WO2000034522A2

WO2000034522A2 - Detection of biomaterial

Info

Publication number: WO2000034522A2
Application number: PCT/US1999/029000
Authority: WO
Inventors: Andrew Dubitsky; Damien Decollibus
Original assignee: Pall Corporation
Priority date: 1998-12-11
Filing date: 1999-12-06
Publication date: 2000-06-15
Also published as: AU2477000A; WO2000034522A9; WO2000034522A3

Abstract

A method and system for detecting a labeled complex including biomaterial without stringency washing after complexing and/or without amplifying the label is disclosed.

Description

DETECTION OF BIOMATERIAL

This application claims the benefit of provisional patent application nos. 60/111,915, filed December 11, 1998, and 60/163,788, filed November 5, 1999, and this application is a continuation-m-part application of copendmg application no. 09/392,793, filed September 9, 1999, and each of these applications is incorporated by reference.

TECHNICAL FIELD This invention relates to the detection of biomateπals such as nucleic acids and proteins.

BACKGROUND OF THE INVENTION

There are a variety of techniques for detecting (including analyzing) nucleic acids (e.g., RNA, DNA, and mRNA) , antigens, and proteins (e.g., antibodies, and some antigens) m fluid samples or tissues (e.g., culture fluid, cell lysates, organic extracts, bodily fluids such as blood). These techniques are useful for a variety of purposes such as diagnosis (to detect the presence of a pathogen such as a virus, to measure vaccine response and/or drug levels, for example), forensic studies (e.g., to identify the source of material such as blood found at a crime scene) , research (for example, to sequence a gene) , genetic screening, or to determine paternity. Thus, it is essential to accurately determine the presence or absence of the particular nucleic acid sequences, antigens or proteins m the sample or tissue. Typically, these techniques include treating or processing the sample thought to contain the material of interest (e.g., the nucleic acids or proteins), and placing the sample on a polymeric support such as a porous membrane. Treating the sample and placing it on the support can include extraction, electrophoretic separation, amplification, and/or blotting. Subsequently, a binding agent, such as a complementary nucleic acid probe or antibody that will specifically bind to the material, is placed in contact with the sample, and the binding agent is bound to the nucleic acid or protein to form a complex. The binding agent may be labeled before use and/or one or more labels may be added to the complex. Depending on the technique, additional binding agents (e.g., anti-antibodies) and/or labeling reagents can also be utilized. The label, that is bound to the complex, is subsequently detected, thus indicating the presence of the material of interest. Since the complex is formed as a result of the binding between the material of interest and the binding reagent (s) , the label should only be present if the material of interest is present. However, label can be present even though a complex was not formed. Typically, for example, some of the label, the labeled probe and/or the labeled antibody will non-specifically bind to the support, rather than specifically bind to form a complex. Accordingly, it is necessary to wash off the excess and/or non-specifically bound label, since failure to sufficiently remove the label can provide an inaccurate result, e.g., a false positive. In other words, although the label is detected, the complex is not present . Washing off the excess or non-specifically bound label can be a labor intensive effort, and may involve carefully contacting the support with a variety of different washing solutions. For example, the technician may have to wash the support several times, using wash solutions of different stringencies. The problem of inaccurate results can be further magnified in that some of the polymeric supports exhibit characteristics similar to those of the labels (characteristics referred to as "background noise") that make it difficult to distinguish the background noise of the support from the presence of the label. Thus, even when stringency washes are used, the background noise of the support can make it difficult to determine if the material of interest is present.

Additionally, some labels require amplification, e.g., enzymatic amplification. This is also labor intensive, as it requires the use of additional reagents and involves additional processing steps. Some labels are radioactive, requiring special handling and safety precautions during use, and during washing. The present invention provides for ameliorating at least some of the disadvantages of the prior art. These and other advantages of the present invention will be apparent from the description as set forth below.

SUMMARY OF THE INVENTION

In accordance with methods and systems provided according to the invention, a sample thought to contain a biomaterial of interest is combined with one or more binding agents, and a fluorescent labeled complex (including the biomaterial and at least one fluorescent labeled binding agent) is formed, wherein the labeled complex contacts a polymeric support comprising a membrane, and the labeled complex is detected without amplifying the fluorescent label and/or without washing the polymeric support after forming the labeled complex.

Methods and systems according to the present invention also provide for detecting a biomaterial of interest associated with (e.g., placed directly or indirectly in contact with) a polymeric support comprising a membrane, wherein a labeled complex is formed including the biomaterial, and the biomaterial is detected without amplifying the label in the complex and/or without washing the support after forming the complex.

In some embodiments, the biomaterial in the labeled complex is detected without enzymatically amplifying the label in the complex and without washing the support after forming the complex.

In preferred embodiments of the invention, the polymeric support comprises a polyamide membrane such as a nylon membrane, or a polysulfone membrane such as a polyethersulfone membrane, and the label comprises a fluorescent red dye that absorbs and emits light at a wavelength readily distinguishable from the fluorescence (e.g., the background or the auto-fluorescence) of the membrane.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagrammatic plot of fluorescence versus wavelength for several fluorescent labels (including a green label and two red labels) , red and blue lasers, and a nylon membrane .

Figure 2 shows the result of a hybridization assay carried out in accordance with an embodiment of the invention, showing selectivity in the absence of stringency washes. Figure 2(a) shows labeled complexes after hybridization and before stringency washing, Figure 2 (b) shows labeled complexes after stringency washing, and Figure 2 (c) shows labeled complexes using indirect detection.

Figure 3 shows the result of a Western transfer carried out in accordance with an embodiment of the invention, showing a detectable signal with and without washes. Figure 3 (a) shows labeled complexes before stringency washing, and Figure 3(b) shows labeled complexes after stringency washing.

