JPH06508689A - Assays for detecting specific ligands - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Assays for detecting specific ligands Relationship with related applications This invention is based on a patent application filed on June 13, 1991 by Tzeng et al. This is a continuation-in-part of a U.S. patent application with an unknown serial number. Books that are common to the subject matter of the same application We hereby claim priority to the subject matter of this application.

Background of the invention Recent technological advances have enabled us to analyze a wide range of analytes, especially polypeptides, nucleotides, and Performs assays on numerous antigenic molecules such as cells and cell fragments. In general, many of the assays currently in use are liquid phase or tend to use antibodies to "capture" antigenic substances in solid phase form.

However, there are many symptoms and diseases that cannot be diagnosed at an early stage by antigen detection. Therefore, it is desirable to improve the sensitivity of the assay. Often cells within a tissue or It is preferable to be able to detect an increase in a specific population of molecules or molecules, and is produced when a specific antigenic substance enters this tissue. be.

For example, in some viral diseases, there may be sufficient detectable viruses in the blood of the tissue. The amount of virus particles is increasing. Possibly Xo Also, a fairly specific or largely undetectable response by the tissue's immune system The disease causing the answer is current or can be accurately or easily determined depending on the test. It cannot be detected. Additionally, specific populations or small collections of immunoglobulins Agents that cause a group response remain undetected until the resulting disease is in full swing. is often not performed, which limits diagnostic and treatment options. for example, Facilitating the detection of different immunoglobulin species by distinguishing them from each other Duermeyer's U.S. Patent No. 4. 292. No. 403 It is described in. Specifically, this assay can be used for IgMlgG, IgA, IgD, or is an antigen-specific immunoglobulin of a particular class of immunoglobulins, including IgE. Robulin can be isolated by using anti-antibodies against this specific immunoglobulin class. The purpose is to detect Many others disclosed in the prior art using anti-antibodies (U.S. Pat. No. 4,818,688 to Adamich et al., M. aggi.

No. 4,828,981 to Todd et al., and No. 4,962.023 to Todd et al.) The assay described in Duermeyer is complex and uses multiple special reagents. do. In addition, both of these assays are specific for antigen-specific small molecules of certain immunoglobulin classes. the population cannot be easily distinguished from other non-antigen-specific members of its class . That is, prior to this application, there were no methods for accurately sensitizing to a single antigenic substance. For example, it is easy to detect small populations of specific 1gG or IgM molecules. There wasn't.

Regarding the need for assays with improved sensitivity, we discuss available assays for antibody detection, especially specific This becomes clear when considering assays for the detection of immunoglobulins against foreign antigens.

Lyme disease is a classic example.

Lyme disease was first detected in Lyme, Connecticut in 1975. At present including North America, Europe, Russia, Asia, Africa and Australia. Infections have been reported across several continents. This disease is transmitted by ticks. Rocheta Borrelia burgdorferi ri). The infection causes a wide range of symptoms that can be confused with other symptoms. may appear. Therefore, accurate diagnosis is important.

Lyme disease is the most commonly reported tick-borne disease in the United States. This disease is It is fairly pronounced in the Northeast, Upper Midwest, and West Coast states. The timing of this disease The onset coincides with the nymph stage of the tick. Therefore, this disease occurs in summer and early Often seen in autumn.

Antibody titers specific for Borrelia puljolferi are typically found in the early stages of the disease. The test was negative on the floor. Antibody titers are almost always elevated even in patients with only erythema migrans. do not. Specific 1 gM Lehelebe begins to rise 2 weeks after onset of disease and from 3 weeks It reaches its peak in 6 weeks. Specific 1gG level approximately 2 weeks specific IgM tie In the late clinical stages of erythema migrans, although it tends to be later than that of tar, It is often positive and indicates symptoms such as carditis, neurological disease, or arthritis. Usually remains positive in the second and third clinical stages.

In general, the "natural history" or progression of Lyme disease can be divided into three clinical stages: can. The first stage is erythema migrans, which are spreading, circular, red, eczema-like patches of skin. This erythema occurs at the site of the tick bite and is typically It typically lasts between two to four weeks. After erythema migrans, the second stage is the heart and joints. and neurological abnormalities, which last for 1 to 4 months after the onset of the disease. occurs. The final stage involves damage to several large joints that can occur from 3 months after onset. characterized by arthritis including This stage may last for several years It can also become chronic. Diagnosis at an early stage can halt the progression of the disease. Prompt treatment with appropriate antibiotics can be given.

Spirochetes are often difficult to culture from affected skin areas or body fluids of patients. So, for Lyme disease, the best thing that can be done is to detect antibodies in serum. It is considered a diagnostic method. Specific against Borrelia brugiolferi IgM is rarely detected within the first two weeks and is usually detected between three and six weeks after initial infection. It peaks in a week and then persists or decreases. against spirochetes A specific 1gG response is rarely detected after 4 to 6 weeks, and is often detected at the stage of arthritis. It often reaches a peak and remains elevated for several years.

Therefore, various polypeptides, polynucleotides, and immunoglobulins Various applications that allow for highly sensitive detection of specific ligands, including In response to a clear need for a method of assaying that has the following characteristics, Applicant discloses the present application.

In addition, our test avoids the agglomeration problem of other tests, thereby improving accuracy. This facilitates the goal of increasing resolution and resolution. Concise and precise main application This disclosure is made by the applicant herein, including the scope of equivalents thereto.

Summary of the invention In one embodiment of the present application, applicant provides a method for detecting or determining test antibodies in body fluids. An analytical device is disclosed, wherein the device includes a plurality of analyzers adjacent to each other and in absorbent contact with each other. including a layer consisting of a substantially flat zone of sample application zone and a substantially flat zone of a binding zone containing antigen bound to moving particles and a detection zone containing immobilized antigen and the antigen is the same in both the binding and detection zones and the antigen is the same in both the binding and detection zones and The antigen that binds and the liquid sample passes through the binding zone to the sample presentation zone. to the detection zone to ensure that the test antibody is present in the sample. If so, it will be detected in the detection zone.

In another embodiment, the mobile particles are colored plastic particles or metal sol. It is. Another variation is that the antigen binds to the antibody and the antibody in turn A device is disclosed for binding to an epitope of a microorganism. In other examples, mobile grains The particles are colored polystyrene microparticles. In yet another embodiment , this layer is made from nitrocellulose.

