WO1995021942A1

WO1995021942A1 - Immunoassay kit comprising universal reagents

Info

Publication number: WO1995021942A1
Application number: PCT/US1995/001529
Authority: WO
Inventors: Georges H. Parsons
Original assignee: T Cell Diagnostics, Inc.
Priority date: 1994-02-15
Filing date: 1995-02-06
Publication date: 1995-08-17
Also published as: AU1744495A

Abstract

An immunoassay kit containing a universal solid phase and a universal conjugate. A first derivatized reagent may be immobilized onto said solid phase and bind a selected analyte component from a sample under analysis. The universal conjugate is capable of detecting a second derivatized reagent bound to the selected analyte component or bound to the first derivatized reagent. The kit is adaptable for measurement of selected component. Methods for measuring selected components with the kits of the present invention are also provided.

Description

IMMUNOASSAY KIT COMPRISING UNIVERSAL REAGENTS

BACKGROUND OF THE INVENTION

Microtiter plate enzyme immunoassay is one of the primary tools used in biomedical research today. A number of kits are now commercially available for measuring selected analyte components in a sample under analysis, which kits come complete with antibody coated microtiter plates, standards, enzyme-labeled antibody wash buffers and substrate reagents. In general, these commercial kits contain optimized, reliable assays developed by trained experts which will function well over the months or sometimes years required for many research projects. However, the most interesting research is often ahead of the commercial kit developers' ability to predict which kit detection components will be in demand next, to detect analytes of interest. Thus, researchers are often forced to develop their own assays for detection of such new analytes of interest, taking time and resources away from the researcher's primary goal. Oftentimes these assays are not optimized, nor are they reliable, as researchers lack the expertise and training needed to develop assays which will function reproducibly over long periods.

An immunoassay kit has now been developed which comprises a universal solid phase and a universal conjugate. The researcher supplies reagents, in the form of selected binding partners capable of detecting a selected analyte component in a sample, which binding partners may be derivatized such that a first derivatized reagent may be immobilized on the universal solid phase and a second derivatized reagent is capable of recognition by the universal conjugate. Thus, the researcher can take advantage of the efficiency and quality control of the universal solid phase and universal conjugate provided with the kit and still maintain the flexibility and responsiveness necessary to perform state of the art research. SUMMARY OF THE INVENTION

An object of the present invention is to provide a kit which comprises a universal solid phase and a universal conjugate, as well as the capability of producing first and second derivatized reagents. The universal solid phase of the invention is capable, through specific interaction with a first derivatized reagent, of immobilizing said first derivatized reagent. The universal conjugate is capable, through specific interaction with a second derivatized reagent, of detecting the presence of said second derivatized reagent. In certain embodiments, the first and second derivatized reagents are members of at least a pair of binding partners capable of detecting the presence of a selected analyte component in a sample in a traditional sandwich assay format. In other embodiments, the first derivatized reagent is a binding partner capable of binding selected analyte components in a sample and the second derivatized reagent is capable of competing with the selected analyte component for binding to the first derivatized reagent.

Another object of the present invention is to provide a method for detecting the presence of a selected component in a sample kit comprising a universal solid phase for immobilizing a first derivatized reagent and a universal conjugate for detecting the presence of a second derivatized reagent. In one embodiment, the method provides a means for derivatizing a first reagent and a second reagent. In another embodiment the invention provides a kit which comprises: a universal solid phase; a means of attaching one or more first derivative moieties to a reagent to form a first derivatized reagent wherein the one or more first derivative moieties facilitate immobilization of the first derivatized reagent to the universal solid phase; a means of attaching one or more second derivative moieties to a reagent to form a second derivatized reagent wherein the one or more second derivative moieties facilitate recognition by the universal conjugate; and, a universal conjugate capable of detecting the presence of the second derivatized reagent, optionally all together with the standard buffers, accessories, and accessory reagents of standard immunoassay kits.