SPECIFIC DESCRIPTION OF THE INVENTION

Embodiments of the invention address an unmet need in the art for a simplified and accurate detection protocol that can be utilized with nucleic acids and proteins, and does not require extra handling such as stringency washes and/or label amplification. In particular, embodiments of the invention provide a simplified, accurate protocol that does not require a radioactive label, and can be carried out using conventional equipment without extensive retraining of laboratory personnel and technicians. Methods and systems according to the present invention also provide for detecting a biomaterial of interest that is placed in association with a polymeric support comprising a polyamide membrane or a polysulfone membrane, wherein a labeled complex is formed including the biomaterial, and the biomaterial is detected. Illustratively, an embodiment of a method for detecting a biomaterial of interest comprises forming a fluorescent labeled complex comprising the biomaterial of interest and a binding agent including a fluorescent label, the fluorescent labeled complex being in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, wherein the fluorescent label comprises a fluorescent dye that absorbs and emits light at a wavelength of about 600 nm or more, and detecting the labeled complex. In accordance with some embodiments of the invention, a method for detecting a biomaterial of interest comprises forming a labeled complex including the biomaterial, the complex contacting a polymeric support comprising a polyamide membrane or a polysulfone membrane, and detecting the biomaterial without amplifying the label.

In accordance with additional embodiments of the invention, a method for detecting a biomaterial of interest comprises forming a labeled complex including the biomaterial, the complex contacting a polymeric support comprising a polyamide membrane or a polysulfone membrane, and detecting the biomaterial without washing the support after forming the labeled complex.

In some embodiments of the invention, the biomaterial is detected without washing the support after forming the labeled complex, and without amplifying the label. In accordance with preferred embodiments of the invention, fluorescent labels (even more preferably, red fluorescent labels) are utilized wherein the labels have emission maxima that correspond to low or minimal fluorescence produced by the polymeric supports. In some preferred embodiments, wherein the polymeric support comprises a polyamide (preferably nylon) membrane or a polysulfone (e.g., a polysulfone, a polyethersulfone, a polyphenylsulfone, or a polyarylsulfone) membrane, a red fluorescent label is utilized. Illustratively, as shown in Figure 1, red fluorescent labels have emission maxima that are readily distinguishable from the fluorescence produced by the reference nylon membrane. Accordingly, a labeled complex including a red fluorescent label can be directly detected without amplification.

Moreover, particularly in those embodiments wherein non-specific binding is low or reduced, the labeled complex can be directly detected without washing the polymeric support after the labeled complex is formed. Additionally, or alternatively, as the label can be directly detected without amplification (and since amplification can create "noise" or spurious signals) , embodiments of the invention provide for directly detecting the labeled complex without washing the polymeric support after forming the labeled complex.

Illustratively, the biomaterial can be placed in contact with (e.g., placed in and/or on) the support, and a labeled binding agent that specifically binds to the biomaterial (to form a complex) can be applied to the biomaterial to allow binding to occur. Since the binding agent is labeled, the labeled complex, that includes the biomaterial, is detectable without amplifying the label after forming the complex. Alternatively, a binding agent that is capable of binding to the biomaterial can be placed in contact with the support, and the biomaterial can be added to form a complex (e.g., wherein the biomaterial may indirectly contact the support) , that can be detected as described below.

If desired, a "sandwich assay" or a "double binding agent assay" can be carried out m accordance with the invention, e.g., wherein a biomaterial and a first

(unlabeled) binding agent form a first complex contacting the polymeric support, and a second (labeled) binding agent binds to the first binding agent or the biomaterial of interest to form a second (labeled) complex, that is subsequently detected, without amplifying the label after forming the complex.

Alternatively, or additionally, the labeled complex formed m accordance with any of these embodiments can be detected without washing the support after forming the labeled complex.

In some embodiments, a method for detecting a biomaterial of interest comprises placing a sample, thought to contain the biomaterial of interest, m contact with a polymeric support, adding a labeled binding agent that specifically binds with the biomaterial of interest to form a labeled complex, wherein the labeled binding agent and the biomaterial form the labeled complex contacting the support, and detecting the labeled complex without washing the support after forming the complex and/or without amplifying the label after forming the complex.

In some other embodiments, a method for detecting a biomaterial of interest comprises placing a sample, thought to contain the biomaterial of interest, m and/or on a polymeric support, adding a first binding agent that binds with the biomaterial of interest to form a first complex, wherein the first binding agent and the biomaterial form the first complex, adding a labeled second binding agent that binds with the first binding agent m the first complex to form a labeled (second) complex, and detecting the labeled complex without washing the support after forming the labeled complex and/or without amplifying the label after forming the labeled complex.

In variations of any embodiments of the invention (e.g., the embodiments described above) , the first binding agent can be placed in contact with the polymeric support, and the sample thought to contain the biomaterial of interest is added to form a labeled complex (or to form a first complex) in contact with the support. The labeled complex can be detected, or a second (labeled) complex can be formed and detected, as described above.

In accordance with an embodiment of the invention, a method for detecting a biomaterial of interest comprises combining, in contact with a polymeric support comprising a nylon membrane, a sample thought to contain a biomaterial of interest and a fluorescent red dye-labeled binding agent adapted to specifically bind to the biomaterial and form a fluorescent red dye-labeled complex, forming the labeled complex in contact with the membrane, and detecting the labeled complex without amplifying the fluorescent red label and/or detecting the labeled complex without washing the membrane after forming the labeled complex.

A method for detecting a biomaterial of interest according to another embodiment of the invention comprises placing a sample thought to contain the biomaterial of interest in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, adding a binding agent that specifically binds with the biomaterial of interest to form a complex, the binding agent having a label, wherein the labeled binding agent and the biomaterial form the complex, and detecting the complex without washing the support and/or without amplifying the label after forming the complex.