This invention also discloses a method of using the device of this disclosure, which method includes a test antibody. Apply the suspected sample to the sample application zone and allow the sample to pass through the detection zone. Wait enough time to cross the lever layer and read the result within the detection zone. include. In some variations, the mobile particles are colored plastic particles or metal sols. . In another variation, the antigen used binds to an antibody, which in turn causes Borrelia Binds to the epitope of the B. brujolferi microorganism. In other aspects, mobile particles Disclosed are colored polystyrene microparticles. In other embodiments, this layer is Made from trocellulose.

The invention also provides a method for determining the presence or concentration of a test antibody in a sample fluid. The method includes contacting a fluid sample with a first antigen for a test antibody. a step in which the first antigen is present between the first antigen and the test antibody. labeled to form a soluble complex and contacting the soluble complex with a second antigen; the second antigen, the first antigen, the test antibody, and the second antigen. binds to a solid phase that does not dissolve in the fluid to form an insoluble complex with the antigen of and a step to separate this solid phase from the fluid sample and unreacted first antigen. a first labeled antigen bound to a solid phase or an unreacted portion of the first labeled antigen. step of measuring the amount of reaction and the test antibody prepared according to the first four steps. Correlating the amount of first labeled antigen measured with respect to the control sample without Determine the presence of test antibodies in a body sample or on a fluid sample The amount of first labeled antigen prepared according to the first four steps, including The amount of labeled antigen measured on a sample in which the amount of test antibody present is known. associated with determining the concentration of the test antibody in the fluid sample; and a second antigen, respectively, before being labeled or attached to a solid phase. It's the same.

Those skilled in the art will appreciate that the label is a gold sol or colored or colorable microparticles. When the particles are visible, such as those formed by the labeled antigen and the test antibody, It will be appreciated that the complex may be insoluble.

In other embodiments, the label is an enzyme or a radioisotope. Still others In this example, the antigen reacts with an antibody, which in turn Reacts with the epitope of Jolfueli. Another example is when the label is alkaline phosphatase and the solid phase is beads, the inner wall of a test tube or the wafer of a microtiter plate. or the label is alkaline phosphatase and the solid phase is a non-chromatogram. It is a device.

The invention further provides a first antigen bound to a solid phase that is not soluble in the fluid to be tested; A solid phase and a reagent containing a second antigen bound to a solid phase are disclosed. is present in a sufficient amount to perform at least one assay of the test antibody in the fluid; , and before the first and second antigens are respectively bound to the solid phase or labeled. is the same. In other examples, the label is an enzyme or a radioisotope. Ma In another example, the antigen binds to an antibody, and the antibody in turn binds to Borrelia purjo. It is an antigen that binds to the epitope of rufeli. In other variations, the solid phase on the inside of a test tube or on a non-chromatographic device, and the label is alkaline. It is a phosphatase. In other embodiments, the label is alkaline phosphatase. and solid phase or non-chromatographic equipment.

In other embodiments, an assay kit is disclosed that is insoluble in the fluid being tested. a reagent containing a first antigen bound to a solid phase and a second antigen bound to a label. The solid phase and reagents are sufficient to perform at least one assay of the test antibody in the fluid. the first and second antigens are each bound to a solid phase. or are the same before being labeled.

In another embodiment, a method is disclosed, according to which the second method is performed in a fluid. A method for determining the presence or concentration of a test antibody comprising: a) sampling a fluid; simultaneously contacting the first and second antigens; an antigen is bound to a solid phase that does not dissolve in this fluid, and a second antigen is labeled and the first and second antigens are labeled. and the formation of an insoluble complex between the second antigen and the test antibody. b) a fluid sample containing an unreacted second labeled antigen; C) separating the amount of second labeled antigen bound to the solid phase; or measuring the amount of unreacted second labeled antigen; and d) measuring the amount of second labeled antigen. the amount of antigen containing the test antibody prepared according to steps (a) to (C). The amount of labeled antigen measured on control samples known to be Determine the presence of a test antibody in a fluid sample by associating or Prepare the amount of labeled antigen measured according to steps a) to C). A standard that is measured on a prepared sample containing a known amount of test antibody. A step that determines the concentration of analyte antibody in a fluid sample in relation to the amount of antigen identified. the first antigen and the second antigen are each bound to a solid phase, or They are the same before they are labeled.

In other embodiments, the label is an enzyme or a radioisotope; The first and second antigens bind to the antibody, which in turn binds to Borrelia purpura. It may be something that binds to the epitope of rufeli. In another variant, The label is alkaline phosphatase, and the solid phase is plastic beads and test tubes. The inner wall is the well of the microtiter plate. In yet other embodiments, the label is alkaline phosphatase and the solid phase is a non-chromatographic device.

Another example is a method for determining the presence or amount of a test antibody in a fluid sample. Disclosed is a method comprising: a) contacting a sample specimen with a first antigen; There, the first antigen forms an insoluble complex between the first antigen and the test antibody. b) the test antibody and the first A step that separates a fluid sample containing unreacted test antibodies from insoluble complexes with antigen. and C) adding a measured amount of the second labeled antigen to the first antigen and the test antibody. An insoluble complex consisting of the first and second antigens and the test antibody is prepared by reacting with the insoluble complex. d) separating the solid phase from unreacted second labeled antigen; and e) determining the amount of second labeled antigen associated with the solid phase or unreacted. measuring the amount of the second labeled antigen; f) Confirm that the measured amount of second labeled antigen does not contain the test antibody. measured on control samples prepared according to steps a) to e), determining the presence of the test antibody in the fluid sample in relation to the amount of labeled antigen or the amount of labeled antigen measured in the fluid sample from a) to e) For samples prepared according to the steps described above and containing a known amount of test antibody. and determine the concentration of the test antibody in the fluid sample in relation to the amount of labeled antigen measured. the first and second antigens are each bound to a solid phase. or the same before being labeled.

In various alternative embodiments, the label is an enzyme, a radioisotope, or an amine. Lucary phosphatase. In other variations, the first and second antigens are both It is an antigen that forms a complex with an epitope of the microorganism Borrelia burgiolferi. . Yet another example is where the solid phase is beads, the inside wall of a test tube or a microtiter. In other variations, the solid phase is a non-chromatographic device. It is.

detailed description This invention is simple and quick to distinguish between specific molecules such as antibodies. Concerning an apparatus and method for testing. Solid materials suitable for solid supports may be used to allow capillary transport of liquid from the air-fluid interface. any of the hygroscopic or absorbent solid materials that can be used for or more) is preferred. Various hygroscopic materials include paper, cell Contains loin particles, silica gel, cellulose beads, glass fiber, filter paper, etc. Ru. The surface of a solid support has a high concentration of particles, e.g. in the form of sharp bands or dots. It needs to be relatively smooth so that it can be formed into sections.