In another embodiment the invention provides a method of detecting the presence or absence of a selected analyte in a sample suspected of containing such an analyte comprising: providing a universal solid phase; derivatizing a first reagent to form a first derivatized reagent, contacting the first derivatized reagent with the universal solid phase such that the first derivatized reagent is immobilized; contacting a sample with the universal solid phase and the immobilized first derivatized reagent such that a selected analyte component may bind to the immobilized first derivatized reagent; derivatizing a second reagent with a second derivative moiety capable of reacting with a universal conjugate such that a second derivatized reagent is formed; contacting the second derivatized reagent with the universal solid phase and immobilized first derivatized reagent such that the second derivatized reagent may bind the selected analyte or compete with the selected analyte for binding to the first derivatized reagent; adding the universal conjugate to the universal solid phase and first derivatized reagent such that the universal conjugate may bind to the second derivatized reagent; and, detecting the presence or absence of the selected analyte component .

In one embodiment the method provides for a sandwich assay format wherein the detection of the selected analyte is accomplished by virtue of the analyte binding to the first derivatized reagent and being bound by the second derivatized reagent to form a sandwich. In another embodiment the presence of a selected analyte is detected by competition between said analyte and the second derivatized reagent for binding to said first derivatized reagent.

In another embodiment the invention provides for standard reagents which facilitate the methods of the instant invention. Thus, in a preferred aspect, the invention includes standard reagents which allow the user of the kit to ascertain that appropriate reagent preparation and additions have been performed. In a preferred embodiment, the invention includes a standard reagent such as a derivatized protein or polypeptide. Such derivatized protein may be a protein that is derivatized to contain both the first derivative moiety and the second derivative moiety. Such a protein can serve, in certain embodiments, as a bridge between the universal conjugate and the universal solid phase such that the user can determine whether the two universal reagents are functioning properly. The invention may also include standard reagents, such as proteins, which have been derivatized to contain the derivative moiety of either the first or the second derivatized reagent. The invention thus provides a means of determining whether the derivatization chemistry performed as part of the method of the invention are functioning properly.

In a preferred embodiment, the invention thus provides for a kit which comprises a universal solid phase, a means for derivatizing a first and a second reagent, a multiplicity of standard reagents, and a universal conjugate, along with the appropriate buffers and accessory reagents .

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, a kit has been developed which provides a researcher with the flexibility to assay or detect the presence of a selected analyte component in a sample to be assayed while maintaining the high quality control and reproducibility of a commercially developed assay. In a first embodiment, a kit is provided which comprises a universal solid phase onto which a first derivatized reagent may be immobilized and a universal conjugate capable of detecting a second derivatized reagent bound to a selected component in a sample or bound to the first derivatized reagent. Any selected analyte component in a sample for which at least one "binding partner" is available can be detected or measured according to the present invention. The selected analyte may be for instance, a cytokine, cell adhesion molecule, phenotypic marker, activation marker, or nucleic acids. Non-limiting examples of selected analytes include proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, pharmacological agents and their metabolites, and the like. "Binding partner" within the scope of the present invention is meant to include, but is not limited to, antibodies to and receptors for the selected analyte component. In particular, binding partners which are not antibodies include cell surface or soluble receptors for the selected analyte component. Binding partners which are not antibodies or receptors include those binding partners described in the sections below. Particular non-limiting examples of binding partners are, for instance, complementary nucleic acids; in an assay for a CD4 auto-antibody, a CD4 molecule; in an assay for CD4, an HIV gpl20 or an gpl60; in an assay for T cell antigen receptor, a superantigen or Concanavalin A.

In the present invention, the term "universal solid phase" refers to any solid surface such as a microtiter plate, slide or bead, which is coated with a reasonably high affinity derivative moiety with feasible derivatization chemistry. Thus, the solid surface within the scope of the present invention is meant to include any solid support including but not limited to a glass or a plastic bead, membrane, a plastic stick, a microwell, a glass or plastic test tube, or other solid support known in the art for immunoassays .

The derivative moiety used to coat the solid surface within the scope of the present invention includes any member of a derivative pair such as a hapten and anti- hapten antibody; nucleic acids, and complementary nucleic acids; lectins and their complimentary ligands; and derivative pairs known in the art of immunochemistry such as avidin and biotin.

In a preferred embodiment the universal solid phase is a microtiter well and the derivative moiety is streptavidin. Therefore, in a preferred embodiment, the universal solid phase comprises a streptavidin coated microtiter plate.