In an embodiment, a method for detecting a biomaterial of interest comprises placing a sample thought to contain the biomaterial of interest in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, adding a first binding agent that binds with the biomaterial of interest to form a complex, wherein the first binding agent and the biomaterial form the complex, adding a labeled second binding agent that binds with the first binding agent in the complex to form a labeled complex, and detecting the labeled complex without washing the support after forming the labeled complex.

In accordance with another embodiment, a method for detecting a biomaterial of interest comprises placing a sample thought to contain the biomaterial of interest in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, adding a first binding agent that binds with the biomaterial of interest to form a complex, wherein the first binding agent and the biomaterial form the complex, adding a second binding agent that binds with the first binding agent in the complex, the second binding agent having a label, and forming a labeled complex, and detecting the labeled complex without amplifying the label after forming the labeled complex.

In yet another embodiment of the invention, a method for detecting a biomaterial of interest comprises placing a sample thought to contain the biomaterial of interest in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, adding a first binding agent that binds with the biomaterial of interest to form a complex, wherein the first binding agent and the biomaterial form the complex in contact with the support, adding a second binding agent that binds with the first binding agent in the complex, the second binding agent having a label, and forming a labeled complex, and detecting the labeled complex without either washing the support or amplifying the label after forming the labeled complex.

Another embodiment of the invention comprises forming a fluorescent labeled complex comprising a biomaterial of interest and a fluorescent labeled binding agent, the labeled complex being in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, and detecting the labeled complex without washing the support after forming the labeled complex and/or without amplifying the label after forming the labeled complex. In one embodiment, the labeled complex is detected without washing the support after forming the labeled complex and without amplifying the label after forming the labeled complex. Embodiments of the invention also include a system and/or a kit for carrying out the methods.

For example, an embodiment of a system for detecting a biomaterial of interest according to the invention comprises a polymeric support comprising a polyamide (e.g., nylon) membrane or a polysulfone (e.g., polysulfone, polyethersulfone, polyphenylsulfone, or polyarylsulfone) membrane, a fluorescent label that absorbs and emits light at about 600 nm or more (preferably a fluorescent red label that absorbs and emits light at about 650 nm or more) , wherein the fluorescent label is capable of attachment to a binding agent that is adapted to specifically bind to the biomaterial of interest and form a complex with the biomaterial of interest, wherein the system is arranged to detect a labeled complex without washing the support after forming the labeled complex and/or to detect a labeled complex without amplifying the label.

Another embodiment of a system for detecting a biomaterial of interest comprises (1) a polymeric support comprising a nylon membrane or a polysulfone membrane for receiving the biomaterial of interest, or for receiving a first binding agent capable of binding to the biomaterial of interest, (2) a first binding agent capable of binding to the biomaterial and forming a first complex contacting the polymeric support, (3) a second binding agent capable of binding to the first complex, thus forming a second complex, by binding to the first binding agent or the biomaterial of interest, and (4) a fluorescent red label, wherein the fluorescent red label is capable of attachment to the second binding agent so that the second complex is a labeled complex, wherein the system is arranged to detect the labeled complex without washing the support after forming the labeled complex and/or to detect a labeled complex without amplifying the label.

A system for detecting a biological material of interest according to an embodiment of the invention comprises a polymeric support comprising a polyamide membrane or a polysulfone membrane, and a fluorescent red label, wherein the fluorescent red label is capable of attachment to a binding agent that is adapted to specifically bind to the biomaterial of interest and form a complex with the biomaterial of interest in contact with the polymeric support, wherein the system is arranged to detect a labeled complex without washing the support and to detect the labeled complex without amplifying the label after forming the labeled complex.

In accordance with another embodiment, a system for detecting a biomaterial of interest comprises a polymeric support comprising a polyamide membrane or a polysulfone membrane, a first binding agent that is capable of binding to the biomaterial of interest to form a first complex, a second binding agent that is capable of binding to the biomaterial of interest or the first binding agent to form a second complex, a fluorescent red label, wherein the fluorescent red label is capable of attachment to the second binding agent so that the second complex is a labeled complex, wherein the system is arranged to detect a labeled complex without washing the support after forming the labeled complex.

Another embodiment of a system for detecting a biomaterial of interest comprises (1) a polymeric support comprising a polyamide membrane for receiving the biomaterial of interest, or for receiving a first binding agent capable of binding to the biomaterial of interest, (2) a first binding agent adapted to bind to the biomaterial and form a first complex in contact with the polymeric support, (3) a second binding agent adapted to bind to the first complex, thus forming a second complex, by binding to the first binding agent or the biomaterial of interest, and (4) a fluorescent label, wherein the fluorescent label is capable of attachment to the second binding agent so that the second complex is a labeled complex, wherein the system is arranged to detect the labeled complex without washing the support after forming the labeled complex and/or wherein the system is arranged to detect the labeled complex without amplifying the label . In some embodiments, the system includes a test device including the polymeric support, wherein the test device includes at least one additional element, e.g., a chip such as a biochip.

As used herein, the term "biomaterial" includes, but is not limited to, nucleic acid sequences (e.g., natural or synthetic DNA, RNA (including mRNA) , and/or PNA (peptide nucleic acids); mixtures and/or hybrids thereof, as well as oligonucleotides, modified nucleic acids, fragments and/or derivatives of nucleic acids) , antigens, proteins (including antibodies, and some antigens), peptides, bacteria, viruses, protozoans (as well as components of bacteria, viruses, and protozoans), and one or more analytes of interest (e.g., recombinant nucleic acid products and/or byproducts, drugs, pollutants, and poisons) . The biomaterial can be obtained from a variety of sources. A sample containing the biomaterial can comprise one or more cells, or an aqueous or aqueous miscible solution that is obtained directly from a liquid source or as a wash from a solid material, a growth medium or buffer solution in which biomaterial is present or has been introduced. In some embodiments, the sample is obtained from a biological fluid, including separated or unfiltered fluids such as blood or blood components, urine, cerebrospinal fluid, lymph fluids, tissue homogenate, cell extracts, saliva, sputum, stool, or physiological secretions. The sample can be obtained from an environmental source, e.g., taken from the air (including, for example, the air at a battlefield, or a site where chemical warfare may have taken place) , a waste stream, a water source, a supply line, or a production lot. Industrial sources include fermentation media, such as from a biological reactor or food fermentation process such as brewing, or foodstuffs, such as meat, produce, or dairy products.