The size and shape of the hygroscopic material can vary widely depending on the purpose of the assay. parable For example, this hygroscopic material may be formed into elongated strips approximately 1 mm to 5 mm wide. or any other convenient geometric or non-geometric shape. obtain. In either case, the hygroscopic material usually has a support that provides structural strength.

Water-impermeable layers or coatings are conceivable as non-hygroscopic materials.

One embodiment of the second invention has an antigen as a capture antigen bound to a porous membrane. porous Membranes can include glass fibers, microfibers, and natural or synthetic materials. A flexible or or a rigid matrix. Fluid flows easily through this porous membrane It is necessary to come in such a way that it is both inclusive and passing. The membrane also preferably has at least 0. It has a pore size of not more than 1μ and preferably not more than 20μ.

Porous membranes can be used alone or as part of more sophisticated equipment. This kind of outfit were published on December 20, 1986 and February 23, 1988, respectively. , U.S. Pat. Nos. 4,632,901 and 4,727,019 to Valkirs et al. It is a device such as a non-quamadography ICON (registered trademark) described in the No. These documents are hereby incorporated by reference. IC0N (registered trademark) is the V listed above. The device described in the patent of Alkirs et al. San Diego, USA) trademark.

More specifically, Valkirs et al. described a device for the detection of target antigens in liquid samples. the device is (a) a porous membrane or filter and has anti-target antigen properties; a first member having an upper and a lower surface to which an antibody is attached; , the sample is applied to the top surface, and the antibody is applied to the top surface of the member to which the sample is applied. and (b) the first member is placed thereon. bristles having a surface and passing in a direction generally transverse to the surface on which the first member is placed; a second member being a body of absorbent material having capillaries, the capillaries being connected to the first part; the first to draw the added liquid to the upper surface through the material to the capillary of the member. a capillary tube between said first and second members in communication with a hole on a lower surface of the member; Communication must be established before applying fluid to the device during the immunoassay process. maintained during this certification process.

Other such devices are described in European patent application no. No. 217,403, Abbott Laboratories (North, Illinois) - TestPack (registered trademark) device from Chicago). main application Other such devices including porous membranes available in U.S. Pat. No. 3,811,840 to Bauer et al., issued April 1, 1990. Brown [[I et al. U.S. Pat. No. 4,916,056 and I98 No. 4,407°943 to Coile et al., 1981, issued on 04/03/1981. U.S. Patent No. 4,246,339 to Coile et al. Intengan U.S. Pat. No. 4,440,301, issued April 3, 1987. No. 4,704,255 to Jolley, issued November 3, 1985. 1 (Atz et al. U.S. Pat. No. 4.496.654, issued January 29, No. 4,366,241 issued Dec. 28, 1982. , all of which are hereby incorporated by reference.

The method of the present application is also described, for example, in U.S. Pat. No. 4,740,468 (herein incorporated by reference), and July 2, 1986. Such as those described in Published European Patent Application No. 186,100 of Yue et al., published in Japan. It can also be achieved by chromatographic methods.

The porous membrane of the present application is described, for example, in Fr1esen et al., published August 29, 1989. Rosenste, U.S. Pat. No. 4,861.711, issued August 8, 1989. No. 4,855°453, issued August 15, 1989, Ull. U.S. Pat. No. 4,857,453 to Man. May et al., EPO Publication, published November 17, 1988. No. 291,194, published January 18, 1989, Ching et al. EPO Publication No. No. 299,428 and Devereaux et al.'s EP released on July 12, 1989. Also used in the chromatography assay described in O Publication No. 323,605. can be done.

The capture antigen can be bound to the membrane directly or indirectly.

Direct bonding can be covalent or non-covalent to glutaraldehyde and aminosilane methods known in the art, such as using [chiro Immobilized Enzyme J (Imm.

bilized Enzymes) Halstead Brace, NY (+978 ( (1974). Non-covalent bonding is non-synthesis of antibodies and especially exploiting natural adhesion to synthetic fibers. This ensures a properly buffered solution. Mixing the liquid with the membrane and allowing it to evaporate, leaving a coating of the desired ligand on the membrane. Can be done.

The non-direct method of adding this ligand to the membrane is to bind it to the membrane or confine it in the membrane. If the microparticles are present on the surface of the membrane within the membrane matrix, or bind to other particles such that these other particles in turn bind to the membrane. slight The particles may have a similar shape, but are preferably spherical. Particle size matters must be prevented from migrating through the membrane to some extent. Particle sizes vary However, it is generally slightly larger than the minimum pore size of the membrane and smaller than the maximum pore size. and may additionally or alternatively be larger than the maximum pore size. like this The particles can be bound on the surface of the membrane, within the matrix of the membrane, or bound to the membrane. The particles are bound to other particles. Particles can be made from a variety of naturally occurring or synthetic materials. can be made. Examples of such particles are polyethylene, polyacrylate, and polyethylene. lyacrylamide, and preferably polystyrene, or agarose, dex Naturally occurring materials such as cross-linked polysaccharides such as tolan, cellulose and starch It is a fee. The main requirement is that this particle is located in a zone other than that of the membrane. signal, usually optical, that can cause a signal to be different from the part of the This means that it does not contribute to absorption.

Ligands can be covalently or non-covalently attached to the microparticles. The binding of the ligand to the particle is , by methods similar to those described above for binding the ligand directly to the membrane. or by other methods known to those skilled in the art.

Preferred methods for coating particles with ligands are described in the Examples section herein. Describe it.

The particles may be applied (or "spotted") to the membrane in zones within the surface area of the membrane. stomach. Therefore, by dotting the antigen-coated particles, they can be separated on the membrane. antigen-coated microparticles are localized on or within the membrane. Become a custodian. Any method known in the art may be used. the According to one such method, a mostly aqueous ("latex") suspension is applied to the membrane. The Sandy Springs Subbot Ink Machine (S andy Springs Spotting & Iachine) (Mary (Land, Germantown), etc. using various mechanical means.

Methods of preparing and using such microparticles in connection with the present application are described in the April 26, 1988 No. 4,740,468 to Weng et al. (particularly columns 13-15) (incorporated herein by reference), published April 8, 1987, Brown [ Other European Patent Applications Section 217. '403, and November 1986 European Patent Application Section 200 of A. S. Rubenstein published on the 5th, It is described in detail in No. 381.