The term "universal conjugate" within the scope of the present invention refers to a component capable of binding a second derivatized reagent and producing a detectable signal. The universal conjugate therefore is capable of detecting the presence of the second derivatized reagent. The universal conjugate detects the presence of the second derivatized reagent by virtue of its reactivity with the derivatized moiety of the second derivatized reagent. In one embodiment, the universal conjugate is an antibody capable of reacting with the derivatized moiety of the second derivatized reagent. For instance, when the second derivatized reagent is an antibody capable of detecting the presence of a selected analyte component in a sample, which antibody has been derivatized to contain a derivatized moiety such as fluorescein isothiocyanate (FITC) , then the universal conjugate can be an antibody which is capable of detecting the presence of FITC, such as an anti-FITC antibody. All that is required is that the universal conjugate be capable of detecting the presence of the derivatized moiety of the second derivatized reagent.

The universal conjugate should also be detectably labelled. Detectable labels are known in the art of immunoassays and include but are not limited to enzymes, fluorescent compounds, fluorescence emitting metals, and chemiluminescent compounds. In certain preferred embodiments, the universal conjugate is an antibody labelled with an enzyme . Enzymes that can be used to label the universal conjugate include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose-6- phosphate dehydrogenase, malate dehydrogenase, staphylococcal nuclease, β-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease, urease, catalase, glucoamylase and acetylcholinesterase. In a preferred embodiment the enzyme is horseradish peroxidase (HRP) . Examples of universal conjugates with in the scope of the present invention include, but are not limited to, anti-FITC-HRP conjugated antibodies . A first derivatized reagent within the scope of the present invention is any binding partner capable of binding to a selected analyte component in a sample, which binding partner can be derivatized according to the methods of the present invention, and those known to one of skill in the art, to be immobilized on the universal solid phase. Derivatized reagents suitable for use in the present invention include any binding partner for a selected analyte component. Binding partners suitable for derivatized reagents within the scope of the present invention include, but are not limited to those listed in preceding sections, antibodies, soluble receptors, bacterially derived binding proteins such as enterotoxins, haptens, nucleic acids, lectins and lectin receptors. In one embodiment, the first derivatized reagent is a member of a pair of derivatized reagents, which pair of reagents are selected by the user of the kit, and which pair are capable of detecting the presence of a selected analyte component in a sample. For example, a first and second derivatized reagent may be a pair of antibodies capable of detecting the presence of a certain lymphokine in a sample. In another embodiment the derivatized reagent is a hapten, and the selected analyte is an anti-hapten antibody.

Within the present invention, the first derivatized reagent is derivatized to form the first derivatized reagent. The first derivatized reagent is, for example, an antibody capable of detecting the presence of a component in a sample, which antibody has been derivatized to contain the complement of the derivatized moiety on the universal solid phase. Therefore, if the universal solid phase is coated with streptavidin, then the first derivatized reagent is derivatized with biotin NHS ester; if the universal solid phase is coated with an anti-hapten antibody, the first derivatized reagent is derivatized with the complement hapten; if the universal solid phase is coated with a nucleic acid, the first derivatized reagent is derivatized to a complementary nucleic acid. The chemistry of derivatizing reagents is well known in the art. For instance, the chemistry of adding biotin to various proteins is discussed in Updyke, T.V. and Nicolsen, G.L., (1984) , J. Immunol. Methods 73:83-95 and Savage, M.D., et al. , (1992) "Avidin-Biotin Chemistry: A Handbook" Rockford, Illinois, Pierce Chemical Company

Catalog, P.O. Box 117 Rockford Illinois. Additionally, the first derivatized reagent may be purchased prederivatized from vendors known to those of skill in the art, for example, T Cell Diagnostics, Inc., Cambridge, MA 02139; or Pierce Chemical Company, Rockford, IL.

In a preferred embodiment the universal solid phase is a streptavidin coated microtiter plate and the first derivatized reagent is an antibody labelled with a biotin NHS ester. The labelling or derivatization of the first

(and second) derivatized reagent may be performed by the researcher by methods known to those skilled in the art or the derivatized reagents may be purchased from well-known vendors. The kit of the present invention may include the appropriate reagents required to perform this derivatization. The derivatization is such that it facilitates immobilization of the first derivatized reagent to the universal solid phase. One of skill in the art will recognize that derivatization may result in one or more derivative moieties being added to the derivatized reagent. The terms first and second derivative moieties and reagents are meant to include these forms. Examples of appropriate labels include, but are not limited to, biotin NHS ester, photoactivable biotin, p- aminobenzoyl biotin, and NHS-iminobiotin.

