JPH06505803A - Multitest immunochemical reagents and methods for their use - 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] 51, wherein the secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin; The method according to claim 47.

One fortune 1 Multi-test immunochemical reagents and methods of use Cross-reference of related applications This application is now U.S. Pat. No. 4,517,288 filed January 23, 1981. Wish “5olid Phase system for Ligand As5 1/1985, a continuation in part of U.S. Application No. 227.664 entitled Continuing from U.S. Application No. 693.569(7) filed January 22, 1989, January 3, 1989 Application No. 07/294.931(7), filed in Japan, is a partial continuation of the pending application.

Background of the invention Field of invention: The present invention is directed to methods and compositions. More specifically, the present invention itself, a multi-test solid phase or Regarding supports (i.e. porous media).

Description of prior art: Techniques related to immunoassays and the various techniques previously described in the patent literature. Techniques generally involve isolation/detection of reaction products (generally containing analytes of interest). It involves four basic steps. The usual form of immunoassay is the analysis of interest. involves immunochemical interaction with a ligand (antigen or antibody) of a substance, thereby immunizing This results in the formation of a precipitate, and the presence of an immunoprecipitate indicates the presence of the analyte. immunity If the complex remains in solution, addition of a second antibody (i.e. One of these immunoassays is that it can be easily precipitated (double antibody precipitation method). The layer refinements (semi-quantitative and quantitative) are homogeneous immunoassays (homo genetic immunoassay); and classical heterogeneous systems solid-phase immunoassays (i.e., U.S. Pat. No. 3.654.090 ), multiple zone solid phase system (+nu 1 tiple zone 5 solid phase system) phase system) (i.e., U.S. Pat. No. 4.258.001) and solid phase radial fractionation immunoassay system (solid phase radio). al partition immunoassay system) (U.S. Pat. No. 4,517,208). currently used In homogeneous immunoassay systems of this type, the entire assay is 1, essentially carried out in the liquid phase. Reactants, samples and indicators are in the same liquid fraction. Monitoring of the amount of indicator contained within the immunochemically bonded material without any separation between the material and the material remaining free in the liquid. It will be done.

In classical heterogeneous immunoassays described in the technical and patent literature, immunoassays A component of a chemical reaction is often immobilized on a solid phase or support. Such a solid The immunochemically bound components of such systems are expected to be quantified. (presumed to be bound to the solid phase) from unbound components. Depends on the need. This system consists of bound materials (solid phase) and unbound materials (solid phase). It is traditionally assumed that the liquid phase) should be separated into two mutually exclusive fractions. Ru. The immobilization of one of the immunochemical reactants on the solid phase in this system is , provides the most practical means to achieve such separation.

The multi-zone solid phase system described in the above-referenced patent is based on the classical heterogeneous system described above. The assay involves similar immunochemical interactions, however, the more mobile Separation of the immobilized component from the other components - from adjacent areas (i.e. adjacent layers) This is achieved by its transfer or elution to.

The fourth type of immunoassay system is a very recent development, but it is based on the classical immunoassay system. It is a hybrid of both a homogeneous immunoassay and a homogeneous immunoassay, and also is distinguished from the multi-area system of

This hybrid was further characterized descriptively as a “solid-phase, radially resolved immunoassay.” method, but first involves immobilization of the immunoreagent (ie, antibody) within a solid phase. that Subsequently, aliquots of the patient sample are transferred to a solid phase containing the immobilized immunoreagents. Applies to that part. After a short incubation period, the second immunoreagent (general (ligand conjugated to an indicator) is applied to the solid phase in a similar manner and analyzed. Depending on the nature of the protocol (i.e. competitive or sandwich assay), analysis from infectious reagents (which are immobilized within the solid phase) or patient samples. be made to interact with any object.

The manner in which various materials are applied to a solid phase containing immobilized immunoreagents Define the reaction area within the Following such interaction, on this reaction zone A cleaning solution can be added to the solution, thereby removing unbound material (i.e. (i.e., materials that did not immunochemically react directly or indirectly with the immobilized reagent) are eluted from the reaction zone in a radial pattern, thereby unbound A region essentially free of material is created within the reaction zone. This type of assay The ability to achieve such redistribution of material entirely within a single solid phase is unique. materials that could interfere with the monitoring and quantification of analyte amounts in the reaction zone. to effectively eliminate the part of

Classical heterogeneous immunoassay systems and more recently developed solid-phase radial partitioning As is clear from the above discussion of assays, such systems are Success requires a clean separation of bonded and unbonded materials. depends on. In radially fractionated immunoassays, such separation occurs entirely inside the solid phase, whereas in classical heterogeneous systems In this case, the solid and liquid phases are separated into two mutually exclusive fractions.

In such solid phase systems, the immobilized immunoreagent is It is desirable, and sometimes necessary, for the phase to be essentially inert. There is also. This requirement avoids non-specific binding of proteins and other materials, thereby to prevent interference with or distortion of the binding selectivity of the immunochemical reaction. Technically recognized.

Various mechanisms by which immunoreagents can be immobilized on the surface of a solid phase include adsorption, absorption or can involve any mechanism including covalent bonding or a combination thereof. US patent See No. 4.168.146 (Grubb). Grubb is a different species various substrates (i.e., cellulose-containing materials, cellulose acetate films or silica to covalently bind antibodies to a microporous plastic sheet containing For this purpose, various reagents including cyanogen bromide and glutaraldehyde have been used. Ru.

Unfortunately, many of these same substrates are relatively immunochemically reactive and They tend to adsorb proteins indiscriminately from samples. Relatively inert like glass When trying to use a substrate that is suitable for use in a same-phase immunoassay (e.g. The amount of adsorption (for example, antibodies or other immunoreagents) is generally unsatisfactory. Lack of glass substrate For a discussion of characterization, see U.S. Pat. No. 3,790,663 (Garrison) checking ...

Due to the desirability of inert substrates as matrices for immunochemistry, Therefore, efforts continue to adapt such inert materials to immunoassay procedures. I've been kicked. One such attempt is to develop anti-resistance within a glass fiber matrix. It was accompanied by coprecipitation of the body. U.S. Patent No. 3.888.629 (Bagsha see). When dealing with the problem of nonspecific adsorption of proteinaceous materials, B agshawe to prevent non-specific binding of the immunogen to the matrix. Pretreating the solid phase or matrix with a polymeric substance such as albumin I tried. In one alternative embodiment of his invention, Bagshawe also Immune his matrix by forming immunoprecipitates within the interstices of the matrix. A procedure for sensitization to epizootic reagents was also described. Thus, the immunoprecipitate The particles were physically trapped within the matrix. Therefore, Bagshawe relies on the adsorption properties of an inert matrix for the retention of his immunoreagents. rather than a relatively inert matrix (the glass is held up in a suitable medium). **, line 41) in that he is now able to use The invention was conceived to overcome some of the shortcomings of prior art systems.

