were assayed using six different standard concentrations of europium (note: the numbers below correspond to those used in the figures: (1) 5x10 ^-6 M; (5) 5x10 ^-5 M; (3) 5x10 ^-5 M; (6) 5x10 ^-4 M; (2) 5x10 ^-4 M and (4) 5x10 ^-3 M. Europium was surrounded in acrylic acid and cut into pieces of different sizes to provide different concentrations. In Figure 33A Up to Fig. 33H, the measured value is the number per meter x 10 ^-4 .

In another example, seven 1mm wide europium hard standards were mounted on 4mm centers, Figures 34A and 34B.

Example 8. Archive samples.

Influenza virus: inactivated influenza A/Texas 1/77 (H3N2), 40960 HA units/mL, from Microbix Biosystem Ltd., Ontario, Canada, classification number EL-13-02; inactivated influenza B/Hong Kong , 40960 HA units/mL, from Microbix Biosystems, Inc., Ontario, Canada, Cat. No. EL-14-03.

Viral RNA transcription positive control; Influenza A RNA control, PN: 606161, LN: RH2174P73, 50,000 copies/μL; Influenza B RNA control, PN: 606162, LN: RH2174P73, 50,000 copies/μL.

Extraction buffer: 3x extraction buffer

The table below lists the formulation of the 3x extraction buffer and the corresponding content (part number), lot number and supplier of each component.

Table 22.

Tris(2-carboxyethyl)phosphine hydrochloride (Tris(2-carboxyethyl)phosphine hydrochloride TCEP) 200 mL, prepared by HR on 7-6-06, ordered from PIERCE, product number: 20491, lot number: GL102548.

Prepare fresh 1x extraction buffer: combine 200 µL of 3x extraction buffer with 6 µL of 0.2M of TCEP and 394 µL of water to obtain 600 µL of 1x extraction buffer. The formulation of 1X extraction buffer is: 50mM Tris, 1% BSA, 0.1% block polyether F68, 0.5% casein, 0.02% NaN ₃ , 0.75M NaCl and 2mM TCEP.

Sample packaging and storage materials: Whatman FTA Elute Micro Card, Cat. No. WB120401, LN: FE6231106; Whatman Harris UNI-CORE 2.00mm Hole Punch, Cat. 0540, LN: P34H4QA11, ABI.

Extraction material: distilled water, DNase, RNase-free, Cat. No. 10977-015, LN: 1317578, Invitrogen.

Heat source: GeneAmp PCR System 9700, ABI, QA2126

RT and PCR reagents and materials:

Table 23

component supplier Classification number batch number RT Mix Prodesse GLS01 050105RM MgCl21M Ambion 9530G 064R37A MulV reverse transcriptase, 50u/μL ABI N808-0018 H03264 RNase inhibitor, 20u/μL ABI N808-0119 H05526 RVA Primer Mix Nanogen No RH2174P100 AmpliTaq Gold Polymerase, 5u/μL ABI N808-0249 G03670 96-well PCR reaction plate ABI 4306737 P03F6QA41

Materials for evaluating RT-PCR reactions: Agilent DNA 1000 Reagent: PN: 5067-1504, LN: 0623; Agilent DNA Fragment: PN5067-1522, LN: JA21BK01

method

Preparation of RT master mix (Master Mix)

Table 24

component Volume of each reaction (μL) RT mixture 11 1M MgCl ₂ 0.1 MulV reverse transcriptase (50u/μL) 1 RNase inhibitor (20u/μL) 1 DNase, RNase-free water 1.9 Total 15

Add 15 µL of the RT total mix to wells of a 96-well PCR plate.

Table 25. Preparation of PCR master mix

component Volume of each reaction (μL) RVA Primer Mix 39.5 AmpliTaq Gold Polymerase (5u/μL) 0.5 Total 40

Add 40 µL of the PCR total mix to wells of a 96-well plate.

Table 26. Dilution of RNA Positive Control: Influenza A and B RNA was diluted with DNase, RNase-free water as described in the table below.

Add 5 µL of the diluted RNA to the appropriate RT reaction.

Table 27. Dilution of Inactivated Influenza A and B Viruses with 1X Extraction Buffer

Apply samples to FTA Elute cards

Apply 5 μL of the sample below to the sample area of the Whatman FTA Elute Micro card; use 1X extraction buffer as a control; dilute virus with 1X extraction buffer; air dry the FTA Elute card at room temperature for 15-20 minutes; Use a puncher to punch a 5mm to 6mm diameter piece from the area coated with the sample, and then put it into a 0.2mL MicroAmp reaction tube;

Extract RNA from FTA Elute tablets: add 150 μL of water to the tube containing the tablet and vortex 3 times for 5 seconds; remove water by using sterile tips; repeat process of adding/removing water once; centrifuge for 5 seconds, then aspirate excess liquid; add 50 μL of water, heat at 65°C for 30 minutes, then vortex tube for 5 seconds; remove 5 μL of liquid and add to appropriate well of 96-well RT plate.

RT-PCR assay

Thermal cycling process of RT step

Temperature (°C) time (minutes) Cycles twenty two 10 1 42 60 1 95 5 1 4 Keep

Transfer 10 µL of the RT-completed reaction to the appropriate PCR well containing the PCR master mix. Pipette up and down to mix.

Table 28. Thermal Cycling Process for PCR Steps

Using the Agilent Bioanalyzer to Evaluate PCR Reactions of Amplicons

RT-PCR Agilent results: The control reaction without template did not produce any detectable amplicons.

RNA Positive Control

Table 29.

The bands observed for the influenza A and influenza B RNA positive controls were of appropriate size.

Table 30. Influenza viruses

Conclusions: RNA was successfully detected from lysed influenza A and influenza B viruses using the Whatman FTA Flute.

Cards and RT-PCR amplification

The assay detected as little as 0.2 HA units/tablet of influenza A virus and 0.002 HA units/tablet of influenza B virus. Whatman FTA Elute Cards can be used to archive viral RNA for later testing in RT-PCR assays to detect influenza A and B.

Example 9. Multiplex Analyte Detection.

To show evidence for the theory that a multiplexed-analyte influenza diagnostic test can differentiate influenza H5N1 from seasonal influenza, a rapid and highly sensitive assay was developed and compared to Gold in comparison to traditional lateral flow tests.

The use of lanthanide labels in the case of latex microparticles filled with europium complexes, in which lanthanide labels are combined with wash buffers, shows improved sensitivity. Using membranes of anti-influenza A, anti-influenza B, and anti-H5 antibodies that band directly at different areas on the test strip, seasonal influenza (e.g., influenza B in this example) was compared with avian influenza H5N1 differentiate.

Data show that using pRNA and MAb directly stripped on nitrocellulose as a capture system on the same lateral flow test strip enables independent detection of influenza A H1N1 and H5N1 subtypes located on different test areas on a single nitrocellulose test strip and beta and alpha nucleoproteins.