Separate screens for various test formats (e.g. forward, simultaneous, and inverse) Steps may be performed using any method known in the art. membrane and For filters and filters, further cleaning with a buffer solution is often sufficient. , preferably by applying a vacuum to the opposite side of the membrane or filter or Bringing the opposite side of the membrane into contact with a liquid absorbing member, etc. that draws liquid through capillary action, etc. draws liquid through a membrane or filter. Use in one embodiment of this application The IC0N® device (Hybritech California) is preferably used for San Diego, California) uses the latter method.

Additionally, non-chromatographic devices and assays are suitable for use according to the present application.

For example, bead assays are an alternative method to chromatographic assays. 19 Ge i ge published on May 14, 1985! Other U.S. Patent No. 4,517,288 This document is incorporated herein by reference.

Moderate temperatures are usually used to perform the assay. The test is generally performed with a control sample. A constant temperature is required during the measurement period only if it is carried out without comparison. Decision Temperatures for determination generally range from about 15°C to 45°C.

[The term labeled antigen j refers to any antigen that has specific reactivity to the particular antibody of interest.] Refers to antigen. Such antigens are labeled using conventional methods to create a signal generation system. may constitute all or part of. For example, antigens can be radioisotopes, enzymes, Hyotine, avidin, chromogenic or fluorogenic substances, chemiluminescent labels, colloidal gold Labeled with genus particles, colored microparticles, colorable particles, and colorable latex particles can be done.

Thus, the labeled antigen can be labeled with tritium, carbon, etc. using methods well known in the art. Shared with radioactive isotopes such as 14, phosphorus-32, iodine-125, and iodine-131 Can be combined. For example, 7125 can be treated by treatment such as chloramine-T treatment. Enzymatically or prelabeled Porton-Hunter by lactoperoxidase treatment - (Bolton-Hunter) technology. these and other techniques, Van Vunakis and J. J. Lan. Go'ne Edited Methods in Snzymology 7 Bart A ( (1980).

and for other examples of radioactive labels, incorporated herein by reference, February 1972. U.S. Patent No. 3,646°346, issued on December 29, 1979; See line no. 4,062.733. Chromogenic labels emit light at visible ultraviolet wavelengths. It is a compound that absorbs Such compounds are usually dyes and quinoline dyes , triarylmethane dyes, phthalein dyes, insect dyes, azo dyes, anthracite dyes Includes moid dyes, cyanine dyes, and phenazoxonium dyes.

Fluorescent substances include those that emit light in ultraviolet or visible wavelengths following irradiation with light. Ru. Fluorogens can be used alone or with quenchers. Major fluorescence generation The substance is from the group rhodamine, fluorescein, and umbelliferone . Methods of conjugation and use for these and other fluorogenic substances are well known in the art. can be found in For example, Langone and Van Vunakis' M methods in Enzymolog L: par hc (1981) especially See pages 3-105. Representatives of other suitable fluorogens No. 4,366,241 to Tom et al., issued December 28, 1982. In particular, columns 288 and 29 and U.S. Pat. No. 3,996,345. and both of these documents are hereby incorporated by reference.

Chemiluminescent labels may also be used herein. For example, issued on August 1, 1978 Maier et al. U.S. Pat. No. 4,104.029 (herein incorporated by reference) The mentioned labels may be used as detection signals in this application.

Alternative methods of detection include colored latex particles and colloidal metal particles , the use of colored microparticles and colorable particles. known in the art For colored microparticles and their use in assays, see e.g. October 1987. No. 4,703.017 to Campbell et al., issued on the 27th. This document is incorporated herein by reference. Ko in the test The use of loid-like metal particles is also well known in the art. For example, in 1982 U.S. Pat. No. 4,313.734, issued Feb. 2, 198 No. 4,775,636 to Moermaguchi S. et al., issued Oct. 4, 1998 ( (both hereby incorporated by reference), and published on January 11, 1989. See EPO Publication No. 298,368. Colorable particles and coloring It is known in the art that possible latex particles are also effective as markers. As known and e.g. Gribnau et al., published February 15, 1983, U.S. No. 4,373,932, issued June 6, 1989, to deJaeger et al. U.S. Pat. No. 4,837,168, and both of these documents This document is incorporated herein by reference.

These non-enzymatic signal stems are sufficient for this application. However, those skilled in the art In humans, enzyme-catalyzed signal systems are generally less sensitive than non-enzymatic systems. You will recognize that it is preferable because it is expensive. Catalytic labels are well known in the art. Rikatsu alkaline phosphatase, horseradish peroxidase, luciferase , β-galactosidase, glucose oxidase, lysozyme, malate dehyde single and double (such as drogenase, glucose-6-phosphate dehydrogenase) “Channel J) Contains enzymes. Dual catalytic systems include e.g. glucose-6 - Alkaline phosphatase and glucose phosphatase using phosphate as initial material Contains oxidase. A second example of a dual catalytic system is glucose oxidase oxidizes glucose to hydrogen peroxide, and this hydrogen peroxide A signal-generating species is generated by reaction with a leuco dye. About the catalyst system A more detailed description can be found in U.S. Pat. No. 4,366,241 to Rom et al., particularly at column 27. No. 4,843 to Itman et al., column 40, issued June 27, 1989. No. 000, and U.S. Pat. No. 4,8 to Itman et al., issued July 18, 1989. No. 49,338, all of which are hereby incorporated by reference. use Also incorporated by reference, U.S. Pat. No. 4,740 to Weng et al. ．． Especially the 2ndfli of issue 468! See column 6-8m.

Although the process of binding enzymes to antigens is well known in the art, In Kennedy et al. Cl1n, Chim, ACTA 70:I (1976) Are listed. Reagents that can be used for this treatment include glutaraldehyde, p-Toluene diisocyanate, various carbonimide reagents, p-benzoyl Contains non-, m-periodate, N, N'-o-fujidimaleimide, etc. Ru.

Materials for the catalyst system include para-nitrophenyl phosphate (PNPP) simple chromogenic and fluorogenic substances such as β-D-glucose (plus suitable redox dyes), homovanillic acid, 0-dianisidine, bromox Resol purple powder, 4-alkyl-umbelliferone, luminol, pigment para-dimethylaminolophine ine), paramethyloxylopine hine), with indoxyl phosphate being the preferred substrate.