A second derivatized reagent within the scope of the present invention is any binding partner capable of detecting the presence of a selected analyte component in a sample or capable of competing with the selected analyte component for binding to the first derivatized reagent. In certain embodiments, the second derivatized reagent can be an antibody capable of detecting the presence of an analyte in a sample. The second derivatized reagent is derivatized with a second derivative moiety capable of reacting with the universal conjugate. This derivatization may be performed by the researcher by methods well known in the art or the second derivatized reagent may be purchased prederivatized with the second derivative moiety in place from well-known vendors as discussed supra for the first derivatized reagent . The kit of the present invention may include the appropriate reagents required to perform this derivatization. Examples of appropriate second derivative moieties include, but are not limited to, fluorescein isothiocyanate (FITC) , rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and o- phthaldehyde.

It will be recognized, that any derivative moiety with feasible derivatization chemistry can be used to derivatize the second derivatized reagent. The derivative moiety used to derivatize the second derivatized reagent within the scope of the present invention may include any member of a derivative pair such as a hapten and anti-hapten antibody; nucleic acids, and complementary nucleic acids; lectins and their complimentary ligands; and binding pairs known in the art of immunochemistry such as avidin and biotin. All that is required is that the universal conjugate is capable of detecting the presence of the second derivatized reagent by virtue of the second derivative moiety. In a preferred embodiment the second derivatized reagent is derivatized to include a fluorescein isothiocyanate molecule and the universal conjugate is an anti-FITC enzyme linked conjugate.

It will be recognized by one of skill in the art that the present invention can be used in conjunction with preexisting immunoassay kits. All that is required is that the binding partners of the preexisting kits be derivatized to form the first and second derivatized reagents of the present invention. Thus, such assays as those described in International Publication No. WO92/08981 entitled "Therapeutic and Diagnostic Methods Using Total Leukocyte Surface Antigens" by Rittershaus, C.W., are particularly useful within the context of the present invention, the disclosure of which is hereby incorporated by reference. Also within the scope of the present invention are standard reagents that will allow the user of the kit to ascertain that appropriate reagent preparation and addition have been done. A protein that is derivatized with both biotin and FITC, for instance, can be used as a control that the universal solid phase, the universal conjugate reagents were functional when the universal solid phase is a streptavidin coated surface and the universal conjugate is a detectably labelled anti-FITC antibody. A protein, such as bovine serum albumin (BSA) could be provided along with the kit or methods of the invention pre-derivatized to contain biotin or another member of a binding pair. Such a protein could be derivatized with the second derivatizing reagent, for instance, FITC, at the same time and with the same reagents employed by the user when he labels his own reagent with FITC. Conversely, in the above scenario, a protein pre-derivatized with FITC and supplied with the kit, could be derivatized by the user with the same reagent and at the same time the user derivatizes the corresponding reagent with biotin. If all the derivatization chemistries are done correctly, all three controls should give similar signals.

Such a method or kit with controls is very valuable for doing a wide variety of immunological research and serves to expedite both the development and manufacture of reagents based on these methods.

In one embodiment the invention provides a method for detecting a selected analyte component in a sample which comprises: providing a universal solid phase; contacting a first derivatized reagent with the universal solid phase such that the first derivatized reagent is immobilized; contacting a sample suspected of containing a selected analyte component with said immobilized first derivatized reagent; contacting a second derivatized reagent with the immobilized first derivatized reagent and the sample; contacting the second derivatized reagent with a universal conjugate capable of binding the second derivatized reagent; detecting the presence of the universal conjugate. The method is varied according to whether a sandwich assay format is used or a competitive assay format is used. Standard assay conditions known to those of skill in the art can be used in conjunction with the present invention.