Table 31. Tables providing evidence for opinions

Material

1. The pre-activated pRNA is provided by the Nanogen Bothell development team.

a.102a10 (10mer), lot number 825-079A

b.102b10 (10mer, complementary pRNA of 102a10), lot number 825-065D, lot number 825-065D

c.3a10 (10mer), lot number 825-079C

d.3b10 (10mer, complementary pRNA of 3a10), lot number 825-65F, lot number 825-065F

2. Antibodies

a. Anti-influenza A 7304 from BiosPacific

b. Anti-influenza A M4090913 from Fitzgerald

c. Anti-Influenza B 2/3 from HyTest

d. Anti-influenza B M2110171 from Fitzgerald

e. Anti-H1 influenza M322210 from Fitzgerald

f. H1 type anti-influenza 8252K from Chemicon

g. Anti-H510F7 influenza from Xiamen University

h. Anti-H53G4 influenza from Xiamen University

3. OptiLink carboxylate modified particles (europium beads)

Europium beads with a diameter of 0.3 μm were purchased from ThermoFisher Scientific (Seradyn), Cat. No. 83470750010250, production lot No. C020512, package lot No. 201154.

4. Viruses

Influenza B Hong Kong/5/72 from Microbix, B/Shanghai/2002 from Dr. Bovian's lab, A/Taiwan/1/86(H1N1) from NIBSC, A/Beijing from Dr. Bovian's lab /95(H1N1), Bar-headed goose (Bar-headed) GS/QH/15/2005(H5N1) and DK/VNM/568/2005(H5N1) from the laboratory of Dr. Guan, University of Hong Kong.

5. Triple extraction buffer, containing 150mM Tris-Cl, pH 8.0, 2.25M NaCl, 3% BSA, 1.5% endocasein, 0.3% block poly (pluronic) F68 and 0.15% ProClin 300, Lot No. 2277-038.

6. Solution: 0.2M solution of tris(2-carboxyethylphosphine) hydrochloride, batch number 2277-067.

7. Recombinant streptavidin.

8. Non-specific antibody 5G4 made by Nanogen Toronto facility for conjugation to pRNA and used to form strips on nitrocellulose.

9. Strip liner card, 0.1” super white polystyrene from G&L.

10. Absorbent pad, grade 222 filter paper from Alstrom Filtration.

11. Wick material pad, polyester pad treated with 0.05% Tween-20.

process

Preparation of antibody europium conjugates

1.1 Antibodies M4090913, 2/3 and 3G4 were conjugated to 0.3 μm europium by ThermoFisher Scientific (Seradyn).

1.2 Antibody 8252K europium conjugate was prepared by an indirect procedure by conjugating biotinylated 8252K to streptavidin coated with europium microparticles. Biotinylated 8252K and streptavidin coated with europium microparticles were prepared at Nanogen Toronto facility. The streptavidin europium stock containing 1% solids was sonicated for 5 minutes using a bath sonicator. It was then diluted to 0.02% with Europium Conjugate Dilution Buffer and sonicated with a probe proximity sonicator for 20 seconds. Dilute biotinylated 8252K to 10 μg/mL with PBS buffer. Add an aliquot of 5 µL to each tube. A 5 μL aliquot of 0.02% solid streptavidin europium conjugate was then quickly mixed with biotinylated 8252K. The conjugate was blocked with 2.5 μL of 100 mM biotin. The 8252K europium conjugate was then used directly in the assay.

2. Preparation of antibody-pRNA conjugates: All antibodies were concentrated to approximately 20 mg/mL using Microcon 50 from Amicon. The buffer was changed to 0.1M sodium borate, pH 9.0. Add an aliquot of 1 mg of non-specific antibody 5G4 to the tube containing 33.5 nmol of preactivated pRNA 102b10 or 3b10 (dry powder). Aliquots of 0.5 mg M322210 and 0.5 mg 10F7 were added to pRNA 102a10 and 3a10 containing 16.7 nmol preactivated, respectively. The reaction was carried out at room temperature for 15 hours. Antibody pRNA conjugates were purified using a Sephadex G-50 column equilibrated with 10 mMPBS buffer, pH 7.0. Two peaks were identified, the first being the conjugate and the second being the unactivated pRNA (confirmed by HPLC, data not shown).

3. Prepare a nitrocellulose membrane forming a strip with four test lines. For this membrane, 1.5 mg/mL of anti-influenza A 7304 and anti-influenza B M2110171 were directly formed into test line "A" and test line "B", respectively. Conjugates 102b10-5G4 and 3b10-5G4 formed "H1" and "H5" test lines at 0.75 mg/mL. A control line was formed with 0.5 mg/mL of rabbit anti-mouse antibody. All lines are spaced 5mm apart. Then, the membrane was blocked with a solution containing 0.35% PVP, 0.25% Triton X-100 and 0.5% endocasein, and cured at 37°C.

4. Preparation of test strips: Laminate a nitrocellulose membrane with 4 test lines and a control line on the target card, and stack the nitrocellulose membrane with the treated polyester pad and absorbent pad for 1-2 mm. Strips were cut to 5mm width using a Kinematic 2360 strip cutter.

5. Test Procedure: Assay mixtures were prepared as follows. Add 16.7 µL of 3x extraction buffer, 0.5 µL of 0.2M TCEP, 0.1 µg of 102a10-M322210 conjugate, 0.1 µg of 3a10-10F7 conjugate, antibody europium conjugate, and different viruses to the tube. The final concentration of all antibody europium conjugates was 0.002% solids in a final volume of 50 [mu]L. Start the test by inserting the test strip into the assay mix. After 15 minutes, add another 50 μL of 1X extraction buffer to wash the strips. At 20 minutes, the strips were read with a fluorescence reader.

result.

Table 32. The table below compares the signal-to-noise ratio of the different test peaks when tested with different viruses.

The above data clearly show that the test system is capable of discriminating multiple analytes. In this example, the analytes detected include: seasonal H1N1 subtype influenza, avian influenza B H5N1, which has a good type A signal. In this test, two pairs of pRNAs were used as the capture system, thereby distinguishing the H1 test line from the H5 test line. When detecting H5 virus, some hyporeactivity was observed at the position of the H1 test line, indicating a small amount of hybridization of pRNA 3a10 conjugated to the anti-H5 antibody to 102b10 forming a band on the test line H1. When detecting H1N1 subtype viruses, no false positive reactions were observed on the H5-specific test line.

In addition, the report clearly shows that the assay showed good reactivity with good separation of analyte signals when a single multi-analytical reaction mixture was used for all tested samples. Additionally, when testing a single test sample at an intermediate viral load, the assay can simultaneously detect all suitable analytes.

Conclusions: The results show that with multiple subtypes of virus, clear signal separation for each analyte was achieved for each virus strain and subtype tested (Figure 36-38). To ensure the validity of the results, two separate lines of each subtype were tested. Furthermore, the results clearly show that the application of the pRNA-like capture system to the H1 and H5 detection systems enables the use of the pRNA capture system for other assay series for different analytes.

Furthermore, the assays can be readily adapted to include additional analyte-specific binding agents. Additionally, the devices/assays of the invention can be used in methods of screening for different analytes. For example, a large number of anti-H3 expressing clones can be screened to determine.

Of course, the device/method of the present invention can be used to screen new virus strains (important examples are new Fugan-like strains). For example, new strains of the H5N1 subtype are obtained as new MAbs, which can be included in the SCD and the test device of the present invention for screening.

The results shown above have clearly demonstrated that a new fully integrated assay technology has been found that allows the individual detection of different virus types and subtypes on the same test strip with high sensitivity. In addition, this assay can be performed in a point-of-care setting.