The amount of avidity per unit of immunoreagent in the immunoprecipitate, as can be readily recognized. The comparison is the activity per unit of the immunoreagent when the same immunoreagent is adsorbed to a solid phase. significantly lower. Therefore, Bag While shawe's approach appeared to be effective, from a reagent utilization perspective It is highly inefficient and at best a stopgap solution to a long-standing problem. It was nothing more than As is also clear, (of the type used by Bagshawe) The preferred form of combination of immunoreagents with matrices fully utilizes high surface area properties. Such materials can be adsorbed onto the surface of the matrix for use. Dew. Therefore, the use of immunoprecipitates in the context of a solid phase by Bagshawe The use of immunoreagents and/or inert matrices is, at best, a compromise. have not yet been put to the most effective use.

Thus, for adsorption of immunoreagents onto the surface of an inert solid phase support or medium, There continues to be a need for methods. Ideally, such a method would look like this Consistent with the utilization of the high surface area properties of type supports, yet minimizing the consumption of immunoreagents. It must be limited.

For the above and related purposes, immunoreagents are adsorbed onto essentially inert porous media. an essentially soluble immunoreagent consisting of an immunoreagent and an antiserum against the immunoreagent to the improved porous medium under adsorption conditions. (U.S. Pat. No. 4,517,288) issue). The amount of soluble immune complex adsorbed onto the porous media will depend on the specific immunoconjugate. In an assay, an analyte of interest is placed within a finite area of the porous medium. where it has been experimentally determined to be effective in selectively binding and retaining drugs. It was based on Therefore, the definition of effective amount varies between immunoassays and is more It may depend on the physical geometry of the phase and how the assay results are monitored. death However, one of the drawbacks of this method is that for each immunoassay of a different type, requires different soluble immune complexes adsorbed onto a porous medium. As such, there are a number of matrices that can be used in various immunoassays. There is a need to develop multitest immunoreactants.

Summary of the invention It is therefore an object of the present invention to eliminate the above-mentioned drawbacks. In particular, the present invention is an immunochemical reagent made from a secondary antibody adsorbed onto an inert porous medium. provide. More specifically, the present invention relates to a specific The present invention provides immunochemical reagents made from antisera against specific species. The antiserum can be used in e.g. For example, goat anti-mouse, goat anti-rabbit, goat anti-sheep, sheep anti-goat, rabbit anti-goat, It may be donkey anti-goat or more generally a second species anti-first species. Purpose of the invention The antiserum may herein be referred to as "secondary antibody J". It may be refined or unrefined. In addition, various specific animal species combinations can be applied to the solid phase. You can also. These antibodies adsorbed onto the surface of an inert solid phase or support The reagents can be used in a large number of immunoassays.

In particular, the present invention provides solid-phase immunoassays of liquid samples for unknown amounts of analytes. 1. A method for carrying out a method, the method comprising: A method for performing a solid phase immunoassay of a sample, the solid phase comprising , a second antibody immobilized on a solid phase, and the method comprises a second analyte-first antibody complex. binding conditions to form a liquid mixture of primary antibody and uncomplexed primary antibody. Combine a liquid sample containing the analyte with the antibody under the conditions: analyte - to bind the secondary antibody complex and the uncomplexed primary antibody. applying the liquid mixture to the solid support under binding conditions: binding, complex formation; to bind to an unbound antibody or to bind to a bound analyte, applying an indicator to the solid phase under binding conditions: an acid indicator is present on the solid support; and observe the extent to which the indicator is present on the solid support. A method is provided that involves correlating the amount of an unknown analyte in a pull.

The indicator may also be added to the liquid sample, which allows each component to be added at the same time. or allowing the analyte and antibody to incubate briefly before adding the indicator. This can be done by The joining conditions are further defined as P, Tijssen, La borato■Teahniques in Biochemistry, M o1ecular Biology, Practice and Theor y of Enzyme Immunoassay 123-145. (4th edition 1987) (incorporated herein by reference).

In the context of the present invention, the term "indicator" means a labeled conjugate. The contact The conjugate is either the antibody or the analyte depending on the assay format. The label indicates that the zygote a fluorescent, enzymatic, colorimetric or radiometric compound bound directly or indirectly to be. The label may consist of an enzymatic compound that generates fluorescence upon contact with the substrate. I can do that. The extent to which the indicator is present on the solid support is related to the amount of unknown analyte. can be attached. P, Tijssen, Laboratory Tec hniques in BiOChemiStry, Mo1ecular B iology, Practice and Theory of Enzym e [mmunoassay 173-217 and 368-376 (4th edition 19 87) (incorporated herein by reference).

The invention also provides solid-phase immunoassays of liquid samples for unknown amounts of analytes. A method for carrying out the method, wherein the solid phase is an essentially solid, inert, porous support. The method consists of a secondary antibody immobilized within a finite area of the gap of the support. is: Liquid mixture of analyte-primary antibody complex and uncomplexed primary antibody a liquid sample containing the analyte under binding conditions to form a primary Combine with antibody: analyte--next antibody complex and uncomplexed antibody. applying the liquid mixture to the center of the finite area under binding conditions to : the solid support under binding conditions to bind uncomplexed antibody. applying an indicator to the support; the extent to which the indicator is within a limited area of the reaction zone; and the extent to which the indicator is present in the defined area of the sample. A method is also provided that involves correlating the amount of an unknown analyte in a sample.