As used herein a "sample" refers to a sample suspected of containing a selected analyte. Selected analytes may be measured according to the present invention in samples derived from biological fluids, e.g., whole blood, plasma, serum, blood cells saliva, urine, synovial fluid, pleural effusion, tumor and tissue infiltrates, amniotic fluid, spinal fluid or cranial fluid. In another embodiment the sample may be a cell culture fluid, preferable when the sample is a tissue sample, the tissue is treated to disrupt the connective tissue matrix, e.g. by homogenization. The components of the kit of the instant invention can be provided as lyophilized preparations for reconstitution, or as a concentrated solution. The components can be provided in a container having sufficient volume to hold the amount of solution necessary for the number of assays contemplated by the kit . The kit can further comprise a reagent reactive with the universal conjugate. For example, when the detectable label is an enzyme, the kit can provide the enzyme substrate in the form of a substrate solution or preparation for reconstitution. When the detectable label is horseradish peroxidase, then the working substrate is a tetramethyl benzene (TMB) solution, or an O-Phenylenediamine solution. The kit can also provide a dilution buffer, in a final concentration, high concentration for dilution, or dry for reconstitution.

In a particularly preferred embodiment the kit comprises a streptavidin coated universal solid phase microtiter plate and plate sealers; a purified stock solution of anti-FITC-HRP universal conjugate; a control such as a stock solution of biotin-BSA-FITC; a control such as a stock solution of biotin-BSA; a control solution such as a BSA- FITC; a working substrate solution, preferably a TMB working substrate solution or an OPD solution, more preferably a TMB working substrate solution which consists of a 1:1 ratio of 2 provided bottles, the first bottle containing peroxidase substrate solution and the second bottle containing TMB peroxidase substrate; a Stop Solution, preferably 0.18M H₂S0₄; a conjugate diluent which comprises PBS containing 10% fetal bovine serum, 0.05% NP-40, 100 μg/ml mouse IgG, and 0.01% Gentamicin Sulfate; and standard or sample diluent which comprises PBS containing 4% bovine serum albumin, 1% sucrose, and 0.01% Gentamicin Sulfate; and directions for use. This kit may further comprise a biotin labeling kit and/or a FITC labeling kit. The biotin labeling kit comprises biotin, sample diluent and reaction buffers, a test control sample, NAP 5/10 columns and directions for use. The FITC labeling kit comprises FITC, sample diluent and reaction buffers, test control sample, NAP 5/10 columns and directions for use. In this embodiment the kit can be used to measure a vast variety of selected components in a sample, preferably antigens or antibodies including, but not limited to, cytokines, interleukins, such as IL-8, IFN, T cell antigen receptor proteins (TCAR) , CD3, and DNA amptamers. Other selected components which can be measured through use of the kit will become obvious to those skilled in the art upon this disclosure.

In this embodiment, an assay for determining concentrations of a selected component in a sample such as a cytokine, for example IL-4, could be performed by selecting pair of antibodies specific for IL-4 and labeling one antibody with biotin NHS ester (BNHS) and the other antibody with fluorescein isothiocyanate (FITC) . The biotinylated antibody will react with and be immobilized on a streptavidi coated surface. A sample or standard containing IL-4 can then be placed in contact with the surface under conditions which would enhance binding of the IL-4 to the immobilized antibody. The FITC labelled antibody can then be placed in contact with the surface so that this antibody will bind wit the IL-4. The universal conjugate, antifluorescein antibody labeled with an enzyme, will bind to the FITC label on the second antibody and provide a signal which is detected by the TMB Working substrate solution.

The kit of the present invention could also be used to measure a binding protein or antibody by derivatizing the hapten for the binding protein or antibody with a derivative moiety which immobilizes it to the universal soli phase. In a preferred embodiment the universal solid phase is a streptavidin coated plate and the derivative moiety is biotin NHS ester. When the sample or standard is placed in contact with the universal solid phase, a portion of the immobilized hapten reacts with any corresponding binding protein or antibody in the sample or standard. Detection of this reaction can be accomplished by addition of a labelled reagent, preferably an FITC labeled reagent, that is capable of either binding competitively with the immobilized hapten or binding with the binding protein or antibody in a sandwic type assay.

For example, antibodies to CD4 can be measured in the competitive assay in the following manner.