Example 10. Multi-analyte detection: Marker proteins

The devices and methods of the present invention may also be used to detect various analytes associated with disease conditions or nutritional status or any other relevant property. For example, a variety of cardiac markers can be detected and/or quantified:

(1) Troponin marker: the capture antibody is a polyclonal rabbit anti-Troponin-1, and is conjugated to a specific pRNA sequence (SEQ ID NO: 98). The detection antibody was a mixture of two murine MAbs, conjugated to biotin, and reacted with streptavidin coated with 0.3 μm europium latex particles.

(2) CK/MB marker: The capture antibody is polyclonal and conjugated to a specific pRNA sequence (SEQ ID NO: 99). The detection antibody was also a MAb, conjugated to biotin, and reacted with streptavidin coated with 0.3 μm europium latex particles.

(3) Myoglobin marker: The capture antibody is a rabbit anti-myoglobin polyclonal, and is conjugated to a specific pRNA sequence (SEQ ID NO: 100). The detection antibody was also a MAb, conjugated to biotin, and reacted with streptavidin coated with 0.3 μm europium latex particles.

(4) Control line composed of rabbit anti-mouse

Thus, a multi-analyte "point-of-care" assay system can quantify each marker individually as described above in a single assay format, as follows:

Utilize readers with hard europium standards (europium standards that are locked in acrylic and produce uniform readings over a long period of time under a uniform set of conditions) to ensure that the reader meets the fluorescent signal strength requirements reproducibility. Therefore, each analyte can be quantified individually.

Example 11. Multi-analyte detection: Viruses

As noted herein, the devices of the present invention may also be configured to detect multiple different viruses, or multiple different subtypes of viruses. For example, devices of the present invention can be configured to provide influenza multi-analyte plus RSV quantitative assays, wherein the multi-analyte can be detected from a single sample:

(1) Influenza A: the capture antibody is a MAb, and is conjugated to a specific pRNA sequence (SEQID NO:99); the detection antibody is also a MAb, is conjugated to biotin, and is coated with 0.3 μm of europium latex Streptavidin response of microparticles.

(2) Influenza B: The capture antibody is a monoclonal mouse MAb and is conjugated to a specific pRNA sequence (SEQ ID NO: 100); the detection antibody is also a MAb, is conjugated to biotin, and is coated with 0.3 μm Streptavidin Reaction of Europium Latex Microparticles.

(3) H1 and H3 influenza: The capture antibody is a MAb specific for H1 and H3 hemagglutinin, and is conjugated to a specific pRNA sequence (SEQ ID NO: 101). The detection antibody was also a MAb, conjugated to biotin, and reacted with streptavidin coated with 0.3 μm europium latex particles.

(4) H5 influenza: the capture antibody is a monoclonal mouse MAb, and is conjugated to a specific pRNA sequence (SEQ ID NO: 102); the detection antibody is also a MAb, is conjugated to biotin, and is coated with 0.3μm Streptavidin Reaction of Europium Latex Microparticles.

(5) RSV: The capture antibody is a monoclonal mouse MAb, and is conjugated to a specific pRNA sequence (SEQ ID NO: 103); the detection antibody is also a MAb, conjugated to biotin, and is coated with 0.3 μm europium Streptavidin reaction of latex microparticles.

(6) Control line: the capture antibody is a rabbit anti-mouse MAb bound to europium latex particles.

Example 12. Expression of single chain antibodies 10F7 and 4D1 and testing of their activity.

The variable region genes of the heavy and light chains of each antibody are linked to nucleic acid encoding a short peptide (GGGGS), thereby forming DNA encoding a single-chain antibody. 10F7VHF/10F7VHR was used as a primer pair to amplify the variable region DNA fragment of the 10F7 heavy chain. 10F7VKF/10F7VKR was used as a primer pair to amplify the variable region DNA fragment of the 10F7 light chain. 4D1VHF/4D1VHR was used as a primer pair to amplify the variable region DNA fragment of the 4D1 heavy chain. 4D1VKF/4D1VKR was used as a primer pair to amplify the variable region DNA fragment of the 4D1 light chain.

10F7VHF/10F7VKR was used as a primer to amplify a single-stranded DNA fragment superposed with 10F7. 4D1VHF/4D1VKR was used as primers to amplify the heavy chain DNA fragment superimposed with 4D1. The amplified DNA fragment was recovered, cut with BamH I and Sal I, and cloned into the prokaryotic expression vector pTO-T7 cut with the same restriction endonuclease. Using ER2566E.coli as a host cell, standard methods were used to express the single chain antibody protein. The expressed protein is in the form of an insoluble inclusion body. The inclusion bodies were broken by sonication and the resulting precipitate was purified using standard methods. The purified sediment was dissolved in 8M urea. The urea solution was slowly dialyzed against 1X PBS solution and centrifuged at 12000 rpm for 10 minutes to remove remaining sediment. The final purified scFv solution was used to test activity.

26 strains of H5N1 virus were selected, and the activities of the above purified 10F7 and 4D1 single chain antibodies were tested using the HI assay described above. The concentration of 10F7 single chain antibody was used at 1.06 mg/mL. The concentration of 4D1 single chain antibody was used at 0.34 mg/mL. The 4D1 scFv showed HA inhibitory activity against 23 virus strains. The 10F7 scFv showed HA inhibitory activity against 14 virus strains (see Table 33 below).

Table 33. HA inhibitory activity of three single-chain antibodies against 25 H5N1 viruses

HI titers are diluted by 2 to the nth power. n is the number shown in the table. The activity of the above purified 10F7 single chain antibody was tested by neutralization method. Seven virus strains isolated from chickens, ducks and various wild birds in Hong Kong, Indonesia, Qinghai and other regions were tested for the activity of 10F7 using the HI assay. Antibodies showed good neutralizing activity against 5 virus strains (Table 34). At a 64-fold dilution, the antibody was still able to inhibit viral infection of host cells.

Table 34. 10F7 single chain antibody neutralization test results.

Example 13. Detection of 7aa serine activity

Three antibiotics 8H5A, 8H5E and 3C8A containing 7aa serine were greatly amplified. After precipitation of the three antibiotics with PEG, the antibiotics were dissolved in PBS. Phage titers were between 10 ¹¹ and 10 ¹² . Pre-coat microplates with 5 µg/mL of monoclonal antibodies 8H5, 4A1, 9N7, and 4D11. The plates were blocked with PBS containing 5% milk. Three phages were serially diluted and added to the plate. React for 1 hour. Then, the plate was washed 5 times. Mouse anti-M13/HRP antibody (Amersham Phamarcia Biotech, USA) diluted 1:5000 was added as a secondary antibody and kept for 0.5 hours. Read the result after the reaction is complete. The results, shown in Table 35 below, show that the specific reaction between peptide 8H5A and monoclonal antibody 8H5 is good, and the reaction between 8H5A and the other three monoclonal antibodies is weak. The specific reactivity between 8H5E and mAb 8H5 was relatively poor.

Table 35.7 The detection results of specific binding activity of aa serine and monoclonal antibody

Monoclonal antibodies 8H5A(1:1000) 8H5E(1:1000) 8H5 0.559 0.25 4A1 0.158 0.142 9N7 0.062 0.065 4D11 0.118 0.078

Example 14. MAb capture system and pRNA capture system tested with Nanogen Influenza A.

To compare the sensitivity of the Nanogen Influenza A test using two different capture systems, and to compare the pRNA capture system using two different test strips.