If desired, the biomaterial can be amplified and/or enriched (e.g., allowed to reproduce in growth medium) before placing it in contact with the membrane. Alternatively, the biomaterial can be placed in contact with the membrane without previous amplification and/or enrichment. For example, a sample containing the biomaterial (e.g., a liquid sample, a solid sample, or a gaseous sample such as air) can be placed in contact with the membrane. In some embodiments, a sample is passed through the membrane (e.g., the membrane "filters" the sample so that some portion of the biomaterial is retained) , and the biomaterial is subsequently detected. Embodiments of the system and/or kit can include additional components, e.g., binding agents, other reagents, solutions (e.g., buffer solutions) and/or blocking agents.

As used herein, the term "binding agent" includes, but is not limited to, one or more ligands and receptors, e.g., nucleic acid probes, antibodies (including a monoclonal antibodies, polyclonal antibodies, and anti-antibodies), and specific binding proteins (e.g., surface membrane protein receptors) . Preferably at least one binding agent, e.g., the binding agent that is capable of binding to the biomaterial of interest, is a specific binding agent. For example, in some embodiments wherein the biomaterial of interest is a nucleic acid sequence, the specific binding agent has at least about 30% (more preferably, at least about 50%) complementarity with a region in the biomaterial of interest . The desired specificity can vary, depending on the particular nature of the biomaterials to be detected, the information desired about the nature of the sample, and the like.

Typically, in those embodiments including a plurality of binding agents, e.g., a first binding agent and a second binding agent, the first binding agent is capable of binding to the biomaterial, and the second binding agent is capable of binding to either the first binding agent (e.g., as in a double antibody assay), or to the biomaterial (e.g., as in a sandwich assay) .

Embodiments of the invention can be carried out with a variety of techniques and assays, such as hybridization assays and immunoassays , including, but not limited to, arrays (e.g., microarrays, including but not limited to those described in "Microarrays: biotechnology's discovery platform for functional genomics," Schena, M. et al . , Trends in Biotechnology, (1998), vol. 16, July, pp. 301-306), mRNA abundance analyses, Southern, Northern, Western, Southern-Western, dot, slot, and colony blots. Embodiments of the invention are compatible with automated and semi -automated protocols, as well as high throughput applications.

In accordance with the invention, detecting the biomaterial can include confirming the presence of the biomaterial, as well as (if desired) identifying the biomaterial, analyzing the biomaterial, and/or quantifying the biomaterial. Biomaterial can be detected as part of an on-going or monitoring process, e.g., as part of a quality control system, and/or to monitor the appearance/removal (or rate of appearance/removal) of the biomaterial in the sample, in the material of interest and/or in the product or process fluid being produced. A variety of polymeric supports comprising porous and non-porous media (including porous and non-porous films, such as, for example, free standing films, extruded films, cast films, coated films, composites, and laminates) comprising polyamide (e.g., nylon), or polysulfone (e.g., at least one of polysulfone, polyethersulfone, polyphenylsulfone, and polyarylsulfone) , membranes are suitable for use in the invention. Suitable polymeric supports comprising nylon include, but are not limited to, at least one of nylon 6, 6T, 11, 46, 66, and 610. The polyamide (e.g., nylon) or polysulfone (e.g., polyethersulfone) supports can be unmodified or modified to include a surface charge, e.g., a positive or negative charge, or to alter the polarity or hydrophilicity of the surface. Examples of such modifications include grafting, e.g., irradiation, a polar or charged monomer, coating and/or curing the surface with a charged polymer, and carrying out conventional chemical modification to attach functional groups on the surface. The polymeric support can also include additional material (including additional polymeric material (s) ) and/or can include combinations of materials. The polymeric support can consist essentially of, or even consist entirely of, polymeric material (s) .

In some embodiments, diagnostic and/or analytic test devices comprise a polymeric support and one or more additional elements, e.g., a glass slide, a chip such as a biochip or a microfabricated chip (e.g., a microarray on a silicon based platform, suitable for, for example, microfluidic protocols) . Accordingly, some embodiments of a system according to the invention include a test device comprising a polymeric support and at least one additional element (e.g., a chip) in contact with the support, and a fluorescent label .

Suitable membranes include, but are not limited to, those described in U.S. Patent Nos. 4,340,479, 4,702,840, 4,707,266, 4,900,449, 4,906,374, 4,964,989, 4,964,990, 5,108,607, 5,277,812 and 5,531,893, and International Publication No. WO 98/21588.

As noted above, the polymeric support (for example, the nylon membrane and/or the polysulfone membrane) can be untreated (e.g., unmodified) or treated (e.g., modified), for example, to provide for covalent or non-covalent binding and/or to reduce or minimize non-specific binding.

Illustratively, a polyamide (e.g., nylon) membrane and/or a polysulfone (e.g., polyethersulfone) membrane can be charged, or uncharged. Alternatively, or additionally, the membrane can be activated to produce covalent bonds.

If desired, the membranes can be suitable for binding the biomaterials through covalent interaction, or non-covalent bonds, e.g., hydrophobic and/or ionic attraction.

Typically, in those embodiments wherein the support comprises a polyamide (e.g., nylon) membrane, the membrane has a neutral charge, a positive charge, or is activated to produce covalent bonds. In some of the embodiments wherein the support comprises a polysulfone (e.g., polyethersulfone) membrane, the membrane has a neutral charge, or is activated to produce covalent bonds.

Suitable membranes include, for example, asymmetric or homogenous membranes.