Biotinylated CD4 is added to streptavidin coated plates. Antibodies to CD4 are labelled with FITC. Such antibodies compete with anti-CD4 antibody in the sample for immobilized CD4 on the plate. Detection is accomplished through a labelled anti-FITC conjugate which binds to the FITC-labeled CD4 antibodies and is detected by incubation with the working substrate solution, preferably TMB working substrate solution.

The same kit may be used in the sandwich type mode to detect selected components such as tetanus toxin. Tetanus toxin labelled with biotin is immobilized onto streptavidin coated plates. Samples or standards containing various amounts of human anti-tetanus antibody are allowed to react with the immobilized tetanus toxin. Anti-human immunoglobulins, preferably IgG, IgM or IgA, labelled with FITC and the universal conjugate anti-FITC antibody are used to detect the bound anti-tetanus antibody through reaction with the working substrate solution.

This invention is further illustrated by the following nonlimiting examples.

EXAMPLES Example 1: Coating Plates with Streptavidin

A 100 μl aliquot containing 5 μg/ml streptavidin in PBS with no Thimerosal is added to each well of a microtiter plate. The plate is then incubated overnight at room temperature. The plates is then blocked with a 1% sucrose 4% BSA/PBS blocking solution for 2 hours at room temperature and then dried at room temperature.

Example 2 : Procedure for Biotin Labeling

A 10 mg/ml solution of NHC-LC-Biotin is prepared in dimethyl formamide. A 2 mg/ml solution of a selected first binding partner is prepared in 0.5 M sodium bicarbonate buffer, pH 9.5. The biotin and binding partner solutions are then mixed at a ratio of 150 μg biotin for every 1 mg of binding partner. The solution is mixed until homogenous and incubated at room temperature for with rocking for 1 hour. After incubation, the biotin labeling reaction is terminated by addition of 20 μl of 1M NH₄C1₂ for every 0.25 mg of biotin. The solution is mixed until homogenous and incubated for ten minutes at room temperature. Unreacted biotin is removed from the solution by passing it across a NAP 5, 10 or 25 column depending on the sample size. The column is pre-equilibrated with the appropriate volume of phosphate buffered saline (PBS) based on the column size. The biotin-binding partner solution is added to the column. The biotin labeled binding partner is then eluted from the column with the appropriate volume of PBS based on the column size. The labeled binding partner can be stored in aliquots at -70°C.

Example 3 : Procedure for FITC Labeling

A 10 mg/ml stock solution of FITC is prepared in dimethyl formamide. A 2 mg/ml solution of a selected second binding partner is prepared in 0.5 M sodium bicarbonate buffer, pH 9.5. The FITC solution and the binding partner solution are mixed at a ratio of 125 μg of FITC for every 1 mg of binding partner. The mixture is stirred until homogenous and incubated at room temperature with rocking, for 1 hour. After the incubation, the FITC labeling reaction is terminated by addition of 0.1 ml of 1.5 M hydroxylamine HCl for every 1 ml of reaction. This solution is incubated for 30 minutes at room temperature with rocking. The unreacted FITC is removed by passing the solution across a NAP 5, 10 or 25 column depending on the sample size. The column is pre-equilibrated with the appropriate volume of PBS based on the column size. The FITC-binding partner solution is added to the column. The FITC-labeled binding partner is then eluted with the appropriate volume of PBS based on the column size. The labeled binding partner is stored in aliquots at -70°C.

Example 4: Measurement of Cytokines

Biotinylated anti-cytokine capture mAb is diluted to approximately 0.5 to 4 μg/ml in coating buffer containing 0.1M NaHC0₃, pH 8.2. 100 microliters of this solution is added to each well of a streptavidin coated microtiter plate. The plate is covered and incubated for 30 minutes at room temperature with shaking. The plate is washed two times with PBS/Tween. PBS solution, or phosphate buffered saline solution, contains 80.0 g NaCl, 11.6 g Na₂HP0₄, and 2.0 g KCl in 10 L of water, pH 7.0. PBS/Tween contains 0.5 ml of Tween-20 in 1 L of PBS. For each washing the wells are filled with PBS/Tween and allowed to stand for at least 30 seconds prior to emptying. One hundred microliters of sample or standard is then added to each well. The plate is covered and incubated for 2 hours at room temperature with shaking. The plate is then washed 4 times with PBS/Tween. Anti-cytokine detecting mAb labeled with fluorescein isothiocyanate (FITC) is diluted to approximately 0.1-1 μg/ml in PBS conjugate diluent containing 10% fetal bovine serum, 0.05% NP-40, 100 microg/ml mouse IgG, and 0.01% Gentamicin Sulfate. One hundred microliters is added to each well. The plate is covered and incubated at room temperature for 1 hour with shaking. The plate is then washed at least 4 times with PBS/Tween. One hundred microliters of TMB working substrate solution is added to each well. The plate is covered and incubated at room temperature for 30 minutes. Following this incubation, the reaction is stopped by addition of 100 μl of 0.18 M H₂S0₄. The plate is then read at 450 nm.