Depending on the nature of the label and the catalytic signal generation system, the signal can be both illuminated and fluorescent. Observe the level and see if the dye is visible or fluorescent by eye or spectrophotometer. A catalytic system that produces a dye, fluorescence or chemiluminescence that can be observed with a fluorometer. or in the case of chemiluminescent or radioactive labels, It can be detected by using a line counter. If suitable equipment is not available, It is usually desirable to produce a chromophore that produces visible color. Precise equipment is used Any of these techniques may be used in some cases. For example, alkaline phosph A preferred mixture of indoxyl phosphate and indoxyl phosphate is used as an enzyme. is used as a substrate, a color change is visually detectable for a qualitative positive reaction. It can be done. For qualitative analysis, the IC0N® reader and accompanying software (Hybritech, San Diego, California) and are preferred for use in this application.

Although the use of latex particles tends to be preferred in connection with the present application, other particles may also be used. Note that results can also be obtained using . For example, particles included in the assay may be present in the sample, added as a reagent or formed in situ. may be done. The nature of the particles can vary widely, i.e. naturally derived or synthetic. , a single substance, several substances or a mixture of a wide range of substances.

Naturally occurring particles include nuclei, mycoplasmas, plasmids, blastids, and mammalian particles. cells, single-celled microorganisms (e.g. bacteria). Synthetic particles are polyethylene Naturally derived substances such as polysaccharides such as polyacrylic acid esters or agarose Synthetic or naturally derived materials such as metal colloids or latex particles made from It can be prepared from Non-naturally derived particles may be used in specific assays, assay protocols, or other may be changed by consideration. (e.g., describing the use of various particles, (See U.S. Pat. No. 4,837,168 to Gould et al.).

Uniform latex particles (rULPJ) are generally very uniform spheres of small diameter. It is. Typical diameters range from less than about 1 μm to about 100 μm. 5 Particles smaller than μm are usually prepared by emulsion polymerization. The result of this process is extremely uniform. A series of particles with uniform particle size distribution is obtained.

The basic application of ULP is in the area of medical diagnostics, where particles are latex aggregated. It is used for testing. Various other particles such as amide modified latex (rAMLJ) and carboxylic acid ester modified latex (rcML) Each has an amide and a carboxylic acid group on its surface. These functional groups Covalent attachment of a ligand - such as an antigen or an antibody - to the ULP surface is possible. This improves the agglomeration test.

For example, if you want to measure a specific antibody (rAb), use the appropriate antigen (rA gJ) is coated on the latex particles. Ab is divalent, so two neighbors It may bind to the same location on adjacent particles and connect them together. did Therefore, the Ab present in a person's sample mixes with Ag-coated particles. If the You can see it. This phenomenon is essentially the latex agglutination test (“LATJ”). It is the basis.

One major problem with mobile particles is that coated particles naturally aggregate. The fact is that there is a tendency to collect In particular, latex suspensions contain hydrophobic particles. It is a loid-like suspension. The stability of this suspension depends on the surface active charge, with small amounts of protein Adding white matter (approximately 108 g per milligram of latex) results in agglomery. The stability of the particles tends to increase as larger amounts of protein continue to be added. It's across the street. This type of aggregation can be chromatographed using colored or visible particles. This is also a problem in the Ai-test.

This problem and a variety of problems associated with nonspecific aggregation are included. But long None of these proposals has been shown to yield completely satisfactory results. All of these tend to interfere with the assay, and many of them do so in ways that inhibit aggregation. have a finger in the pie. This is of course a totally unacceptable result for many assays.

A signal detectable at or away from the site of high concentration, A step may or may not be an internal property of a particle. Particles are radionuclides , dyes, fluorescent substances, enzymes, etc., which enable the detection of various materials or visual aids. any other convenient method that provides a detectable signal that can be detected or detected using equipment. can be labeled with a different label. Usually, various signs are suitably linked using linking arms. It is thought that it may covalently bond to particles. Labels can be attached to surfaces or Penetrates the particles if any.

The particle size is usually in the range of about 0.05 to 100 micrometers (μm), and The diameter is generally in the range of about 0.1 to 75 μm. particles are positive or negative It can be either electrically charged or amphoteric, that is, it can lose its charge and become neutral.

The presence or absence of charge can affect other parameters associated with the assay.

Numerous patents have been issued describing various labels that have found use in diagnostic assays. is being carried out. A variety of butrocols may be used to advantage with these labels. It can be developed in various ways. Examples of such patents include U.S. Patent No. 3.850.7. No. 52, No. 4,255,329, No. 4,233.402, and No. 4,20 No. 8.479.

To perform the assay, various reagents are pre-prepared in combination with various auxiliary materials. mixed in defined amounts and mixed with the sample or for other uses in assays. A kit is provided. Due to the diversity of protocols and reagents, a wide range of A kit can be prepared. Generally, if the method involves the addition of particles, the kit will include particles that are covalently or non-covalently bonded to particles or have Gantts.

Labels attached to the surface of the particles or dispersed within the particles, especially those that are visible Dyes that can be colored within a range may also be used. The following documents describe various labeling methods. chemiluminescence (Maier et al., U.S. Pat. No. 4,104,029) ), colored particles (C, amp be l I et al., U.S. Patent No. 4,703,017 ), colorable particles (deJaeger et al. U.S. Pat. No. 4,837,168) or or fluorescent (Langone and Van Vunaki). For example, Langone and Van Vu, who describe methods for labeling radioactive isotopes. Methods in Enzymology 70 edited by nakis: Bart See A. (1980).

) In other examples, the particles may be labeled with an enzyme.

If particles are not included, the reagent usually contains a labeled receptor or ligand. The label will provide a detectable signal and will detect particles present in the assay medium. Movement can be inhibited. In addition to labeled reagents, buffers, stabilizers, surfactants and If appropriate, auxiliary reagents such as substrates for enzymes, fillers, etc. may also be used. Ma It could be in strip form or something that could be added to a kit like an octopus. Wicking substances prepared in other convenient forms are contemplated. It will be done. In a preferred embodiment of the present application, the assay kit includes a desiccant, more preferably The desiccant is contained within the assay device or housing.

The present application may be better understood by the following examples illustrating preferred embodiments thereof, which shall not be construed as limiting the scope of this application.