In addition, the present invention provides solid-phase immunoassays of liquid samples for unknown amounts of analytes. 1. A method for carrying out a method, the method comprising: A method for performing a solid phase immunoassay of a sample, the solid phase being essentially a secondary antibody immobilized on a solid support, the method comprising: applying a primary antibody to the solid support under binding conditions; Apply the liquid sample to the support; combine with the bound, uncomplexed antibody. applying an indicator to the solid phase under binding conditions; observe the extent to which the indicator is present on the solid support; and observe the extent to which the indicator is present on the solid support. involves relating the degree of unknown analyte to the concentration of unknown analyte in the sample. provide a method for

The invention also provides solid-phase immunoassays of liquid samples for unknown amounts of analytes. A method for carrying out the method, wherein the solid phase is an essentially solid, inert porous support. a secondary antibody immobilized within a finite area of a body cavity, the method comprising: Applying the primary antibody to the center of a finite area of the solid support under conditions; complex formation An indicator is attached to the solid support under binding conditions to bind to the uninitiated antibody. Apply: the liquid sump to the center of a finite area of the cough solid support under binding conditions. the extent to which the indicator is present within a limited area of the reaction zone; ; analyzing the extent to which the indicator is present in the defined region in the sample; A method is also provided that involves relating quantities of things.

Brief description of the drawing Figure 1 shows the percent (%) goat anti-matrix adsorbed on an inert porous matrix. Figure 3 shows the response of calibration standards to immunoglobulin.

Figure 2 shows the percent (%) response to goat anti-rabbit in normal rabbit serum. vinegar.

Figure 3 shows titration of rabbit anti-estradiol.

Figure 4 shows 5TRATUS @ (Baxter Diagnostics In c,) Comparison of estradiol and multi-test calibration curves.

Figure 5 shows 5TRATtJS @ (Baxter Diagnostics I nc,) of the response to estradiol concentration by performance and multi-test method. DETAILED DESCRIPTION OF THE INVENTION Including preferred embodiments for comparison. Multitest immunoreagents are used to adsorb species-specific antisera or antisera from homologous species. It is a porous, inert medium. These reagents do not contain specific antibodies , any secondary antibody can be bound. In particular, adsorption to the porous inert medium Goat anti-rabbit and goat anti-mouse antisera work well.

Antisera are produced by injecting immunoglobulins from a first species into a second species. prepared. The second species contains an antiserum (i.e. or, more generally, a second species against a first species). For the purpose of this invention , the antiserum can be referred to herein as a secondary antibody. The secondary antibody is purified It may also be unrefined.

A primary antibody is an antibody that is specific for an analyte, e.g. anti-hCG The antibody is specific for hCG. - The following antibodies were prepared using standard techniques: It can be made from seeds, but the most commonly used seeds are rabbit and mouse. . - Any primary antibody raised in a species of It is recognized as For example, primary antibodies raised in mice may be goat anti-mouse or domestic Recognized by antisera such as rabbit anti-mouse. Rabbits and mice were given - The most common antiserum is the 20th antiserum, as it is the most common species used to make it. Seed anti-mouse or anti-rabbit.

Due to the homology that exists between primary antibodies of a given strain, secondary antibodies made against - species will also react with other closely related species. For example, goat anti-mouse is , often reacts with primary antibodies prepared from rats. For the purposes of this invention, this These species are herein referred to as "homologous species."

The term “antibody J” used here refers to an antibody that immunologically reacts with an appropriate antiserum. primary antibody from a specific species or any homologous species. Including the body. The advantage of this is that it provides a wider selection of antibodies.

A solid phase compatible with the foregoing purposes of the invention may be "inert." more specifically The characterization of a solid phase as “inert” (in the context of the present invention) The relative non-reactive nature of the surface of the solid phase with respect to its ability to indiscriminately adsorb materials It is intended to be a sign. As stated earlier in the background section of this disclosure, As shown, glass and other materials of similar chemical composition have a similar ability to adsorb proteins. relatively inert when compared to other more reactive materials. It is generally believed that there is.

In preferred embodiments of the invention, the physical form of the solid phase is gaps or bores between which the reaction liquid is retained and transported by capillary action. It is small enough so that it can be used. The solid phase is advantageously glass or composite. Composed of a mat of compressed fibers, such as grown fibres. This solid phase is also calcined Constructed from other porous components such as glass, ceramics and synthetic polymeric materials. may be completed. Glass fiber filter paper is useful for its inert characteristics and for immunoassays. The soluble compound of the present invention is added in an amount sufficient to sensitize the glass fibers to make them suitable for is a preferred solid support of the invention due to its ability to adsorb immune complexes of .

The multi-test immunochemical reagents are suitable for analytes in various biological and industrial fields. can be used in a wide variety of analytical protocols. single gala fiber filter paper can be used for any assay and is very It must be noted that antisera against multiple species can be included. stomach. For example, therapeutic agents, natural or synthetic steroids, hormones, antibodies and other interests. such liquids for immunoassay of blood or urine for the presence of certain analytes. can be advantageously used in rapid and quantitative analysis of

Examples of therapeutic agents that can be analyzed with such protocols include digoxi Silantine, silantin, phenobarbital, theophylline, Includes gentamicin, quinidine, and others. Multitetra prepared in the manner described above The immunochemical reagents also contain cortisol, aldosterone, testosterone, Steroids such as progesterone and estriol, or ferritin It can also be used in immunoassays for the detection of serum proteins. suitable hardness By the use of appropriate immunochemical reagents immobilized on the phase as described above, the hormonal Mon levels can also be quantified. These hormones include thyroid hormone , i.e. triiodothyronine or thyroid stimulating hormone (TSH); peptide Hormones, namely insulin, corticotropin, gastrin, angiotene Syn, and proangiotensin; polypeptide hormones, i.e. pin, levteotropin, and somatotropin , human chorionic gonadotropin (HCG).

Assay formats include both competitive and sandwich assays. Conflict In a specific assay, the conjugate is combined with a sample containing the analyte and a specific antibody. Bodies are added at the same time.

Example 1 Goat anti-mouse multi used in estradiol assay test immunochemical reagents Below is the multi-test immune system that should be used in the estradiol assay. Chemical materials and optimal concentrations of goat anti-mouse and rat anti-estradiol antibodies Describe the selection means for determining the degree.

material Estradiol conjugate: Estradiol (E2) and alkaline phospha The estradiol conjugate consisting of Tase (ALP) was prepared using the standard mixed anhydride method. (B, Van Weeman, FEBS LETTER 3Vol, 24. No, 1. July 1972). Obtained E2-ALP The conjugate contains 0.50% protein with 0.1% azide as a preservative. mM) Lis (pH 8,0), 0.04μg/m in 50mM NaCl Diluted to L.