Procedure for cutting test strips: Membrane cards were prepared by placing absorbent pads (3.5 cm wide) and pre-treating with Tween-20; pre-cut polyester wicking pads (1.4 cm wide) on the adhesive side, polyester A pad of wicking material overlapped the membrane 1 mm on which the appropriate antibody was banded. This prepared card was cut into 5 mm wide test strips with a paper cutter.

Virus Dilution: Dilute A/Texas/1/7740960 HA units/mL virus in PBS containing 1% BSA according to the dilution procedure below.

Table 36: Dilution Procedures for Influenza A Viruses

Europium Dilution: Dilute 17-10-06-1 Europium Conjugate to 0.02% with Europium Dilution Buffer; 245 μL of Europium Dilution Buffer + 5 μL of 1% stock. The diluted europium conjugate was sonicated using a probe proximity sonicator (Fischer model 550) at a power setting of 3 for a total of 20 seconds (10 seconds each with 5 seconds in between).

pRNA conjugate dilution: 102a10-7304 (0.39 mg/mL) conjugate was diluted to 0.1 mg/mL with 10 mM PBS, pH 7.2; 37.2 μL of PBS + 12.8 μL of 102a10-7304 conjugate.

Assay Procedure: Assay mixtures were prepared according to the table below.

Table 37: Assay Mixtures for MAb Capture Systems

Table 38: Assay Mixtures for pRNA Capture Systems

Table 39. Materials

A test strip of 7304 (1.5 mg/mL) was placed into the assay mix of the MAb capture system.

102b10-5G4 (0.75 mg/mL) w/o blocking and w/0.5% casein blocking test strips were placed in the pRNA capture system assay mix.

Repeat multiple trials.

Keep at room temperature for 15 minutes.

Wash the tube by adding 50 µL of 1x extraction buffer to the same tube.

After 5 minutes, the test strips were read using a fluorescent reader.

result

Table 40. MAb capture system (see Figure 39)

HA unit/test 0 0.01 0.02 0.04 0.1 1 1 528.1 793.7 935.7 1421.6 2550.1 21111.3 2 569.6 776.0 1037.5 1440.0 2395.4 19961.8 AVG 548.8 784.9 986.6 1430.8 2472.8 20536.5 S/N 1.4 1.8 2.6 4.5 37.4

Table 41. pRNA capture system with 0.75mg/mL membrane blocked with 0.5% casein (see Figure 40)

HA unit/test 0 0.01 0.02 0.04 0.1 1 1 114.6 434.1 703.0 1360.9 3222.7 21209.1 2 222.6 628.5 804.6 1385.1 2891.4 19440.1 AVG 168.6 531.3 753.8 1373.0 3057.1 20324.6 S/N 3.2 4.5 8.1 18.1 120.5

Table 42: pRNA capture system with 0.75mg/mL membrane without blocking (see Figure 41)

HA unit/test 0 0.01 0.02 0.04 0.1 1 1 284.4 737.9 940.0 1880.4 3336.0 22387.5 2 318.1 612.2 814.6 1471.3 3560.8 22383.6 AVG 301.3 675.1 877.3 1675.9 3448.4 22385.6 S/N 2.2 2.9 5.6 11.4 74.3

Conclusions: As shown by the data, the pRNA capture system has the highest test sensitivity with a signal-to-noise ratio of 120 for 1 HA unit when using the Toronto 0.5% casein-blocked test strip, but using the MAb dotted directly on the test strip, The S/N ratio was only 37.4, which indicated that the pRNA capture system significantly improved the sensitivity of the assay system.

SEQ ID NO: 1 8H5Vh nucleotide sequence

caggttcagc tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata

tcctgcaagg ctactggcta cactttcagt aactactgga tagagtggat aaagcagagg

cctggacatg gccttgagtg gattggagag attttacctg gaagcgatag aacaaactac

aatgggaagt tcaagggcaa ggccacattc actgcagata catcctccaa cacagcccac

atgcaactca gtagcctgac atctgaggac tctgccgtct attackgtgc aaatagatac

gacgggtatt attttggttt ggattactgg ggtcaaggaa cctcagtcgc cgtctcctca

gcc

SEQ ID NO: 2

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thy Gly Tyr Thr Phe Ser Asn Tyr

20 25 30

Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Asp Arg Thr Asn Tyr Asn Gly Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala His

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Asp Gly Tyr Tyr Phe Gly Leu Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Ala Val Ser Ser Ala

SEQ ID NO: 3 8H5Vk Nucleotide Sequence List

gaaatcgtgc tcacccagtc tccagcaatc atgtctgcat ctctagggga gaaggtcacc

atgagctgca gggccagctc aagtgtaaat ttcgtttact ggtaccagca gaggtcagat

gcctccccca aactattgat ttactattca tccaacctgg ctcctggagt cccacctcgc

ttcagtggca gtgggtctgg gaactcttat tctctcacaa tcagcggctt ggagggtgaa

gatgctgcca cttattactg ccagcacttt actagttccc cgtacacgtt cggagggggg

accaacctgg aaataaaacg g

SEQ ID NO: 4 8H5Vk amino acid sequence

Glu Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Arg Ala Ser Ser Ser Val Asn Phe Val

20 25 30

Tyr Trp Tyr Gln Gln Arg Ser Asp Ala Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Tyr Ser Ser Asn Leu Ala Pro Gly Val Pro Pro Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Gly Leu Glu Gly Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln His Phe Thr Ser Ser Pro Tyr Thr

85 90 95

Phe Gly Gly Gly Thr Asn Leu Glu Ile Lys Arg

100 105

SEQ ID NO: 5 3C8Vh nucleotide sequence

cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc

tcctgcaagg cctctgggta cagcttcaca aactatggaa tgaactgggt gaagcaggct

ccaggaaagg gtctaaagtg gatgggctgg ataaacacct acaccggaga gccagcctat

gctgatgact tcaagggacg gtttgccttc tctctggaaa cctctgccag cactgcctat

ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc aagatggaat

agagatgcta tggactactg gggtcaagga acctcggtca ccgtatctag c

SEQ ID NO: 6 3C8Vh amino acid sequence

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Ala Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Trp Asn Arg Asp Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

SEQ ID NO: 7 3C8VK nucleotide sequence

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctcttgggca gagggccacc

atatcctgca gagccagtga aagtgttgat agttctgaca atagtcttat gcactggtac

cagcagaaac caggacagcc acccaaactc ctcatctatc gtgcatccaa cctagaatct

gggatccctg ccaggttcag tggcagtggg tctaggacag acttcaccct caccattaat

cctgtggagg ctgatgatgt tgcaacctat tactgtcagc aaagtattgg ggatcctccg

tacacgttcg gaggggggac caagctggaa ataaaacgg

SEQ ID NO: 8 3C8VK amino acid sequence

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Ser Ser

20 25 30

Asp Asn Ser Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Arg Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Pro Val Glu Ala Asp Asp Val Ala Thr Tyr Tyr Cys Gln Gln Ser Ile

85 90 95

Gly Asp Pro Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

SEQ ID NO: 9 10F7Vh nucleotide sequence

caggtccaac tgcagcagcc tggggctgaa cttgtgaagc ctggggcttc agtgaagctg

tcctgcaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg

cctggacagg gccttgagtg gatcggagag attgatcctt ctgattctta tactaactac

aatcagaagt tcaagggcaa ggccacattg actgtagaca aatcctccag cacagcctac

atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagggggggt

acaggagact ttcactatgc tatggactac tggggtcaag gcacctcggt caccgtatca

tcg

SEQ ID NO: 10 10F7Vh amino acid sequence

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Thr Gly Asp Phe His Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