A variety of porous and non-porous membranes, including commercially available membranes, are suitable for carrying out the invention. Suitable membranes include, but are not limited to, those available from Pall Corporation under the trade names BIODYNE^® PLUS, BIODYNE^® A, BIODYNE^® B, BIODYNE^®

C, POSIDYNE^®, LOPRODYNE^® LP, SUPOR^®, SUPOR^® 30Q, SUPOR^® 30 PLUS, PREDATOR®, ULTRABIND™, and IMMUNODYNE^® ABC.

A variety of fluorescent labels and fluorescent label detection protocols are suitable for carrying out the invention. The fluorescent label (e.g., a fluorescent dye) should be attachable to a binding agent, wherein the labeled binding agent remains capable of binding, e.g., to the biomaterial, or to another binding agent.

The label comprises a fluorescent dye, more preferably a fluorescent red dye, and has an emission maxima that corresponds to low or minimal fluorescence produced by the membrane utilized.

For example, Figure 1 shows examples of fluorescent red dyes having emission maxima corresponding to low or minimal fluorescence produced by a commercially available nylon membrane. In this Figure, "Red Dye 1" corresponds to Cy5™ dye (Amersham Life Science, Inc., IL) , and "Red Dye 2" corresponds to IRD 700™ dye (Licor, NE) .

The fluorescent label should have sufficient Stokes' shift that it is discernable from reflection and excitation signals. In a more preferred embodiment, the fluorescent dye has an excitation and emission wavelength that is compatible with commercially available detection instruments.

The fluorescent label preferably comprises a fluorescent dye that can absorb and emit light at a wavelength of about

600 nm or more. More preferably, the fluorescent label comprises a fluorescent red dye that can absorb and emit light at about 650 nm or more. For example, in some embodiments, the fluorescent red dye absorbs and emits light at about 700 nm or more (e.g., the fluorescent red dye comprises a fluorescent near infra-red dye) .

Exemplary fluorescent red dyes include, but are not limited to, phycocyanin, allophycocyanin, cyanine and related polymethine dyes, as well as phycobiliproteins . For example, the cyanine and related dyes disclosed in U.S. Patent No.

5,268,486 are suitable, as are cyanine dyes commercially available from Licor (Lincoln, NE) , e.g., IRD 700™ dye, Amersham Life Science, Inc. (Arlington Heights, IL) e.g.,

Cy5™ dye, and phycobiliproteins available from ProZyme® (San Leandro, CA) , e.g., phycobiliprotein PB22 GT5™ Allophycocyanin .

The fluorescent emission can be detected qualitatively or quantitatively, using, for example but not limited to, visual detection, laser scanning devices, video cameras, photographic film, microscopes (including but not limited to confocal microscopes), and fluorometers .

Typically, embodiments of the invention are carried out without amplifying the label, e.g., without amplifying the label with enzymes, or without using primary and secondary labeled probes. However, label amplification can be carried out if desired (e.g., for detecting luminescence), and additional reagents can be utilized.

A variety of processing conditions are suitable as are known in the art . For example, in some embodiments involving the detection of nucleic acids, the hybridization time is typically about 10 minutes or more, at an incubation temperature of between about 30° C to about 70° C. The pH of a typical hybridization solution is about 7, wherein the solution preferably includes a buffer, salt and a surfactant. The hybridization solution may include at least one blocking agent (e.g., to reduce non-specific binding), including, but not limited to, milk casein or milk protein. In some embodiments of the invention, after hybridization is carried out, subsequent washes are preferably omitted. Thus, there is no need or one or more subsequent washes, e.g., a series of stringency washes (typically including low ionic strength buffer and surfactant) at room and then elevated temperature, to remove unbound probe from the membrane . If desired, prehybridization can be carried out.

Illustratively, after the nucleic acid sample is fixed to the membrane, but before the probe is added, the membrane can be prehybridized with, for example, buffer including salt and a surfactant. The prehybridization solution may also include at least one blocking agent as described above. After prehybridization (e.g., about one hour or less), hybridization (e.g., adding the labeled probe) can be carried out .

In accordance with some of the embodiments including the detection of antigens and/or proteins, the antigens and/or proteins are immobilized in and/or on the polymeric support (e.g., the membrane), and the membrane is blocked using a buffer solution including at least one blocking agent such as, for example, milk casein or milk protein. Subsequently (for example, after a few minutes to about an hour) a labeled binding agent (e.g., an antibody) is added and incubated, illustratively, for a few minutes to about an hour, to form a complex. In some embodiments of the invention, after the labeled binding agent is added, there is no need to use one or more washes to remove unbound binding agent from the membrane .

Typically, in those embodiments including the detection of antigens and/or proteins utilizing first and second binding agents, the first binding agent is not labeled, and the second binding agent is labeled.

EXAMPLE 1 A reverse dot blot assay for a β-globin sequence is performed, wherein the polymeric support is a porous nylon membrane. An unlabeled oligonucleotide sequence from the β-globin locus (Research Genetics, Huntsville, AL) is diluted in 2X SSC and applied as 0.2 μl spots to a BIODYNE® PLUS positively charged nylon membrane having a pore size of 0.45 μm (Pall Corporation, East Hills, NY) . Twelve dilutions of DNA are applied as duplicate columns of spots, with the spots containing 60, 20, 6, 2, 0.6, 0.2, 0.06, 0.02, 0.006, 0.002, 0.0006, and 0.0002 ng oligonucleotide per spot. The membrane is contacted with a solution containing 50 mM phosphate, pH 7, 0.5% Hammersten casein, and 10% sodium dodecyl sulfate (SDS) , and the membrane is air dried, baked at 80 °C, and exposed to UV light.