Example I Lyme antibody assay The extracted Lyme antigen was purchased from the American Type Culture Collection. Borrelia puljolferi strain B-31. This microorganism is The cells were grown in modified BSKII medium as described below. Incubate this at 33℃ and collected by centrifugation. The collected cells were placed in a solution containing 5mM MgCL. Tris containing 1mM EDTA was washed three times with phosphate buffered saline (pH 7,4). Extracted with 2% Sarkosyl in buffer (pH 8,2) I 0mM. This extraction process This was done until all Sarkosyl-dissolved material was extracted. Insoluble substances are further Mixed with a muni mixer. This mixture of substances will be referred to as ``Lyme antigen'' from now on. I'll do it. Please note that the same Lyme antigen or Lyme antigen from the same source can be used in cattle, dogs, cats, etc. Can be used to detect Lyme-specific antibodies in a variety of mammals, including humans and humans Although point-distilled latex particles are used in the above experiments, other particles can also be used. Note that good results can also be obtained. Particles that do not bind to the ligand are trapped. Although in many cases it is added to the ligand-particle conjugate, such additions are not included in the present application. It is to be understood that there are various embodiments of the present application that are not necessary to the practice of the present invention.

Uniform-tS latex particles (rULPj) are generally extremely uniform spheres with a small diameter. be. Typical diameters range from less than about 0.1 μm to about 100 μm. 5 Particles below .mu.m are usually prepared by emulsion polymerization.

a. Preparation of ligand-particle conjugates For example, the basic principle of glue-Gantt-particle bonding, via simple absorption or covalent bonding, process is well known in the art, as is the use of colored latex particles. improve assay resolution and readability. 1989 American Society of Clinical Chemistry Annual Meeting By Bangs, L, B, submitted at the workshop of the 41st National Convention ``Homogeneous latex particles'' is a general term that describes various methods. Serage Diagnostics in Indianapolis, Indiana It is available in print from the company, or from Galloway, R.J. Development of child testing and immunoassays” Indianapolis, Indiana 5eratyn It will be listed in the company. About these documents and the documents cited in these documents. This is incorporated herein by reference.

For example, one method of preparing coated latex particles is absorption. Ru. Generally speaking, 1) pure reagents are used and 2) particles are cleaned before coating. and 3) determine the quantitative surface coverage of the chemical properties of the particle collection Gantt. It is necessary to

For example, the ligand-latex conjugate (rL-latex) is In the simplest case, a suitable ligand can be prepared by Dissolve in buffer, add to latex suspension, and leave for a few minutes to over 24 hours. Stir for a range of times. After equilibration, centrifuge the latex. Discard the supernatant that is not absorbed or contains Gantt. Latex is the new buffer resuspended in liquid, centrifuged, and the supernatant discarded again.

These steps ensure that any remaining unabsorbed ligand is washed away from the latex. It is repeated until it is determined that the

The latex coating process may be completed at this stage and this latex The sample is ready for use in latex agglutination assays.

Covalent bonding is the permanent or covalent attachment of a ligand or other substance to the surface of a latex particle. Including cases. If covalent bonding is selected, the ligand is first attached to the latex particle. It is necessary to maintain the stability of the latex particle suspension, and then the protein It is necessary to prevent quality deterioration. (General discussion of covalent techniques and more details For further references, see Bangs, L. B., [Uniform Latex Particles J] I want to be This document is incorporated herein by reference. ) so far Although the explanation is not limited to latex particles, naturally occurring (e.g. plasmids) or synthetic particles (e.g. polymers), and Other particles including metal colloids or particles (e.g. gold sol particles) may also be used. I hope you understand the point. These particles and their extraction or preparation methods are Well known in the art.

b. Preparation of BSA-latex conjugate Preparation of bovine serum albumin-latex conjugate (rBSA-latex J) was performed as described above. This is similar to the Gantt latex (L-latex) preparation, and the preparation The difference is that no ligand is used and BSA is used instead.

Alternatively, other albumins (lactal alcohols) that can prevent non-specific binding are available. bumin), casein, globulin, non-specific immunoglobulin (antigen-antibody Other proteins, such as those that do not participate in the reaction, may be used in place of BSA.

C. Mixture of ligand-latex and BSA-latex - Latex and BSA - The latex is mixed in different ratios depending on the test to be performed. for example , when preparing mixtures for use as shown in some of the examples, L- Latex and BSA-latex are mixed in a ratio of approximately 2:1 to 5:1 by volume. and used for the test. Depending on the nature of the test, this ratio may vary substantially. It is often said that the greater the amount of protein-labeled latex, the more the nonspecific binding will be reduced. Get results. of unliganded latex (or other particles) The amount is an amount effective to appreciably reduce nonspecific binding or false positive reactions. Any amount is acceptable. This quantity can be easily determined using a self-evident empirical method. simply determined.

d. Mixture of ligand-latex and "naked" latex - latex and naked latex Tex and beam Gantt-latex/BSA-latex mixtures as described above. As before, they are mixed in various ratios depending on the test being performed. above As such, the ratio of L-latex to bare latex can be varied substantially. It is said that the larger the amount of protein-labeled latex, the greater the reduction in nonspecific binding. results. Liganded psfllt’ latex (also (or other particles) is reduced enough to assess nonspecific binding or false positive reactions. Any amount may be used as long as it is effective to reduce the amount. This quantity is self-evident. easily determined by empirical methods.

Example III Preparation of Borrelia conjugates 10ml of blue with 50mg/ml solids obtained from Magsphere Carboxylated latex (preferably about 0.4tt in diameter) with 10% sucrose Dilute to 0.5 mg/ml in 20 mM MES buffer pH 5.5. vinegar Lujo-NH3 (Pierce) and EDAC (St. Louis, Missouri, Sig. ma) with a final concentration of 1. 087mg/m 1 and 2mg/ml Add up to. Mix for approximately 30 minutes with end-to-end rotation. activated latte The box was an Amicon concentrator with a 0°2μm nylon membrane and a 20mM MESp8 Wash 5 times with 10% sucrose, 0.02% SDS in 6.5 ml. 10% scro each time, resuspend in 2mM MES (pH 6,5) to approximately 50mg/ml. Sonicate 4 times for 15 seconds.

Approximately 0.5 mg of extracted Horeria puljolferi antigen was added to 10% sucrose and 50% 0.01% SDS, 2mM MES buffer containing 0mg active latex suspension Mix in liquid (pH 6.5). Latex and extraction Borrelia brugiolferi is allowed to react for 2 hours at 22°C.