Estradiol calibration standard = 100 μg/mL est in 80% ethanol An estradiol stock solution consisting of radiol was prepared. 0 (A), 20 ( B), 50 (C), 200 (D), 500 (E), 2000 (F) pg /mL of the estradiol to create a set of estradiol calibration standards. A portion of the stock solution was added to activated charcoal-treated human serum. Calibration standards contain specific amounts of analyte It is a standard containing

Substrate/washing solution: Stabilizer, along with 0.1% sodium azide as a preservative. In jetanolamine buffer containing surfactant and blue dye (pH 9,0) A solution containing 1 mM 4-methylumbelliferyl phosphate in it.

Rat anti-estradiol monoclonal antibody (rat anti-E2) is available from OEM C Obtained from Oncepts, Inc.

Goat anti-mouse monoclonal antibody (CAM) was prepared using conventional procedures. .

Preparation and optimization of goat anti-mouse multitest reagent 0.5% ZONLY FSN (DuPont surfactant) in 50mM Tris buffer (pH 8.0). ) of zero to 8% goat anti-mouse (CAM) in 0.5% increments. A liquid was prepared. The solution was then passed through a 0.8 μm filter to remove particles. It was filtered with Each of the many tabs of glass fiber filter paper (Whatman GF/F) Spot within 76 μL of the solution and dry C to immobilize the antibody onto the tab. Tabs spotted with the increasing solution of AM were evaluated as follows. sand That is, 100 μL of a 1/100 dilution of rat anti-E2 was added to 100 μL of zero (A ) added to the calibration standard. Add a 150 μL aliquot of the mixture containing the tab CAM. (reaction zone). After a short (2 min) incubation, the rat Anti-E2 binds to CAM. Next, 5oμL of E2-ALP conjugate was added to the reaction zone. Added near the center of the area. The E2-ALP binds rat anti-E2. Then about 75 μL of substrate/wash solution was applied near the center of the reaction area. The substrate/washing solution will elute any unbound material from the reaction zone and at the same time remove the bound material. A reaction between the released ALP and the substrate is initiated to form a fluorescent product. reaction area This fluorescence response from a finite portion of the area was measured using a front surface fluorometer.

The solution that gave the highest response captured the greatest amount of antibody per unit CAM. It was selected as the most suitable one because of its uniqueness. Here the solution is 2.5 %CAM. See Figure 1 and Table 1. By the method described above, the glass fiber filter Multiple tabs of paper were spotted with this 2.5% CAM solution.

Table 1 %GAM and response to calibration standards This technique allows the identification of the same species by performing simple binding or affinity tests. or can be used with antibodies from homologous species o A, Cabat, 5 Ttructural Concepts in Immunology and Immunochemistry, (2nd edition 1976): 80-94 here Although the antibody against estradiol is rat monoclonal, First, goat anti-mouse immunoglobulin was immobilized on the tab. This is the interspecific phase Utilizing homogeneity to make immunoreactant solid phases (“tabs”) even more truly “versatile” Ru. Other examples of interspecies homology are between sheep/goats or horses/donkeys. this is, Expanding the range of available antibodies so that the best antibodies can be selected. Also, immediately As can be seen, the immunoglobulin spotted on the tab is also from the rat. so estradiol-specific antibodies can be from mice (as described above) (the reverse of the method), this also increases the choice of antibodies available.

Rat anti-estradiol One-time optimization Optimal rat anti-estradiol antibody ( Rat anti-E2) was determined using 2.5% CAM tabs prepared as described above. determined.

Rat anti-E2 was 0. 50mM) containing 1% bovine serum albumin (pH 8,0) and diluted to various concentrations. The dilution was as follows. That is, l: 50, l:60 and 1:5° depending on the antibody being evaluated and the nature of that antibody. wider or narrower dilutions (i.e. 1:10, i:100.1, respectively) : 1000 and 1:10000 or! =2, l:3, l:5) It is immediately clear that These concentrations were evaluated as follows = 10 Cup containing 0 μL A (zero) calibration standard and 100 μL B (20 pg /mL) Add 100 μL of rat anti-E2 of each dilution to the cup containing the calibration standards. I got it. The mixture was incubated for 15 minutes at room temperature. incubation Time and temperature can be varied depending on the requirements of the assay. preferred range The range is 0-4 hours and 18-37°C. After incubation, mix A 150 μL aliquot of the combined solution was added to the area of the tab containing the CAM (reaction area).

During a short (2 min) incubation, rat anti-E2 binds to CAM.

Next, 50 μL of E2-ALP conjugate was added near the center of the reaction area. Said E2 - ALP binds rat anti-E2. Next, add approximately 75 μL of substrate/wash solution to the It was applied near the center of the reaction zone. The substrate/washing solution can be any unbound material. is also eluted away from the reaction zone, and at the same time the reaction between bound ALP and substrate is initiated. initiated to form a fluorescent product. This fluorescence response from a finite portion of the reaction zone The response was measured using a front surface fluorometer.

The optimal dilution of rat anti-E2 was selected by measuring two parameters: Ta. The first parameter is A (zero) calibration standard by rat anti-E2 for each dilution. This is the fluorescent response that occurs when assaying. This fluorescence response is caused by CAM Directly proportional to the amount of rat anti-E2 captured. Therefore, these responses Indirectly proportional to the amount of E2 in the pull (a feature of competitive assays). The second measured The parameters of B calibration standard (2) relative to the fluorescence response from A (zero) calibration standard 0 pg/mL) (B/A ratio). A low B/A ratio is indicates that there is a measurable difference between the levels of these calibration standards. smallest B/A rat anti-E giving the highest fluorescence response using the A (zero) calibration standard, with the ratio 2 dilution was chosen as optimal.

The results are shown in Table 2 below.

Table 2 Rat anti-E2 dilution 1:50 1:60 1 near 5 fluorescence response 9012 68 98 from 6024A (zero) calibration standard Fluorescence response 8351 7097 6234B (20 pg/mL) from calibration standard B/A ratio 0.93 1.02 1.03 As is immediately clear from Table 2 above, The highest calibration standard A ratio with the lowest B/A ratio was rat anti-E2 at a l:50 dilution. seen when using. Other dilutions are between these two levels of calibration standards. Does not show any difference and is therefore useful in diagnostic assays for estradiol Not. Using the above data, make a l:50 dilution and assay for estradiol. Selected for implementation.

A standard curve was constructed using the calibration standards described in the Materials section above.

Assay method In this assay format, the antibody-antigen complex is 5TRATUS (Bax ter Diagnostics Inc.) cup in 100 μL of solution. 1 = 50 diluted rat strradiol monoclonal antibody and 100 μL serum formed by adding a sample or calibration standard.