Complementary oligonucleotide probes end-labeled with a commercially available fluorescent cyanine dye, IRD 700™ (Licor, Lincoln, NE) , m a solution containing 50 mM phosphate, pH 7 , 0.5% Hammersten casein, and 10% SDS, are applied to the membrane to allow hybridization to occur. Hybridization is carried out overnight at 45 °C. After hybridization, no stringency washes are carried out, and the label is not amplified using an enzyme. The fluorescent signal of the hybridized probes is detected using a STORM™ imager (Molecular Dynamics, Inc., Sunnyvale, CA) , to scan the wet membrane. The imager uses a red diode laser (emitting red light at 635 nm ± 5 nm) for excitation of the label. Spots containing down to 60 pg are detected.

This example shows that hybridized DNA is detected, using fluorescence as part of a homogenous assay, without stringency washes and without enzymatically amplifying the label .

EXAMPLE 2 Serial dilutions of 17mer oligonucleotides end-labeled with Cy5™ red dye, IRD 700™ dye, or fluorescein, are applied to the following membranes (each available from Pall Corporation, East Hills, NY) : BIODYNE^® PLUS and BIODYNE^® B nylon membranes, and FLUOROTRANS^® polyvinylidene difluoride membranes .

Twelve dilutions of DNA are applied as four replicate spots per location, with the spots containing from 20 ng to 60 fg per 0.2 μl spot. The membranes are air dried and placed in a STORM™ imager. The membranes are scanned with both blue (488 nm ± 5 nm) and red (635 nm ± 5 nm) excitation.

The sensitivity (minimum pg labeled DNA required to produce a positive signal) is shown in the following Table I: Membrane blue scan red scan

TABLE I

This example shows DNA labeled with two different fluorescent red dyes and detected using red excitation is detected with greater sensitivity (for one red dye, by about 3 orders of magnitude greater sensitivity) than DNA labeled with fluorescein and detected using red and blue excitation. This example also shows DNA labeled with the red dyes is detected with greater sensitivity using red excitation than blue excitation.

The red diode laser being used in this example (emitting red light at 635 nm + 5 nm) , does not provide optimal excitation for one of the red dyes, i.e., IRD 700™ dye. It is believed that the IRD 700™ red dye label can be detected with greater sensitivity using a red diode laser that emits red light at about 700 nm.

EXAMPLE 3

Reverse dot blot assays for a β-globin sequence are performed, wherein an unlabeled oligonucleotide sequence from this locus (Research Genetics, Huntsville, AL) is diluted in 2X SSC and applied as 0.2 μl spots to the following membranes (Pall Corporation, East Hills, NY) : BIODYNE^® PLUS and BIODYNE^® A nylon membranes, and a SUPOR^® 30Q polyethersulfone membrane .

Complementary oligonucleotide probes end-labeled with the commercially available dyes fluorescein, IRD 700™, or

Cy5™ in a solution containing 50 mM phosphate, pH 7 , 0.5% Hammersten casein, and 10% SDS, are applied to the membranes to allow hybridization to occur. The membranes having Cy5™ and IRD 700™ labeled probes are promptly scanned in a Molecular Dynamics Inc. STORM™ imager (using red excitation) without exposing the membranes to stringency washes .

The membranes having fluorescein-labeled probes are exposed to stringency washes, blocked with casein, and incubated with anti-fluorescein IgG antibody conjugated to alkaline phosphatase . Signal is generated with a color forming substrate containing 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and 4-nitroblue tetrazolium chloride (NBT) . The sensitivity (minimum pg labeled DNA required to produce a positive signal) is shown in the following Table II :

TABLE II

This example shows DNA labeled with two different fluorescent red dyes can be directly detected without requiring stringency washes and label amplification, and can be detected with at least similar sensitivity to DNA labeled with fluorescein and exposed to stringency washes and label amplification.

EXAMPLE 4 Two unrelated target sequences, β-globin sequence and Lambda Hind III, are applied in alternating rows to BIODYNE^® PLUS nylon membranes. Complementary probes for the β-globin sequence, end-labeled with Cy5™ or IRD 700™, in a solution as described in Example 3, are applied to the membranes to allow hybridization to occur.

The membranes are scanned in a STORM™ imager (using red excitation) before and after exposing the membranes to stringency washes. Subsequently, using these same membranes, complementary probes for the Lambda Hind III, labeled with digoxigenin (DIG) (Roche Molecular Bioproducts) , in Roche EZ Hyb hybridization solution containing .5% casein, are applied to the membranes to allow hybridization to occur.

The membranes are blocked with casein, and incubated with anti-DIG antibody conjugated to alkaline phosphatase

(Roche Molecular Bioproducts) . Signal is generated with the color forming substrate BCIP/NBT.

The results are shown in Figure 2, wherein Figure 2(a) shows the results post hybridization before stringency washing, Figure 2 (b) shows the post stringency washing results, and Figure 2(c) shows the results utilizing indirect Lambda detection. The labeled probes shown in the top row of

Figure 2 (Red Dye 1) are labeled with Cy5™ red dye, and the labeled probes shown in the bottom row of Figure 2 (Red Dye 2) are labeled with IRD 700™ red dye.

There is no cross reactivity between the probe systems, and the sensitivity for the Cy5™ red dye system, and the DIG system, is 20 pg per spot. The sensitivity for the IRD 700 ™ red dye system is 200 pg per spot. As described with respect to Example 2, it is believed that the IRD 700™ red dye label can be detected with greater sensitivity using a red diode laser that emits red light at about 700 nm. This example shows unrelated DNA can be detected selectively, even when stringency washes are omitted. EXAMPLE 5 Protein dot blots are performed using various membranes as described below. Mouse immunoglobulin G (IgG), whole molecule (Rockland, Gilbertsville, PA) is diluted in phosphate buffered saline (PBS) pH 7.2 and applied to the following membranes, each available from Pall Corporation (East Hills, NY) : BIODYNE^®

PLUS and BIODYNE^® A nylon membranes; an ULTRABIND™ SV-450 polyethersulfone membrane; and FLUOROTRANS^® and FLUOROTRANS^® W polyvinylidene difluoride membranes. The IgG is applied using a PlateMate Liquid Handling Station and 96-pin transfer tool (Matrix Technologies, Lowell, MA) .