SEQ ID NO: 11-10F7VK nucleotide sequence

gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc

atcacttgcc atgcaagtca gggcattagc agtaatatag ggtggttgca gcagaaacca

gggaaatcat ttaagggcct gatctatcat ggaaccaact tggaagatgg agttccatca

aggttcagtg gcagtggatc tggagcagat tattctctca ccatcagcag cctggaatct

gaagattttg cagactatta ctgtgtacag tatgttcagt tcccgtacac gttcggaggg

ggcaccaagc tggaaatcaa acgg

SEQ ID NO: 12 10F7VK amino acid sequence

Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly

1 5 10 15

Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105

SEQ ID NO: 13 artificial sequence/unknown organism

catgggatgc tgccggtgta t

SEQ ID NO: 14 artificial sequence/unknown organism

aattctgggc cttggctgac g

SEQ ID NO: 15 artificial sequence/unknown organism

tggccgcctc tgtcgaagaa g

SEQ ID NO: 16 4D1 VH nucleotide sequence

caggtccaac tgcagcagcc tggggctgag cttgtgaagc ctggggcttc agtgaacctg

tcctgtaagg cttctggcta caccttcacc agctactgga tgcactgggt gaagcagagg

cctggacaag gccttgagtg gatcggagag attgatcctt ctgatagttt tactacctac

aatcaaaact tcaaagacag ggccacattg actgtagaca aatcatccag cacagcctac

atgcagctca gaagtctgac atctgaggac tctgcggtct attactgtgc caggggggggt

ccaggagact ttcgctatgc tatggattac tggggccaag gcacctcggt caccgtctcc

tca

SEQ ID NO: 17-4D1 VH amino acid sequence

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asp Pro Ser Asp Ser Phe Thr Thr Tyr Asn Gln Asn Phe

50 55 60

Lys Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Pro Gly Asp Phe Arg Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

SEQ ID NO: 18-4D1VK nucleotide sequence

gacatcctga tgacccaatc tccatcctcc atgtctgtat ctctgggaga cacagtcagc

atcacttgcc atgcaagtca gggcattagc agtaatatag ggtggttgca gcagaaacca

gggaaatcat ttaagggcct gatctatcat ggaaccaact tggaagatgg agttccatca

aggttcagtg gcagtggatc tggagcagat tattctctca ccatcagcag cctggaatcc

gaagactttg cagactatta ctgtgtacag tatgttcagt ttccctacac gttcggaggg

gggaccaagc tggaaataaa acgggct

SEQ ID NO: 19-4D1VK amino acid sequence

Asp Ile Leu Met Thr Gln Ser Pro Ser Ser Met Ser Val Ser Leu Gly

1 5 10 15

Asp Thr Val Ser Ile Thr Cys His Ala Ser Gln Gly Ile Ser Ser Asn

20 25 30

Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile

35 40 45

Tyr His Gly Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser

65 70 75 80

Glu Asp Phe Ala Asp Tyr Tyr Cys Val Gln Tyr Val Gln Phe Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala

100 105

SEQ ID NO: 20-3G4VH nucleotide sequence

caggtccaac tgcagcagtc tggggctgag ctggtgaggc ctggggtctc agtgaagatt

tcctgcaagg gttctggcta cacattcact gattatgcta tgcattgggt gaagcagagt

catgcaaaga gtctagagtg gattggactt attaatactg actatggtga tactacttac

aaccagaagt tcaagggcaa ggccacaatg actgtagaca aatcctccaa cacagcctat

atggaacttg ccagactgac atctgaggat tctgccatct attackgtgc aagatcggac

tatgattact atttctgtgg tatggactac tggggtcaag gaaccacggt caccgaatct

cta

SEQ ID NO: 21 3G4VH amino acid sequence

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Val

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Gly Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ala Met His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Thr Asp Tyr Gly Asp Thr Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Met Thr Val Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Ala Arg Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Tyr Asp Tyr Tyr Phe Cys Gly Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Glu Ser Leu

115 120

SEQ ID NO: 24-2F2VH nucleotide sequence

caggtgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagcg cctgtccatc

acatgcaccg tctcagggtt ctcattaacc ggctatggtg tacactggat tcgccagtct

ccaggaaagg gtctggagtg gctgggaatg atatgggctg agggaagaac cgactataat

tcagttctca aatccagact gagcatcaat aaggacaatt ccaggagcca agttttctta

gaaatgaaca gtctgcaaac tgatgacaca gccaggtact actgtgccag agaggtgatt

actacggaag cctggtactt cgatgtctgg ggccaaggaa cctcggtcac cgaatct

SEQ ID NO: 25-2F2VH amino acid sequence

Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Arg Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Met Ile Trp Ala Glu Gly Arg Thr Asp Tyr Asn Ser Val Leu Lys

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Arg Ser Gln Val Phe Leu

65 70 75 80

Glu Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala

85 90 95

Arg Glu Val Ile Thr Thr Glu Ala Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Glu Ser

115

SEQ ID NO: 26-2F2VK nucleotide sequence

gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct

ctttcctgca gggccagcca gagtattagc gactacttat actggtatca acaaaaatca

catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg gatcccctcc

agattcagtg gcagtggatc agggtcagat ttcactctca ctatcaacag tgtggaacct

gaagatgttg gaatgtatta ctgtcaaaat ggtcacacct ttccgctcac gttcggtgct

ggcaccaagc tggaaatcaa acgg

SEQ ID NO: 27-2F2VK amino acid sequence

Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr

20 25 30

Leu Tyr Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Phe Thr Leu Thr Ile Asn Ser Val Glu Pro

65 70 75 80

Glu Asp Val Gly Met Tyr Tyr Cys Gln Asn Gly His Thr Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile Lys Arg.

Claims (154)