Extract surface carboxyl groups that are not bound to Borrelia puljolferi antigens. To neutralize, add a solution of Tris buffer (pH 8,5) to 20mM, After that, casein solution (Sigma) and Zwittergent 3-12 (Casein solution) 0.5 each of San Diego Calbiochem, California solutions. % and 0.1%. Borrelia purjolferi latex has 20% sc. loin, 20mM) Lisu (pH 8°5), 0.1% Zwittergent3- 12.0, 5% casein, and 0.3% BSA-latex. Diluted to a degree of 1.8 SK- and ready for printing on solid supports or matrices. Preparation of II medium Add the following to 1 liter of fortified PRMI-1640 that does not contain glutamine. Mix well, i.e. 5g of Neopeptone (Dir co) and 50 g of BSA (Sigma A-4503, St. Lewis). After completely dissolving neopeptone and BSA, add the following.

i.e. 2g TC Yeastolate (Dirco) , 6g HEPES (Sigma), 0. 7 g sodium citrate , 5g glucose, 0.8g sodium pyruvate, 0.4g N-acetyl luglucosamine (Sigma), and 2.2 g of sodium bicarbonate.

Adjust pH to 7.6-7.8.

Modified BSK-11 media was manufactured by Barbour, A.G., Yale J.B.M. ed.

57:521-525 (1984); The literature is incorporated herein by reference. Modified medium contains glutamine Prepared from unfortified PRMI-1640 and 7% gelatin.

2. Ingestion and collection of Borrelia culture Equipment used: 2, 2L Erlenmeyer flask, vacuum device with trap, and 0.2 micron 1 liter filter unit (Na1gene 90mm diameter) and laminar flow hood including.

The culture medium was ingested with Borrelia by appropriate and safe laboratory treatments. Cultivation The ground flask was incubated overnight at 33°-35°C to check for contamination. . Signs of contamination include a cloudy appearance and/or an acidic pH shift.

Example V Assay preparation and processing 1. Preparation of solid phase One example of the application's use in a solid-phase assay format proceeds essentially as follows. It can be done. A typical reactor includes a solid support for assaying, typically consists of a plastic m-housing. Generally, nitrocellulose etc. membrane fragments are used, preferably immunochromatography strips are used. Ru. For example, bring the right edge of the membrane into contact with the sample well. sampleware The membrane contains an absorbent pad that provides an even flow of sample fluid from right to left along the membrane. nothing. The mobile particles are applied to the first zone of the membrane - e.g. latex beads. , the particles are bound to the appropriate ligand or antiligand. For example, Borrelia ・Antigens extracted from P. puljolferi were used in the experiments described herein. was used. Various methods of isolation and preparation of viral or bacterial antigens are applicable. known in the technical field. For example, but not limited to, A The antigen for ffi hepatitis is the Markus antigen described in U.S. Pat. No. 4,301,249. It may be prepared according to other methods, which document is hereby incorporated by reference.

In a second zone on the membrane, the ligand or antiligand is immobilized. Third reagents may be used, for example such reagents may be applied to zones on the solid support. can be immobilized at This reagent is removed from the first zone after the sample is added. Any material that can bind moving particles may be used. This third reagent is and can be detected in the zone in which a third reagent is immobilized, for example. The test is complete or properly performed when a response occurs. It can play a role in showing.

In a typical chromatography test process, a sample of liquid is placed close to the first zone. Applied to the adjacent solid support.

The fluid moves by capillary action to the first zone of the membrane, displacing the mobile particles. style The body continues to move particles across the membrane to the next zone (or zones).

If the analyte is present in the sample, the solid-phase antigen/analyte/antigen-binding particle ndwich] structure is formed and a detectable result occurs. the fluid crosses the membrane As the particles continue to move, the fluid/particles become immobilized on the support with a third reagent. The state of contact is reached. A detectable response occurs and the test is valid or should indicate that the test is complete.

2. Test progress Place the solid-phase assay (or just the membrane) on a flat, lighted surface in the first zone. Place 1 illi (50 μL) of liquid sample in/on the adjacent sample application area. Drop it. The application area may contain a buffer to further enhance assay performance. stomach. An example of such a buffer is 2% casei in O. and 05% Zwittergent.

Alternatively, the sample can be applied by applying the sample or sample mixture to the sample application area. It may be mixed with other substances, such as saline, prior to administration. Directly A few drops (preferably about 6 drops) of developer solution are then added to the sample application area. valid The developing agent solution is an aqueous solution, for example, 1% Zwittergent, 1% Try. Triton x - 100.0.02% SDS, 0.2% NaN 3, and 50mM) Squirrel buffer (pH 8.5).

Run this test for approximately 10 minutes and read the results. signal before 10 minutes elapse It may appear in the third zone, but it's much better to wait until 10 minutes. It seems that the results can be read accurately. After an appropriate amount of time, the appropriate signal is detected in the appropriate region (or regions), reading the test result good. For example, if a symbol appears in the third zone, the test results are valid and negative. It can be interpreted as sex. The result is valid if the symbol appears in the second and third zones can be interpreted as positive.

Control solutions may be added for comparison purposes with assay results or to test reagent viability. May be added to. For example, the positive control solution is 2 mg/ml rabbit gamma tuna. About 0.5 mg/ml of rabbit anti-Latin in 50mM) (pH 8.0) immunobodies. Negative control was 50mM ml of rabbit gamma globulin. These solutions are disclosed herein. Any or all of the reagents and assay components may be provided in a kit.

Although this application has been described based on specific examples, the scope of patent rights is based on these specific examples. is intended to be determined with reference to, but not limited to, the following claims. It is something that

International search report e+M/lie 691MLD.

PCT/LIS 92103680 Continuation of front page (81) Designation-EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IT, LU, MC, NL, SE), AU, CA , FI, JP, KR, N○ (72) Inventor Wang, Doumei Silver Pi, Encinitas, California, 92024, United States -ke brace, 2216

Claims (32)