Dilutions of IgG are applied to the membranes as 0.2 μl spots, containing 200, 60, 20, 6, 2, 0.6, 0.2, 0.06, 0.02, 0.006, and 0.002 ng protein. The membranes are incubated in a PBS solution containing 0.3% Hammersten grade casein (Gallard Schlessinger, Westbury, NY) for 30 minutes at room temperature, with gentle agitation. The membranes are next incubated in PBS solution containing 0.05% Hammersten grade casein, and a goat IgG developed against mouse IgG that is crosslinked and conjugated to phycobiliprotein PB22 GT5™ Allophycocyanin (ProZyme^®, San Leandro, CA) for 1 hour at room temperature, with gentle agitation.

The fluorescent signal of the phycobiliprotein conjugate is detected using a STORM™ imager to scan the wet membranes. The imager uses a red diode laser (emitting red light at 635 nm + 5 nm) for excitation of the label. The membranes are washed twice in IX PBS for 5 minutes at room temperature, with gentle agitation. The fluorescent signal of the phycobiliprotein conjugate is detected using the STORM™ imager to scan the wet membranes. The endpoint sensitivity is shown in the following Table

III

TABLE III

Endpoint Sensitivity

This example shows that a fluorescently labeled immune complex (immobilized antigen and complementary antibody labeled with a fluorescent red tag) can be detected in an immunoassay without enzymatically amplifying the label. The example also shows the fluorescently labeled immune complex can be directly detected immediately after the conjugate incubation step, without stringency washes.

EXAMPLE 6 Western transfers of proteins are performed using 2 positively charged nylon membranes.

Mouse immunoglobulin G (IgG) , whole molecule (Rockland, Gilbertsville, PA) is diluted in 4X sample loading buffer (Novex, San Diego, CA) and sterile water for injection USP

(McGaw, Irvine, CA) . Dilutions of IgG, from 20 μg to 200 pg, are added to the wells of a polyacrylamide NuPAGE 4-12% Bis-Tris gel (Novex) with wells containing from 20000 ng to 0.2 ng of protein per well. The protein is electrophoresed in NuPAGE MES SDS Running Buffer (Novex) for 35 minutes at 200 volts. Using an Xcell II blot module (Novex), protein is electro-transferred to 2 different positively charged nylon membranes, for 1 hour at 25 volts.

The membranes are incubated in a PBS solution, pH 7.2, containing 0.3% Hammersten grade casein for 30 minutes at room temperature, with gentle agitation.

The membranes are next incubated in PBS solution containing 0.05% Hammersten grade casein, and a goat IgG developed against mouse IgG that is crosslinked and conjugated to phycobiliprotein PB22 GT5™ Allophycocyanin (ProZyme^®, San Leandro, CA) for 1 hour at room temperature, with gentle agitation.

The fluorescent signal of the phycobiliprotein conjugate is detected using a STORM™ imager to scan the wet membranes.

The imager uses a red diode laser (emitting red light at 635 nm ± 5 nm) for excitation of the label.

As shown in Figure 3 (a) , after incubation with IgG labeled with Allophycocyanin conjugate, signal is detected in all lanes on the transfer membranes (left column shows positively charged nylon membrane 1, the right column shows positively charged nylon membrane 2) . The endpoint sensitivity is less than 200 pg of antigen per lane.

The membranes are washed twice in IX PBS for 5 minutes at room temperature, with gentle agitation. The fluorescent signal of the phycobiliprotein conjugate is detected using the STORM™ imager to scan the wet membranes.

As shown in Figure 3 (b) , after PBS washing, signal is again detected in all lanes on the transfer membranes (left column shows positively charged nylon membrane 1, the right column shows positively charged nylon membrane 2) . This example shows that a fluorescently labeled immune complex (an antigen transferred to a nylon membrane from a separation gel and a complementary antibody conjugated to a fluorescent red tag) can be detected in an immunoassay without enymatically amplifying the label. The example also shows the fluorescently labeled complex can be directly detected immediately after the conjugate incubation step, without stringency washes.

All of the references cited herein, including publications, patents, and patent applications, are hereby incorporated in their entireties by reference.

While the invention has been described in some detail by way of illustration and example, it should be understood that the invention is susceptible to various modifications and alternative forms, and is not restricted to the specific embodiments set forth. It should be understood that these specific embodiments are not intended to limit the invention but, on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.

Claims

What is claimed is:

1. A method for detecting a biomaterial of interest comprising : combining, in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, a sample thought to contain a biomaterial of interest and a fluorescent labeled binding agent capable of specifically binding to the biomaterial and forming a fluorescent labeled complex; forming the labeled complex in contact with the support; and detecting the labeled complex without amplifying the fluorescent label .

2. method for detecting a biomaterial of interest comprising : combining, in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, a sample thought to contain a biomaterial of interest and a fluorescent labeled binding agent capable of specifically binding to the biomaterial and forming a fluorescent labeled complex; forming the labeled complex in contact with the support; and detecting the labeled complex without washing the support after forming the labeled complex.

3. The method of any preceding claim, comprising detecting the labeled complex without amplifying the fluorescent label and without washing the support after forming the labeled complex.

4. The method of any preceding claim, comprising detecting the labeled complex without enzymatically amplifying the fluorescent label.

5. The method of any preceding claim, wherein the fluorescent labeled binding agent comprises a fluorescent red dye-labeled binding agent.

6. The method of any preceding claim, wherein the membrane comprises a positively charged membrane.

7. The method of any preceding claim, wherein the membrane comprises a membrane activated to provide for covalent bonding .

8. The method of any preceding claim, wherein the fluorescent labeled binding agent comprises a fluorescent red dye-labeled binding agent .