1. A sample collection device, said sample collection device comprising a main body, said main body comprising: (a) an upper containment chamber containing one or more solutions, and one or more rupturable seals; (b) a lower chamber in flow communication with an upper chamber containing extraction buffer and/or reagents; (c) a sample collection implement in flow communication with said upper containment chamber; (d) wherein the reagents comprise a plurality of specific binding agent sandwich pairs, each pair comprising: (i) a specific binding agent capture moiety conjugate comprising a specific binding agent that specifically binds the target antigen and the capture moiety together; (ii) a specific binding agent labeled conjugate comprising a specific binding agent that also specifically binds said target antigen together, wherein said pair can form a sandwich with said target antigen; Among them, multiple pairs are directed against different antigens. 2. The sample acquisition device of claim 1, wherein the capture moiety is an oligonucleotide, avidin, streptavidin, pyranRNA, an aptamer, or a combination thereof. 3. The sample acquisition device of claim 2, wherein the oligonucleotide is DNA or RNA. 4. The sample acquisition device of claim 1, wherein the label is a fluorophore, a chromophore, a metal, or a combination thereof. 5. The sample collection device of claim 1, wherein the capture moiety is pyranRNA and the label is europium. 6. The sample collection device of claim 1, wherein the target antigen is one or more influenza viruses. 7. The sample collection device of claim 1, wherein the influenza virus comprises a subtype of the form HxNy, wherein x is 1 to 16, y is 1 to 9, or any combination of xy. 8. The sample acquisition device of claim 1, wherein the extraction buffer is frozen. 9. The sample collection device of claim 1, wherein the reagents are present alone or in combination in the form of pellets, powders, pellets, beads, compressed frozen powders, or dried on a solid support. 10. The sample acquisition device of claim 1, further comprising a mixing region comprising absorbent material in the lower chamber. 11. The sample acquisition device of claim 1, wherein the upper sealed chamber comprises at least two separate sub-chambers. 12. The sample acquisition device of claim 11, wherein the subchambers contain the same or different solutions. 13. The sample acquisition device of claim 1, further comprising a membrane to filter particles from the sample. 14. The sample collection device of claim 1, wherein the sample collection implement is a swab. 15. The sample collection device of claim 1, wherein the sample collection implement comprises foam, brush construction, batt plastic, or spun fiber. 16. The sample collection device of claim 14, wherein the swab is upstream of a mixing zone. 17. The sample acquisition device of claim 13, wherein the reagent is contained within the membrane. 18. The sample acquisition device of claim 1, wherein the seal acts as a valve. 19. The sample collection device of claim 15, wherein the valve is a rupture valve, a flapper valve, a twist valve, or a screw-type valve, or is broken by breaking an ampoule. 20. The sample acquisition device of claim 1, wherein the mixing region contains reagents. 21. The sample acquisition device of claim 1, wherein the reagent is contained in a mixing region. 22. The sample acquisition device of claim 1, wherein the reagents are contained within a solid substrate. 23. A device comprising: (a) the sample chamber upstream of the plunger device; (b) a plurality of sealable holes for releasing one or more solutions into the device; (c) a substrate for filtering one or more compounds, wherein said substrate or said device comprises a reagent specific for at least one analyte. 24. A sample collection device, said device comprising a body, said body comprising: (a) an upper seal chamber containing buffer and valves; (b) a lower chamber in fluid communication with the upper chamber; (c) a swab, contained within said body; (d) a mixing region downstream of said swab comprising an absorbent or absorbent substrate, at least one pair of reagents comprising a label and a binding moiety. 25. A test device comprising a body comprising: (a) a lateral flow membrane, located in the body; (b) Upstream in the direction of cross flow, a fluid containing chamber, wherein the chamber is capable of controlled release of fluid into the cross flow material, wherein a gap exists between the chamber and the cross flow membrane. 26. The apparatus of claim 25, wherein the gap is greater than zero and less than or equal to 3.0 mm. 27. The device of claim 25, wherein the gap is capable of closing if pressure is applied to the chamber. 28. The device of claim 25, wherein the chamber contains a buffer solution with or without a detectable reagent. 29. The device of claim 28, wherein the detectable agent is capable of being detected by instruction. 30. The device of claim 28, wherein the detectable agent is visually detectable. 31. The device of claim 25, wherein said chamber consists of an ampoule. 32. The device of claim 25, wherein said controllably releasing comprises mechanical manipulation of said chamber. 33. The device of claim 25, wherein a cover or flange provides a safety means for said controllably releasing. 34. The device of claim 33, wherein the security device is broken or removed to gain access to the chamber. 35. The device of claim 25, wherein the membrane includes one or more designated test and control regions. 36. A test device comprising a body comprising: (a) a lateral flow membrane located in the body and exposed through single or multiple windows in the body, wherein the membrane comprises: (i) wicking material upstream of the plurality of windows; (ii) absorbent material downstream of the plurality of windows; (iii) a plurality of addressable lines, each line having a capture partner immobilized thereto, each capture partner capable of specifically binding to a different capture moiety; (iv) Does not include any specific reagents for the analyte of interest. 37. The test device of claim 36, wherein the capture partner comprises an oligonucleotide, avidin, streptavidin, pyranRNA, an aptamer, or a combination thereof. 38. The test device of claim 36, wherein the target analyte is an infectious agent. 39. The test device of claim 36, further comprising: in an upstream direction of transverse flow, a chamber comprising a fluid, the chamber controllably releasing the fluid to the wick material. 40. The test device of claim 36, wherein the chamber is breakable or punctureable. 41. The test apparatus of claim 36, further comprising an instrument capable of reading decoded instructions capable of initiating a scheme for detecting signals from one or more addressable lines. 42. The test device of claim 36, further comprising a chamber upstream of the membrane, wherein the chamber includes a fluid solution. 43. A kit comprising the sample acquisition device of claim 1 and the test device of claim 36, wherein each capture partner captures a different capture moiety. 44. A method comprising the steps of: (a) forming a mixture by mixing a sample to be tested for the presence of at least one target antigen with a solution comprising a plurality of reagents comprising: (i) antibody-capture moiety conjugates comprising antibodies that specifically bind together the target antigen and the capture moiety, (ii) labeled antibodies, wherein the antibodies bind together the same target antigen, wherein the reagent is directed against a plurality of different target antigens and forms a complex; (b) applying the mixture to a lateral flow membrane comprising a plurality of detection regions each having a capture partner immobilized thereto, each capture partner capable of binding a different capture moiety specifically combined; (c) the mixture flows through the membrane such that the capture moiety captures a complex corresponding to the capture moiety; (d) detecting the label in at least one of said detection zones, whereby detection of the label indicates the presence of the target antigen in said sample. 45. The method of claim 44, wherein the capture partner is an oligonucleotide, avidin, streptavidin, pyranRNA, an aptamer, or a combination thereof. 46. The method of claim 44, wherein the label is a fluorophore, a chromophore, a metal, a magnetic bead, a Raman particle, or a combination thereof. 47. The method of claim 44, wherein the label is europium. 48. The method of claim 44, wherein the target antigen is influenza virus. 49. The method of claim 48, wherein the influenza virus comprises influenza A and influenza B viruses. 50. A system comprising: (a) a test device as claimed in claim 25 or 36; (b) a reader that reads signals generated by any addressable line of the test device; (c) an instrument capable of reading decoded commands capable of instructing said reader to detect a signal or signals. 51. The system of claim 50, wherein the device is adapted to be inserted into a reader. 52. The system of claim 51, wherein the reader is adapted to perform the insertion only if the chamber is capable of controllably releasing the fluid. 53. A system for detecting one or more analytes in a fluid sample, the system comprising: (a) Sample collection device, including: (i) a swab sample collection device, (ii) chamber containing the fluid used to extract said swab, (iii) multiple pairs of first detection reagents and second detection reagents, each pair of reagents can specifically combine different target analytes in the fluid sample; wherein, the first detection reagent or the second detection reagent also includes a capture moiety ; (b) a test device comprising a substrate defining a lateral flow path, said substrate comprising a plurality of test areas within said flow path, said test areas each comprising an immobilized reagent capable of specifically binding said capture moieties together, wherein each test area comprises a different immobilization reagent capable of binding together different capture moieties; at least one control area is arranged in the lateral flow path, wherein the control area allows determination of the test device suitable performance. 