[Claims] 1. Analyzers for the detection or determination of test antibodies in body fluids, the analyzers being adjacent to each other. and comprising a layer consisting of a plurality of substantially planar zones in absorbing contact with each other. , the layer comprising: a) a sample application zone; b) a binding zone containing antigen bound to mobile particles; and c) containing immobilized antigen. detection zone, the antigen is the same in both the binding and detection zones, and An antigen that binds to the test antibody, allowing the liquid sample to pass through the binding zone and become attached to the sample. is movable from the detection zone to the detection zone and the test antibody is present in the sample. device that is detected in the detection zone if 2. Claim 1, wherein the mobile particles are colored plastic particles or metal sol. The device described. 3. The antigen binds to the antibody, and the antibody binds to the epitope of the Borrelia puljolferi microorganism. 3. The apparatus of claim 2, wherein the apparatus is coupled to a 4. 4. The device of claim 3, wherein the mobile particles are colored polystyrene microparticles. 5. 5. The device according to claim 4, wherein the layer is made of nitrocellulose. 6. Add the sample suspected of containing the test antibody to the sample application zone and Wait enough time for the pull to cross the layer through the detection zone, and 2. A method of employing the apparatus of claim 1, comprising: reading the results at a time. 7. According to claim 6, the mobile particles are colored plastic particles or metal sol. Method described. 8. The antigen used binds to the antibody, and the antibody binds to the Borrelia pulgiolferi microorganism. 8. The method of claim 7, wherein the method binds to an epitope. 9. 9. The method of claim 8, wherein the mobile particles are colored polystyrene microparticles. 10. 10. The method of claim 9, wherein the layer is made of nitrocellulose. 11. A method for determining the presence or concentration of a test antibody in a fluid, the method comprising: a) contacting the fluid sample with a first antigen of the test antibody; The first antigen is labeled to form a soluble complex between the antigen and the test antibody. b) contacting the soluble complex with a second antigen; In order to form an insoluble complex consisting of the test antibody and the second antigen, the antigen is bound to a solid phase that does not dissolve in the fluid; c) separating the solid phase from the fluid sample and unreacted first antigen; d) the first labeled antigen bound to the solid phase or the unreacted amount of the first labeled antigen; and e) measuring the test antibody prepared according to steps a) to d). The amount of first labeled antigen measured relative to the control sample without Determine the presence of a test antibody in a fluid sample or perform measurements on a fluid sample. The determined amount of first labeled antigen was added to the solution containing the first labeled antigen prepared according to steps a) to d). related to the amount of labeled antigen measured on a sample with a known amount of test antibody. determining the concentration of the test antibody in the fluid sample by applying the first and and the second antigen are each the same before being labeled or applied to the solid phase. Law. 12. 12. The method of claim 11, wherein the label is an enzyme or a radioisotope. 13. The antigen reacts with the antibody, and the antibody reacts with the epitope of Borrelia pulgiolferi. 13. The method of claim 12, wherein the method corresponds to claim 12. 14. The label is alkaline phosphatase and the solid phase is beads, the inner wall of a test tube, or the like. 14. The method according to claim 13, wherein the method is a well of a microtiter plate. 15. The label is alkaline phosphatase and the solid phase is a non-chromatographic device. 15. The method according to claim 14. 16. a first antigen bound to a solid phase that is insoluble in the fluid to be tested and a label bound to the label; a reagent containing a second antigen, wherein the solid phase and the reagent are in a fluidic state. the test antibody present in sufficient quantity to perform at least one assay for the test antibody in the sample; The first and second antigens are the same before being bound to a solid phase or labeled, respectively. A test kit. 17. The assay kit according to claim 16, wherein the label is an enzyme or a radioisotope. . 18. An antigen that binds to an antibody, and the antibody is an antigen that binds to an antibody. 18. The assay kit according to claim 17, which binds to a tope. 19. The assay kit according to claim 18, wherein the label is an enzyme. 20. 20. The assay kit of claim 19, wherein the solid phase is a non-chromatographic device. 21. The label is alkaline phosphatase, the solid phase is plastic beads, and the sample is 21. The inner wall of a test tube or the well of a microtiter plate, according to claim 20. test kit. 22. A method for determining the presence or concentration of a test antibody in a fluid, the method comprising: a) simultaneously contacting the fluid sample with the first and second antigens; , to form an insoluble complex between the first and second antigens and the test antibody. an antigen is bound to a solid phase that is not soluble in the fluid and a second antigen is labeled and measured. given in the amount of b) separating the solid support from the fluid sample containing unreacted second labeled antigen; step and c) the amount of second labeled antigen bound to the solid phase or unreacted second labeled antigen; d) measuring the amount of the second labeled antigen; and d) measuring the amount of the second labeled antigen. Controls prepared according to (a) to (c) and known not to contain the test antibody The test antibody in the fluid sample in relation to the amount of labeled antigen measured for the sample. to determine the presence of a body or the amount of labeled antigen measured on a fluid sample. , a sample prepared according to steps a) to c) and containing a known amount of test antibody. The test antibody in the fluid sample in relation to the amount of labeled antigen measured for the sample. and determining the concentration of The first and second antigens are each the same before being bound to the solid phase and sampled. There is a method. 23. 23. The method of claim 22, wherein the label is an enzyme or a radioisotope. 24. The first and second antigens bind to the antibody, and the antibody binds to Borrelia purjolfei. 24. The method of claim 23, wherein the method binds to an epitope of Li. 25. The label is alkaline phosphatase, the solid phase is plastic beads, and the sample is 25. The inner wall of a test tube or the well of a microtiter plate. How to put it on. 26. The label is alkaline phosphatase and the solid phase is a non-chromatographic device. 25. The method according to claim 24. 27. A method for determining the presence or amount of a test antibody in a fluid sample, the method comprising: , a) contacting a sample of fluid with a first antigen; A solid phase in which the first antigen does not dissolve in the fluid because it forms an insoluble complex with the test antibody. b) unreacted target from the insoluble complex consisting of the test antibody and the first antigen; separating the fluid sample containing the analyte; c) an insoluble mixture consisting of a measured amount of the second labeled antigen, the first antigen and the test antibody; An insoluble complex consisting of the first and second antigens and the test antibody is produced by reacting with the sex complex. Steps to form the body; d) separating the solid phase from unreacted second labeled antigen; e) the amount of second labeled antigen bound to the solid phase or unreacted second labeled antigen; f) measuring the amount of the second labeled antigen; A control sample prepared according to steps a) to e) and known not to contain the test antibody. The presence of the test antibody is determined in relation to the amount of labeled antigen measured on the sample. or the amount of labeled antigen measured in the fluid sample in step a). Measurement shall be carried out on a sample prepared according to et. determine the concentration of the test antibody in the fluid sample in relation to the amount of labeled antigen determined. and a step of The first and second antigens are the same before being bound to the solid phase or labeled, respectively. is, the method. 28. 28. The method of claim 27, wherein the label is an enzyme or a radioisotope. 29. The first and second antigens are both Borrelia pulgiolferi microorganisms. 29. The method according to claim 28, which is an antigen forming a complex with an epitope of. 30. 30. The method of claim 29, wherein the label is alkaline phosphatase. 31. The solid phase can be beads, the inner wall of a test tube, or the wells of a microtiter plate. 31. The method of claim 30. 32. 31. The method of claim 30, wherein the solid phase is a non-chromatographic device.