This mixture now contains antigen-antibody complexes as well as uncomplexed antibodies. is then added directly to the center of the MultiTest goat anti-mouse reagent matrix, The matrix contains free estradiol antibody as well as 5TRATLIS@( Baxter Diagnostics Inc.) Capture the tradiol-antibody complex. The reagent matrix is then subjected to a second process. transferred to a working station where 50 μL of E2 conjugate was placed in the center of the matrix. added to. The conjugated E2 is attached to the immobilized estradiol antibody. It also binds to unoccupied binding sites. Then 78 μL of bound and free material The enzymatic action is initiated upon splitting by addition of the substrate/wash solution (2).

All of the above reagent additions are intended to ensure that the reagent addition also removes any unbound material. Goat anti-mouse reagent from Multides 1 to also help separate from the center of the tab. Done directly in the center of the matrix. Finally, to measure the proportion of bound enzyme, For this purpose, front surface fluorometry is used. The observed ratio depends on the concentration of sample E2. clinical units by comparison with a memorized standard curve that is inversely proportional. is converted to

As mentioned above, one factor limiting susceptibility is that the sample is anti-estradiol. This is the time it has for interaction with the antibody. Anti-estradiol antibody sample After being mixed with Le, it can be applied directly to the tab without incubation. Or, if sensitivity must be increased, incubate for a period of time. Good too. This specific example uses a 15 minute incubation.

Example 11 Goat anti-rabbit multi used in estradiol assay test reagent The following uses normal rabbit serum and rabbit anti-estradiol antibody as well as goat anti-rabbit antibody. A description of the multi-test reagent materials and assay format used. Following that The current 5TRATLIS■ (Baxter Diagn.

5ties [nc,) calibration curve and multi-test estradiol calibration curve. It's a comparison.

material E2 Conjugate: E2-ALP conjugate was prepared using standard mixed anhydride method.

It was then diluted in Tris buffer at pH 7.0 to a concentration of 1.2 μg/mL. I interpreted it.

E2 calibration standard = E2 source of approximately 100 μg/mL of E2 in 80% ethanol A liquid was prepared. A portion of the E2 stock solution was added to activated charcoal-treated human serum, and zeo(A) , 50 (B), 100 (C), 500 (D), 1000 (E) and 2500 (F) A set of E2 calibration standards at pg/mL were prepared.

E2 Assay Diluent 20, 1% CHAPSO and 350 μg/mL 8-aniline 100mM acetate containing lino-1-naphthalenesulfonic acid (pH 4,5) .

Substrate/washing solution: 1mM 4-mer in jetanolamine buffer (pH 9,0). Solution Diagnostic Sy containing chilumbelliferyl phosphate rabbit anti-E2 antibody obtained from Stems Laboratories, nc. body.

Tab optimization Solution A was normal rabbit serum at 50mM containing 0.5% Zonyi FSN. was prepared by diluting it to 1% in Tris buffer (pH 8). Solution B is By diluting goat anti-rabbit immunoglobulin to 1.2% in the above buffer. Prepared. Add ImL of solution A to each of 13 test tubes labeled 1 to 13. added. Then, starting with 0.5 mL in tube 1, add solution B to tubes 2 through 13 (titration tubes). ) in increments of 0 and ImL. Goat anti-rabbit/normal rabbit serum complex form After overnight incubation at 25°C to allow formation, particles were removed. The solution was filtered through a 0.8 μm filter to ensure compatibility. from each of the 13 tubes Approximately 75 μL aliquots of were applied to glass fiber paper tabs and allowed to dry. dry here was achieved at 80°C. Zero calibration standards were tested to determine the optimal complex. The sample was used as a human sample and assayed as follows. Optimal goat anti-rabbit/normal breed The rabbit serum complex contains sufficient binding capacity for immobilization of rabbit anti-estradiol antibodies. I'm here. An example of titration is shown in FIG. Tube 5 (80,9 The conditions used in Section A1) were selected for the manufacture of the tabs.

Optimization of rabbit anti-estradiol Use the rabbit anti-estradiol antibody with the optimal goat anti-rabbit/normal rabbit serum tab described above. and titrated. Titration is 0. 1% bovine serum albumin and 0.11% (3-[ku] roamidopropyl)-dimethylammonio2-hydroxy-1-propane Rabbit anti-estra was added to 50 mM Tris buffer (pH 8) containing This was achieved by serial dilution of diol antibodies. rabbit anti-estradiol The optimal dilution was selected by measuring two parameters. first para The meter is fitted with an A (zero) calibration standard by each dilution of rabbit anti-estradiol. This is the fluorescence response that occurs when the sample is analyzed. This fluorescent response is captured by GAR directly proportional to the amount of rabbit anti-estradiol administered. Therefore, these responses are is indirectly proportional to the amount of estradiol in the sample (characteristic of competitive assays). symptoms). The second parameter measured was the fluorescence response of the B (50 pg/mL) calibration standard. The result obtained by subtracting the fluorescence response of the F (2500 pg/mL) calibration standard from the answer (Cal B - Cal F). The dilution that gives the largest difference is taken as a measure of sensitivity. Ta. Gives the maximum calibration standard A response and the maximum "calibration standard B response - calibration standard F response". Determine the desired dilution and consider this dilution as the optimal dilution for rabbit anti-estradiol. I selected it. An example of titration is shown in FIG. The lnea 500 dilution was chosen as optimal.

Assay method In this assay, 25 μL of patient sample, control or calibration standard is combined with 50 μL of Serum estradiol is increased by adding L of E2 assay diluent. Extracted from synthetic proteins. The extracted sample was then treated with rabbit antiestradi Add 25 µL of Oar's 1N 500 dilution, then incubate the sample at 25 °C. The binding reaction is initiated by incubating for 15 minutes. incube After lysis, the goat anti-rabbit/normal rabbit serum complex gave the best response. Approximately 76 μL of sample is applied directly to the prepared glass fiber paper tab. sample Application of is directed to the center of the dry complex. Tab is your second work station where approximately 60 μL of the estradiol conjugate is added near the center. . The conjugate binds to the unoccupied binding sites of the immobilized estradiol antibody. Ru. The tab is then transferred to a third work station and approximately 70 μL of substrate is added to the center. / By addition of the washing solution ■, the enzymatic action is initiated and at the same time the free and bound The material is divided radially. Finally, front fluorescence intensity to measure fluorescence rate. law is used. The response is inversely proportional to the concentration of sample E2 and is stored converted to clinical units by comparison with a standard curve.