54. The system of claim 53, wherein the sample collection device further comprises a reservoir for collecting a portion of the fluid sample. 55. The system of claim 53, wherein the immobilizing reagent is selected from the group consisting of oligonucleotides, avidin, streptavidin, pyranRNA, aptamers, or combinations thereof . 56. The system of claim 53, wherein the capture moiety is selected from the group consisting of oligonucleotides, avidin, streptavidin, pyranRNA, aptamers, or combinations thereof . 57. The system of claim 53, further comprising a reader into which the test device is insertable such that the reader detects the one or more analytes. 58. The system of claim 53, the plurality of first and second detection reagents selected from the group consisting of antibodies, nucleic acids, receptors, ligands, and antigens. 59. The system of claim 53, wherein the sample acquisition device is adapted to positively mate with a test device and allow complete transfer of the sample without spillage or exposure to outside air. 60. The system of claim 53, wherein the plurality of first detection reagent labels are fluorophores, chromophores, metals, or combinations thereof. 61. The system of claim 50, wherein the label is europium. 62. The system of claim 53, wherein the target analytes in the fluid sample comprise viral proteins and/or viral nucleic acids. 63. The system of claim 62, wherein the virus is influenza A and/or influenza B. 64. The system of claim 62, wherein the plurality of first reagents and the plurality of second reagents are antibodies. 65. The system of claim 63, wherein the type A virus comprises a subtype of the form HxNy, wherein x is 1 to 16, y is 1 to 9, or any combination of xy. 66. The system of claim 65, wherein the influenza A is H5N1. 67. The system of claim 53, wherein the testing device further comprises a compartment upstream of the substrate. 68. The system of claim 67, wherein the compartment contains a controllably released solution so as to contact the substrate. 69. The system of claim 67, wherein the compartment is separated from the substrate by a gap. 70. The system of claim 68, wherein the solution is sealed within the compartment by a breachable seal. 71. The system of claim 70, wherein the seal is ruptureable by applying pressure to the compartment. 72. A method for detecting one or more analytes in a fluid sample, said method comprising the steps of: (a) contacting the fluid sample with a system comprising: (i) Sample collection equipment, including: (1) swabs, (2) chamber containing the fluid used to extract the swab, (3) Multiple pairs of first detection reagents and second detection reagents, each pair of reagents can specifically combine different target analytes in the fluid sample; wherein, the first detection reagent or the second detection reagent also includes a capture moiety ; (ii) a test device comprising a substrate defining a lateral flow path, said substrate comprising a plurality of test areas within said flow path, said test areas each comprising an immobilized reagent capable of specifically binding said capture moieties together, Wherein, each test area comprises a different immobilization reagent capable of binding different capture moieties together; at least one control area is arranged in the lateral flow path, wherein the control area allows determination of the sensor's suitable performance; (b) detecting whether one or more analytes bind to said immobilized capture reagent disposed in said test device; (c) determining whether one or more analytes are present in the fluid sample if a signal is detected in at least one of the plurality of test areas. 73. The method of claim 72, wherein the sample collection device further comprises a reservoir for retaining a portion of the sample. 74. The method of claim 72, wherein the result remains steadily manifested for a period of greater than 30 minutes. 75. The method of claim 72, wherein the dilution-based system has a sensitivity equivalent to 2500 copies of influenza nucleic acid and a specificity of 98% or greater. 76. The method of claim 72, wherein the immobilizing reagent is selected from the group consisting of oligonucleotides, avidin, streptavidin, pyranRNA, aptamers, or combinations thereof . 77. The method of claim 72, wherein the immobilized capture agent is pyranRNA. 78. The method of claim 72, wherein each reagent in the plurality of first detection reagents comprises a label. 79. The method of claim 78, wherein the label is selected from the group consisting of a fluorophore, a chromophore, a particle, a metal, or a combination thereof. 80. The method of claim 78, wherein the label is europium. 81. The method of claim 72, wherein the multiplexed one or more analytes are one or more viruses. 82. The method of claim 81, wherein the one or more viruses comprise influenza A and/or influenza B strains. 83. The method of claim 81, wherein the one or more viruses comprise different virus strains and different virus subtypes. 84. The method of claim 83, wherein the different strains or the different subtypes comprise epidemic viruses, non-epidemic viruses, vaccinated viruses, non-vaccinated viruses, or combinations thereof. 85. The method of claim 72, wherein each of said plurality of test areas includes said immobilized reagent specific for said capture object. 86. A method for determining whether a subject is infected with one or more types and/or subtypes and/or strains of influenza virus, said method comprising: (a) contacting a sample from said subject with a system capable of detecting multiple analytes, wherein said system includes components capable of specifically binding to one or more of said influenza strains and/or subtypes together so as to form one or more components of the complex; wherein the complex formed from the sample passes through the system via a lateral flow path, wherein the one or more complexes are capable of The immobilized capture reagents in two or more different test areas of the lateral flow path are specifically bound together, wherein each of the two or more test areas contains a different protein capable of capturing a different analyte complex. The immobilized capture reagent of; (b) determining the presence of said one or more influenza virus strains and subtypes in said fluid sample by observing said two or more test areas, wherein a positive result indicates the presence of a particular influenza virus type and and/or subtype, the positive result remains unchanged for a period of greater than 30 minutes. 87. A method for determining whether a patient is infected with a particular type and/or subtype of influenza virus, said method comprising the steps of: (a) collecting a sample from said patient using a sample collection device, said sample collection device comprising: (i) a plurality of immunological reagents capable of detecting different types and subtypes of viruses, wherein, for any given type or subtype of said virus, said sample collection device contains viruses specific for said type or subtype a pair of said immunological reagents, the members of said pair respectively comprising a capture moiety and a label; (b) inserting said sample collection device into a lateral flow test device, wherein said insertion provides tight flow communication between said sample collection device and said lateral flow test device, wherein said test device comprises one or more test areas, wherein each said test area comprises a specific immobilized capture reagent for one of said capture moieties in (a); (c) detecting said marker in one or more test areas to determine whether said type or subtype of virus is present in said sample; It is thereby determined whether the patient is infected with one or more types and/or subtypes of influenza virus. 88. The method of claim 88, further comprising determining whether there is a subtype of one or more subtypes selected from epidemic, non-epidemic, subtypes with available vaccines, and subtypes without available vaccines. Influenza A. 89. The method of claim 87, wherein said labels for any given said pair are selected from the group consisting of fluorophores, chromophores, metals, or combinations thereof. 90. The method of claim 87, wherein the label is europium. 91. The method of claim 87, wherein the capture moiety is selected from the group consisting of oligonucleotides, aptamers, pyran RNA, avidin, streptavidin, or combinations thereof . 92. The method of claim 87, wherein the capture moiety is pyranRNA. 93. A method of processing a sample from a subject, said method comprising the steps of: (a) collecting a sample from said subject using a sample collection device, said sample collection device including one or more identical identification tags, wherein said sample collection device further includes an archive for storing at least a portion of said sample reservoir; (b) assaying said sample in a test device, thereby detecting one or more analytes by providing a detectable result on said test device; (c) at any time before or after step a or step b, attaching one of said identification tags to said archival reservoir and said test device; Said sample from said subject is thereby processed. 94. The method of claim 93, wherein the identification tag is selected from a bar code, a radio frequency transmitter, a light energy transmitter, an electromagnetic wave transmitter, or a combination thereof. 