9. A method for detecting a biomaterial of interest comprising : placing a sample thought to contain the biomaterial of interest in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane; adding a labeled binding agent that specifically binds with the biomaterial of interest to form a complex, wherein the labeled binding agent and the biomaterial form the complex; and detecting the complex without washing the support after forming the complex and/or detecting the complex without amplifying the label after forming the complex.

ιo. The method of any preceding claim, wherein the polymeric support comprises a nylon membrane, and the method includes detecting the complex without washing the nylon membrane after forming the complex.

ii. The method of any preceding claim, wherein the polymeric support comprises a nylon membrane, and the method includes detecting the complex without amplifying the label after forming the complex.

12. The method of any preceding claim, wherein the polymeric support comprises a nylon membrane, and the method includes detecting the complex without washing the nylon membrane and without amplifying the label after forming the complex.

13. A method for detecting a biomaterial of interest comprising : combining, in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, a sample thought to contain a biomaterial of interest and a first binding agent capable of specifically binding to the biomaterial and forming a first complex; forming the first complex in contact with the support; adding a second binding agent, wherein the second binding agent is labeled and the second binding agent is capable of binding to the biomaterial or the first binding agent to form a second complex, wherein the second complex comprises a labeled complex; and detecting the labeled complex in contact with the support without washing the support after forming the labeled complex.

14. A method for detecting a biomaterial of interest comprising : combining, in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, a sample thought to contain a biomaterial of interest and a first binding agent capable of specifically binding to the biomaterial and forming a first complex; forming the first complex in contact with the support; adding a second binding agent, wherein the second binding agent is labeled and the second binding agent is capable of binding to the biomaterial or the first binding agent to form a second complex, wherein the second complex comprises a labeled complex; and detecting the labeled complex in contact with the support without amplifying the label.

15. The method of any preceding claim, comprising placing the sample thought to contain the biomaterial of interest on the support, adding the first binding agent and forming the first complex, and adding a second binding agent that binds with the first binding agent and forms the labeled complex.

16. The method of any preceding claim, comprising placing the first binding agent on the support, adding the sample thought to contain the biomaterial of interest and forming the first complex, and adding a second binding agent that binds with the biomaterial of interest and forms the labeled complex.

17. The method of any preceding claim, comprising placing the sample thought to contain the biomaterial of interest on the support, adding the first binding agent and forming the first complex, and adding a second binding agent that binds with the first binding agent and forms the labeled complex.

18. The method of any preceding claim, comprising placing the first binding agent on the support, adding the sample thought to contain the biomaterial of interest and forming the first complex, and adding a second binding agent that binds with the biomaterial of interest and forms the labeled complex.

19. A method for detecting a biomaterial of interest comprising: forming a fluorescent labeled complex comprising a biomaterial of interest and a binding agent including a fluorescent label, the fluorescent labeled complex being in contact with a polymeric support comprising a polyamide membrane or a polysulfone membrane, wherein the fluorescent label comprises a fluorescent dye that can absorb and emit light at about 600 nm or more; and detecting the labeled complex.

20. The method of any preceding claim, wherein the polymeric support comprises a positively charged membrane.

21. The method of any preceding claim, including detecting a fluorescent red dye labeled complex.

22. The method of any preceding claim, wherein the fluorescent red dye labeled complex comprises a fluorescent cyanine dye .

23. The method of any preceding claim, comprising a hybridization assay.

24. The method of any preceding claim, wherein the biomaterial of interest comprises a nucleic acid sequence, and the binding agent comprises a nucleic acid sequence that specifically binds to a complementary region of interest in the biomaterial of interest .

25. The method of any preceding claim, wherein the biomaterial comprises an antigen.

26. The method of any preceding claim, wherein the biomaterial of interest comprises a protein.

27. The method of any preceding claim, comprising an immunoassay.

28. system for detecting a biomaterial of interest comprising : a polymeric support comprising a polyamide membrane or a polysulfone membrane; and a fluorescent label that absorbs and emits light at about 600 nm or more, wherein the fluorescent label is attachable to a binding agent that is adapted to specifically bind to the biomaterial of interest and form a complex with the biological material of interest in contact with the polymeric support; wherein the system is arranged to detect a labeled complex without washing the support after forming the labeled complex.

29. A system for detecting a biological material of interest comprising : a polymeric support comprising a polyamide membrane or a polysulfone membrane; and a fluorescent label that absorbs and emits light at about 600 nm or more, wherein the fluorescent label is attachable to a binding agent that is adapted to specifically bind to the biomaterial of interest and form a complex with the biological material of interest in contact with the polymeric support; wherein the system is arranged to detect a labeled complex without amplifying the label after forming the labeled complex.

30. The system of any preceding claim, wherein the polymeric support comprises a polyamide membrane.

31. The system of any preceding claim, wherein the polymeric support comprises a polyamide membrane.

32. The system of any preceding claim, wherein the polymeric support comprises a positively charged membrane.

33. The system of any preceding claim, wherein the fluorescent label comprises a fluorescent red label .

34. The system of any preceding claim, wherein the fluorescent label comprises a fluorescent red label .

35. The system of any preceding claim, wherein the polymeric support comprises a polysulfone membrane.

36. The system of any preceding claim, wherein the polymeric support comprises a polysulfone membrane.

37. The system of any preceding claim, wherein the polymeric support comprises a polyethersulfone membrane.

38. The system of any preceding claim, wherein the polymeric support comprises a polyethersulfone membrane.

39. The system of any preceding claim, wherein the membrane comprises a porous membrane.

40. The system of any preceding claim, wherein the system is arranged to: detect a labeled complex without washing the support after forming the labeled complex; and detect the labeled complex without enzymatically amplifying the label after forming the labeled complex.

41. The system of any preceding claim, wherein the system is adapted for use in a microarray assay.