Example 3 Comparative tab performance In Figure 4, the current 5TRATUS■ (Baxter Diagnostic Inc.) and Multitest Tab calibration curves are compared. multi test Slope of the curve (sensitivity) for estradiol values less than 500 pg/mL for tabs There is a significant improvement in However, the current 5TRATUS @ (Ba The performance of xter Diagnostics Inc.) tab is looopg/ Excellent (larger slope) for estradiol values greater than mL . Figure 5 shows per unit of estradiol for values less than 1100 p/mL. are comparing the responses. The greater the change, the higher the degree of assay. put it here, The multi-test tab is 5TRATUS @ (Baxter Diagnost ics Inc.).

Example 4 Goat anti-mouse multitest reagent used in hCG assays Below are the multi-test reagent materials and goats used in the hCG assay. In a description of the means for determining the optimal concentrations of anti-mouse antibodies and mouse anti-hCG antibodies. be.

Materials: Anti-hCG conjugate: alkaline phosphatase (ALP) and hCG An anti-hCG antibody conjugate consisting of an antibody against sulfo-5IAB (sulfo-5IAB) using imidyl (4-iodoacetyl)-aminobenzoate sodium salt). prepared using standard methods and conjugated to reduced anti-hCG Fab' antibodies. . The resulting conjugate was treated with 0.0% as a preservative. Buffer containing 1% sodium azide It was diluted to approximately 180 ng/mL.

hCG calibration standard: 12000000 m IU/mL hC in distilled water A G stock solution was prepared. Zero (A), 5 (B), 15 (CL 50 (D), 1 hC to make a set of calibration standards of 50 (E) and 500 (F) mIU/mL. Aliquots of the G stock solution were added to normal hCG-free human serum.

Preparation and optimization of goat anti-mouse multi-reagent = 0.5% ZONYL FSN ([ 50mM Tris buffer (pH 8,0) containing 1uPont surfactant). ) of diluted 1 to 10% goat anti-mouse (CAM) in 1% increments. Prepare the liquid. The solution was then passed through a 0.85 μm filter to remove particles. - Filter. Many tabs of glass fiber filter (Whatman GF/F ) is spotted with approximately 76μ of each solution and dried.

Glass fiber filters spotted with increasing solutions of CAM were prepared as follows. evaluated. i.e., a partial 100% dilution of approximately 1:100 of mouse anti-hCG antibody. Add μL to approximately 100 μL of (F) calibration standard. For short incubation The hCG in the F calibration standard binds to the anti-hCG antibody. Approximately 100 μL of in Add the incubation mixture to the CAM-containing area of the tub (reaction area). short During the incubation (approximately 2 minutes), the mouse anti-hCG binds to CAM. Next Then add approximately 50 μL of anti-hCG antibody conjugate near the center of the reaction area. zygote binds to the hCG bound to the anti-hCG antibody, which in turn passes through the glass fiber filter. It is connected to the CAM on the paper tab. Next, add approximately 75 μL near the center of the reaction area. Apply substrate-wash solution. The substrate-wash solution should be free of any unbound material. The ALP elutes from the reaction zone and at the same time the reaction between the bound ALP and the substrate begins. to form a fluorescent product. The fluorescence response from a finite portion of the reaction zone is Measured using a fluorometer.

The solution of maximum calibration standard F response indicates that the maximum amount of antibody is captured per unit CAM. It was selected as the most suitable. The solution that gives the maximum response in the above method is Spot multiple tabs of glass fiber filter paper with the solution.

Optimization of mouse anti-hCG antibody concentration: The optimal mouse anti-hCG antibody is Determine using a CAM tab prepared as follows. Contains 0.1% bovine serum albumin. Mouse anti-hCG antibody was added to various concentrations in 50mM Tris (pH 8.0) with Dilute. The concentrations evaluated are as follows. That is, 1:100.1:2 00 and l: 500°, depending on the antibody being evaluated and the nature of that antibody. are narrower concentrations (i.e. 1:1O11:100.1:1000 and 1:100, respectively). and 1:10,000 or l:2, l:3 and l:5). It is clear that Dilution is evaluated as follows. That is, mouse anti-hCG Approximately 100 μL of each dilution of antibody was added to 100 μL of F (500 mIU/mL ) Cup containing calibration standards, 100 μL of B (5 mIU/mL) calibration standards, and 100 mL of A (zero) calibration standard. Bring the mixture to room temperature Incubate for 15 minutes. The incubation time and temperature are It may be changed according to the requirements of the company. Preferred ranges are 0 to 4 hours and 18 to 4 hours. The temperature is between 37°C and 37°C. After incubation, tab a 100 μL aliquot of the mixture. into the reaction zone. During a short incubation, (bound or bound) Mouse anti-hCG antibody binds to CAM. Next, near the center of the reaction zone Add 50 μL of anti-hCG conjugate to. The conjugate is free from any hCG in the reaction zone. Also combines. Next, apply approximately 75 μL of substrate-wash solution near the center of the reaction area. Ru. The substrate-wash solution elutes any unbound material from the reaction zone. , at the same time, a reaction between the bound ALP and the substrate is initiated, forming a fluorescent product. to be accomplished. Fluorescence response from a finite portion of the reaction zone is measured using a front surface fluorometer be done.

The optimal concentration of mouse anti-hCG antibody was determined by measuring two parameters: selected. The first parameter is F by each concentration of mouse anti-hCG antibody. (500 mIU/mL) is the fluorescence response that occurs when assaying the calibration standard.

This fluorescence response is directly proportional to the amount of anti-hCG antibody captured on the CAM. subordinate Therefore, these responses are also directly proportional to the amount of hCG in the sample (sample). characteristics of the switch assay). The second parameter measured is the A (zero) comparison. Fluorescence from B calibration standard (5 mlU/mL) relative to fluorescence response from authentic standard It is the ratio of responses (B/A ratio). The high B/A ratio is due to the level of these two calibration standards. show that there is a measurable difference between With the highest B/A ratio, F (500m IU/mL) calibration standard to determine the anti-hCG antibody concentration that gives the highest fluorescence response. Select as appropriate.

Alternatively, the anti-hCG antibody was added to a 76 μL excess of CAM (i.e., up to Glasses prepared with a calibration standard (% of CAM greater than % of CAM) giving a response of It can be optimized initially by using a tab of fibrous filter paper. anti-h After the CG is optimized, the GAM can be optimized using the procedure described above.