95. The method of claim 93, wherein the sample collection device further comprises a sample collection implement, the method further comprising attaching the same identification tag to the implement. 96. A lateral flow test device comprising a test strip for determining whether one or more analytes have bands of one or more dispersed pRNAs that are not discretely bound to one or more Indirect specific capture of multiple analytes. 97. The device of claim 96, further comprising a control strip not dispersedly bonded to the strip. 98. The device of claim 96, wherein the pRNA is bound to the strip by a hydrophilic protein. 99. The device of claim 96, wherein the strip comprises a detection zone having multiple dispersed bands of pRNA, each band capable of specifically capturing and recognizing a different The analyte, control zone has a band adapted to show movement of the liquid sample through the detection zone. 100. The device of claim 99, further comprising a sample application region upstream of the detection region. 101. A method of detecting one or more analytes in a fluid sample, said method comprising applying said sample to a lateral flow device, said lateral flow device comprising a nondispersively bound to said device or a plurality of dispersed bands of pRNA capable of indirectly and specifically binding the one or more analytes. 102. The method of claim 101 , further comprising reacting the sample with a first conjugate of each of the analytes prior to being applied to the device, each of the conjugates comprising a The analyte is specifically bound to a specific binding partner of the pRNA, suitable for recognizing one of said isolated bands of the pRNA on said device. 103. The method of claim 102, further comprising, for each of said analytes, adding a second conjugate comprising a specific binding partner capable of binding said analyte to a label. 104. The method of claim 103, wherein the second conjugate is added to the sample together with the first conjugate before applying the mixture to the device, or The first conjugate is then applied to the device with the second conjugate. 105. The method of claim 101, wherein the device comprises an absorbent strip having a sample application area, a detection area upstream of the strip comprising pRNA, and a control downstream of the detection area area. 106. A kit comprising: (a) a lateral flow device comprising an absorbent strip comprising (i) a sample application area, (ii) a detection zone comprising bands of one or more discrete pRNAs not discretely bound to the strip, each band capable of indirectly and specifically capturing a different analyte, and (iii) a control zone; (b) a first conjugate of each of said analytes, each of said conjugates comprising a specific binding partner capable of specifically binding an analyte to a pRNA, thereby recognizing one of said pRNAs on said strip the dispersed bands; (c) a second conjugate of each of said analytes comprising a specific binding partner capable of specifically binding to said analyte and a label. 107. The pRNA of claim 96, 101 or 106, having any one or more of the sequences provided in Table 5. 108. A method of detecting one or more analytes in a sample obtained from a subject, said method comprising applying said sample to an analysis system, wherein said system comprises at least one pair of complementary pRNA Sequences, wherein members of said at least one pair are conjugated to antibodies directed against said one or more analytes. 109. The method of claim 108, wherein the one or more analytes are proteins from an infectious agent. 110. The method of claim 109, wherein the infectious agent is a virus, HIV, hepatitis virus, adenovirus, enterovirus, or parainfluenza virus. 111. The method of claim 108, wherein the one or more analytes comprise at least two different types of infectious agents. 112. The method of claim 108, wherein the one or more analytes comprise at least three different types, subtypes, or combinations of types and subtypes of infectious agents. 113. The method of claim 111 or 112, wherein the infectious agent is influenza virus. 114. The method of claim 113, wherein the influenza virus is selected from influenza A, influenza B, influenza C, and combinations thereof. 115. The method of claim 113, wherein the influenza is selected from the group consisting of influenza A, influenza B, subtypes of epidemic influenza A, and subtypes of non-epidemic influenza A. 116. The method of claim 113, wherein the influenza virus is of the H5N1 subtype. 117. A device configured to detect a test signal from a test sample, wherein said device is also configured to detect a reference signal, wherein said reference signal is permanent and integrated into said device. 118. The apparatus of claim 117, wherein the test and reference signals are emitted by fluorescent or phosphorescent labels. 119. The device of claim 118, wherein the marker comprises a lanthanide metal. 120. The device of claim 118, wherein the label comprises europium, samarium, terbium, or dysprosium. 121. The device of claim 117, wherein the label is Europium III. 122. Apparatus as claimed in claim 117 or 122, wherein the markers are implanted or cast eg in a polymeric material. 123. The device of claim 123, wherein the polymer comprises acrylic, glass, plastic, rubber, or vinyl. 124. The device of claim 124, wherein the polymer comprises acrylic. 125. A fluorescence reader comprising a rigid standard, wherein the reader is configured to read a test sample comprising an analyte labeled with a molecule, wherein, The hard standard and the marker have a large Stokes shift. 126. The reader of claim 126, wherein the hard standard and the indicia comprise lanthanides. 127. The reader of claim 126, wherein the hard standard and the marker are selected from europium, europium, samarium, terbium, and dysprosium. 128. The reader of claim 126, wherein the reader comprises at least two rigid standards. 129. An ultraviolet reader comprising at least one permanent reference standard signal. 130. The reader of claim 130, wherein the reader comprises a laser or a photodiode. 131. The reader of claim 130, wherein the reference signal is a fluorescent signal. 132. The reader of claim 132, wherein the fluorescent light is emitted by lanthanides. 133. The reader of claim 130, wherein the signal is emitted by europium. 134. The reader of claim 133, wherein the reader is configured with light emitting diodes emitting in the ultraviolet spectrum. 135. The reader of claim 135, wherein the reader further comprises one or more filters that emit excitation light. 136. The reader of claim 136, wherein the one or more filters are selected from low pass, band pass, long pass, and combinations thereof. 137. The reader of claim 133, wherein the reader is configured with light emitting diodes that emit primarily light in a range between approximately 390nm and 425nm. 138. The reader of claim 133, wherein the reader is a light emitting diode reader. 139. An ultraviolet light emitting diode reader configured to detect a fluorescent signal from a test strip. 140. The reader of claim 140, wherein the fluorescent signal is emitted from lanthanides. 141. The reader of claim 140, wherein the fluorescent signal is emitted from europium. 142. The reader of claim 140, wherein the reader is further adapted to include at least one hard reference standard. 143. The reader of claim 140, wherein the reader includes a first hard reference standard and a second hard reference standard. 144. The reader of claim 140, wherein the reader comprises a light emitting diode emitting in the visible portion of the spectrum. 145. The reader of claim 140, wherein the reader further comprises one or more filters to confine the excitation light. 146. The reader of claim 146, wherein the one or more filters are selected from low pass, band pass, long pass, and combinations thereof. 147. The reader of claim 146, wherein the reader is configured to include an axis of excitation light at an angle of at least 90 degrees relative to the test strip. 148. The reader of claim 146, wherein the reader is configured to include an axis of excitation light at an angle other than 90 degrees relative to the test strip. 149. A fluorescence reader adapted to specifically and sensitively measure the luminescence of a fluorescent material, the fluorescence reader comprising: (a) light-emitting diodes that emit light in the ultraviolet region of the spectrum; (b) A photodiode capable of detecting the emitted fluorescent signal. 150. The reader of claim 150 adapted to measure fluorescent material on a lateral flow test strip. 151. The reader of claim 150, wherein the light emitting diode and photodiode are positioned such that the axis of excitation light from the light emitting diode is at a 90 degree angle to the test strip, the photodiode The axis of receiving emitted light is at an angle other than 90 degrees to the test strip. 152. The reader of claim 150, wherein wavelengths of the light emitting diodes and photodiodes are optimized to detect europium. 153. A system for detecting one or more analytes, said system comprising: (a) a lateral flow test strip comprising one or more discrete strips capable of directly or indirectly binding a specific binding partner of the one or more analytes; (b) a fluorescent reader capable of distinguishing fluorescent signals from each of the one or more bands on said test strip, said reader comprising a light emitting diode emitting in the ultraviolet region of the spectrum and capable of A photodiode that detects the emitted fluorescent signal. 154. The system of claim 154, wherein the fluorescent signal is produced by europium.

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