Assay format: In this assay format, the antibody-antigen complex is 100 μL of optimal anti-hCG antibody and 100 μL of serum sample, calibration standard or 5TRATUS @ (Baxter Diagnostics Inc, ) formed by adding to the cup. now antigen-antibody complexes and complexes A 100 μL aliquot of this mixture containing unformed antibodies was then added to the unformed antibody. A multi-test that captures antigen-antibody complexes as well as uncomplexed antibodies. Add Toyagi anti-mouse reagent directly near the center of the matrix. reagent matrix is then transferred to a second work station where the centering of the matrix is Next, 50 μL of conjugate is added. The conjugate binds the bound hcG. Next Then, the bound and free material was separated by the addition of 76 μL of substrate/wash solution. At the same time, enzymatic action begins. All of the above reagent additions are , also helps to separate any unbound material from the center of the reaction zone. As such, a multi-test goat anti-mouse reagent is performed near the center of the matrix. most Later, front surface fluorometry is used to measure the bound enzyme ratio. observed The ratio is proportional to the concentration of hCG in the sample and determined by comparison with the standard curve. converted into clinical units.

Example 5 Antibodies on glass fiber filter paper were coated with appropriate final components of GAR from Example 2 and CAM from Example 4. Example 2 is repeated except that the concentration consists of aliquots of 76 μL.

Optimization of the concentration of GAR and GAM applied to glass fiber filter paper (for GAR) determined by the procedures presented in Example 2 (for CAM) and Example 4 (for CAM). determined.

Optimization of rat anti-estradiol antibody concentration is determined according to Example 2. blood The concentrations of the supernatant samples, calibration standards, and controls were determined by the assay format of Example 2. It will be done.

Example 6 The antibodies on the glass fiber filter paper were GAR of Example 2 and GAR of Example 4.

Example 4 is repeated except that it consists of M. GA applied to glass fiber filter paper Optimization of R and GAM concentrations was performed in Example 2 (for GAR) and in Example 2 (for CAM). ) is determined by the procedure presented in Example 4. Mouse anti-hCG antibody concentration The optimization of the degree is determined according to Example 4. Serum sample, calibration standard, or control The concentration of is determined by the assay format of Example 4.

Examples 7-11 Specific antibodies (i.e., anti-hCG antibody, anti- Examples 2 and 4-6 were repeated except that E2 antibody, etc.) was added to the multitest reagent. Repeat each.

Although the invention has been described primarily in the context of particular and preferred embodiments, It will be understood that modifications may be made without departing from the scope of the invention. . The following claims generally follow that principle and relate to the relevant portions of the invention. be within known or customary means in the field or be obvious to a person skilled in the art. It covers any variations, uses, or adaptations of the invention, including any deviations therefrom. is intended to include.

Fig, 1 0 1 2 3 4 5 6’7 8% goat anti-mouse Fig, 2 Goat anti-rabbit in normal rabbit serum 1 2 3 4 1 II 7 1 II 10 Tube number Fig, 3 Titration of rabbit anti-estradiol 2 34 S@711 910111213 dilution (X 1000) +Test l--Test 2 OBoo 1000 1500 2000 215G0 3000 3800 Stradiol (Pg/a+L) -◆-5TRATUS @ + tab + multi test tab 10 20 30 40 ! to H7G 80 10 Estradiol (Pg/IIIL) -φ-8TRATUS @Tab-←Multi-test tab International research report! IM/l le. /1122, continuation of front page (72) Inventor: Reagan, Jeffrey, United States 33140 Florida, MA Yami Beach, North Meridian Avenue (72) Inventor Smith, Dennis 37 Finby Road, San Jose, California 95148, United States 39 (72) Inventor Timmons, Richard United States 33156 Florida; Miami, Southwest 84th Court 10221

Claims (51)

[Claims] 1. An immunochemical reagent consisting of a secondary antibody adsorbed onto an inert porous medium. 2. The set according to claim 1, wherein the secondary antibody is goat anti-mouse immunoglobulin. A product. 3. Claims that the secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulins. Item 1. The composition according to item 1. 4. consisting of an antiserum against a specific animal species adsorbed onto an inert porous medium; Human immunochemical reagents. 5. 5. The composition of claim 4, wherein the antiserum is goat anti-mouse immunoglobulin. thing. 6. 5. The composition of claim 4, wherein the antiserum is goat anti-rabbit immunoglobulin. . 7. Approximately 3% goat anti-mouse immunoglobulin was adsorbed onto an inert porous medium. 6. The composition according to claim 5. 8. The antiserum consists of goat anti-rabbit and goat anti-mouse immunoglobulin. 5. The reagent according to claim 4, which is. 9. Claim 4, wherein the antiserum forms a complex with normal animal serum. Reagents listed. 10. The claim is that the goat antiserum forms a complex with normal rabbit serum. Reagent according to item 6. 11. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase essentially comprises a secondary antibody immobilized on a solid support. the method comprises: a. Prayers - primary antibody complexes and complex formation under binding conditions to form a liquid mixture of primary antibodies without combining a liquid sample containing an analyte with a primary antibody; b. Analyte-primary antibody complex under binding conditions to allow binding of the primary antibody and uncomplexed primary antibody. applying the liquid mixture to the solid support by c. under binding conditions to bind the bound, uncomplexed antibody. applying an indicator to said solid support at d. The indicator is present on the solid support. observe the degree of e. The degree of presence of the indicator on the solid support is determined by A method consisting of relating the amount of analyte. 12. 12. The method of claim 11, wherein the secondary antibody is goat anti-rabbit. 13. 12. The method of claim 11, wherein the secondary antibody is goat anti-mouse. 14. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 11. 15. the indicator consists essentially of an enzyme-labeled analyte and a substrate wash solution; 12. The method according to claim 11. 16. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase is essentially a solid, inert, porous support. a secondary antibody immobilized within a finite area between: a. Liquid mixture of analyte-primary antibody complex and uncomplexed primary antibody together with a liquid sample containing said analyte under binding conditions to form a Combine with the next antibody, b. Analyte-primary antibody complex and uncomplexed antibody In order to bind the bodies, the liquid mixture is placed in the center of the finite area under binding conditions. Apply the compound, c. In order to bind uncomplexed antibody, the above-mentioned applying an indicator to the solid support; d. the indicator within a limited area of the reaction zone; Observe the degree to which exists, and e. The extent to which the indicator is present within the defined region is determined by A method that consists of relating knowledge to the amount of analyte. 17. the indicator consists essentially of an enzyme-labeled analyte and a substrate wash solution; 17. The method of claim 16. 18. 17. The method of claim 16, wherein the secondary antibody is goat anti-rabbit. 19. 17. The method of claim 16, wherein the secondary antibody is goat anti-mouse. 20. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 16. 21. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase essentially comprises a secondary antibody immobilized on a solid support. the method comprises: a. to the solid support under binding conditions. applying the following antibodies; b. applying the liquid sample to the solid support under binding conditions; apply, c. under binding conditions to bind the bound, uncomplexed antibody. applying an indicator to the solid phase at d. the indicator is present on the solid support Observe the extent, and e. The extent to which the indicator is present on the solid support is determined by the unknown in the sample. A method consisting of relating the amount of analyte. 22. 22. The method of claim 21, wherein the secondary antibody is goat anti-rabbit. 23. 22. The method of claim 21, wherein the secondary antibody is goat anti-mouse. 24. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 21. 25. the indicator consists essentially of an enzyme-labeled analyte and a substrate wash solution; 22. The method of claim 21. 26. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase is essentially a solid, inert, porous support. The method consists of a secondary antibody immobilized within a finite area between: a. Applying the primary antibody to the center of the finite area of the solid support under binding conditions. use, b. The liquid sample is placed in the center of the finite area of the solid support under binding conditions. Apply the pull, c. In order to bind uncomplexed antibody, the above-mentioned applying an indicator to the solid support; d. the instructions within a limited area of the reaction zone; Observe the extent to which the drug is present, and e. The extent to which the indicator is present within the defined region is determined by A method that consists of relating knowledge to the amount of analyte. 27. the indicator consists essentially of an enzyme-labeled analyte and a substrate wash solution; 27. The method of claim 26. 28. 27. The method of claim 26, wherein the secondary antibody is goat anti-rabbit. 29. 27. The method of claim 26, wherein the secondary antibody is goat anti-mouse. 30. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 26. 31. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase essentially comprises a secondary antibody immobilized on a solid support. the method comprises: a. Analyte-primary antibody complexes and complex formation under binding conditions to form a liquid mixture of primary antibodies without a liquid sample containing an analyte, said primary antibody, and for binding the antibody; Combined with the indicator, b. Binding the analyte-primary antibody complex and uncomplexed primary antibody applying the liquid mixture to the solid support under binding conditions to c. observing the extent to which the indicator is present on the solid support; and d. The extent to which the indicator is present on the solid support is determined by the unknown in the sample. A method consisting of relating the amount of analyte. 32. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes wherein the solid phase consists essentially of a secondary antibody immobilized on a solid support. The method comprises: a. Analyte-primary antibody complex and complex formation The above components under binding conditions to form a liquid mixture of primary antibodies without combining the liquid sample containing the analyte and the primary antibody; b. Analyte-primary antibody complex under binding conditions to allow binding of the primary antibody and uncomplexed primary antibody. applying the liquid mixture to the solid support by c. to the solid support under binding conditions to bind the bound analyte. apply an indicator, d. observing the extent to which the indicator is present on the solid support; and e. The extent to which the indicator is present on the solid support is determined by the unknown in the sample. A method consisting of relating the amount of analyte. 33. 33. The method of claim 32, wherein the secondary antibody is goat anti-rabbit. 34. 33. The method of claim 32, wherein the secondary antibody is goat anti-mouse. 35. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 32. 36. The indicator essentially comprises an enzyme-labeled antibody directed against the analyte and a substrate. 33. The method of claim 32, comprising a cleaning solution. 37. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase is an essentially solid, interstitial support. The method consists of a secondary antibody immobilized within a limited area, and the method: a. Analyte-primary antibody conjugate and uncomplexed primary antibody liquid mixture together with a liquid sample containing said analyte under binding conditions to form a Combine with the next antibody, b. Analyte-primary antibody complex and uncomplexed antibody In order to bind the bodies, the liquid mixture is placed in the center of the finite area under binding conditions. Apply the compound, c. to the solid support under binding conditions to bind the bound analyte. apply an indicator, d. observing the extent to which the indicator is present within a limited area of the reaction zone; hand e. The extent to which the indicator is present within the defined region is determined by A method that consists of relating knowledge to the amount of analyte. 38. The indicator essentially comprises an enzyme-labeled antibody directed against the analyte and a substrate. 38. The method of claim 37, comprising a cleaning solution. 39. 38. The method of claim 37, wherein the secondary antibody is goat anti-rabbit. 40. 38. The method of claim 37, wherein the secondary antibody is goat anti-mouse. 41. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 37. 42. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase essentially comprises a secondary antibody immobilized on a solid support. the method comprises: a. to the solid support under binding conditions. applying the following antibodies; b. applying the liquid sample to the solid support under binding conditions; apply, c. An indicator is applied to the solid phase under binding conditions to bind the bound analyte. apply, d. observing the extent to which the indicator is present on the solid support; and e. The extent to which the indicator is present on the solid support is determined by the unknown in the sample. A method consisting of relating the amount of analyte. 43. 43. The method of claim 42, wherein the secondary antibody is goat anti-rabbit. 44. 43. The method of claim 42, wherein the secondary antibody is goat anti-mouse. 45. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 42. 46. The indicator essentially comprises an enzyme-labeled antibody directed against the analyte and a substrate. 43. The method of claim 42, comprising a cleaning solution. 47. Perform solid-phase immunoassays on liquid samples for unknown amounts of analytes , wherein the solid phase is essentially a solid, inert, porous support. The method consists of a secondary antibody immobilized within a finite area between: a. Applying the primary antibody to the center of the finite area of the solid support under binding conditions. use, b. The liquid sample is placed in the center of the finite area of the solid support under binding conditions. Apply the pull, c. to the solid support under binding conditions to bind the bound analyte. apply an indicator, d. Observe the extent to which the indicator is present within the limited territory of the reaction zone, and hand e. The extent to which the indicator is present in the defined region in the liquid sample A method consisting of relating the quantity of an unknown analyte. 48. The indicator essentially comprises an enzyme-labeled antibody directed against the analyte and a substrate. 48. The method of claim 47, comprising a cleaning solution. 49. 48. The method of claim 47, wherein the secondary antibody is goat anti-rabbit. 50. 48. The method of claim 47, wherein the secondary antibody is goat anti-mouse. 51. The secondary antibodies are goat anti-rabbit and goat anti-mouse immunoglobulin. The method according to claim 47.