Example 5: IL-8 Assay

IL-8 antibody was purchased from BioSource International, 950 Hynn Road, Suite A, Camarillo, CA 93012 (800)247-0607) , and biotin labeled, catalog number AB-28-010. FITC labeled rabbit anti-IL-8 antibody was purchased from PeproTech, Inc. (Princeton Business Park, G2, P.O. Box 275, Rocky Hill, New Jersey"/ . Recombinant IL-8 was purchased frorr, R&D Systems, Minneapolis MN 55413. All reagents were warmed to room temperature. Stock standard solutions of IL-8 were diluted to appropriate concentration in the standards diluent. The following dilutions were made in conjugate diluent and a 1:1:1 ratio of antibody mix was prepared: Biotin-labeled anti-IL-8 antibody was diluted to 5 μg/ml. FITC labeled anti-IL-8 antibody was diluted to 500 ng/ml . Sheep anti-FITC-HRP antibody was diluted to 1.02 μg/ml.

Streptavidin coated microtiter plates were washed three times with wash buffer PBS/Tween. Fifty μl of the antibody mixture was added to each well followed by 50 μl of the standards material . The plate was then covered and incubated at room temperature with shaking (approx. 150 RPM) for 3 hours . Following the incubation the plate was washed 3 times with wash buffer PBS/Tween. One hundred microliters of TMB working substrate solution was added to each well . The plate was incubated for 30 minutes at room temperature. Following this incubation, the reaction was stopped by addition of 100 μl of 0.18 M H₂S0₄. The plate was then read at 450 nm.

Example 6 : IFN Gamma Assay

Biotin labeled anti-IFN gama antibody, FITC labeled anti-IFN gamma antibody and Human IFN gamma from leukocytes were obtained from Centocor, Inc., 200 Great Valley Parkway, Malvern, Pennsylvania 19355-1307 (215)651- 6000. All reagents were warmed to room temperature. Dilutions of the standard human IFN gamma were prepared in standards diluent. Biotin labeled anti-IFN gamma was diluted to 0.6 μg/ml in conjugate diluent. The antibody mix containing 200 mg/ml FITC labeled anti-IFN gamma and 500 μg/ml sheep anti-FITC HRP was prepared in conjugate diluent. Streptavidin coated microtiter plates were washed three time with wash buffer. One hundred microliters of biotin labeled anti-IFN gamma antibody was added to each well of the plate. The plate was then covered and incubated for at room temperature with shaking (approx. 150 RPM) for 20 minutes. Following the incubation the plates were washed 3 times with wash buffer PBS/Tween. Fifty microliters of the antibody mixture (listed above) was added to each well followed by 50 μl of the standard solution. The plate was then covered and incubated at room temperature with shaking (approx. 150 RPM) for 3 hours. Following this incubation the plates were washed 3 times with wash buffer PBS/Tween. One hundred microliters of TMB working substrate solution was added to each well and the plate was incubated for 30 minutes at room temperature. Following this incubation the reaction was stopped by addition of 100 μl of 0.18 M H₂S0₄. The plate was then read at 450nm.

Example 7: TCAR Assay

Biotin labeled Cβ antibody to the constant region of the β chain of the T cell antigen receptor was purchased from T Cell Diagnostics, Inc., 38 Sidney Street, Cambridge, Massachusetts 02139. FITC labeled Vβ antibody to a variable region determinant of the T cell antigen receptor was purchased from and was prepared as described in Example 3. Rabbit anti-FITC HRP conjugate was purchased from Gene- Trak Systems, Inc., 31 New York Avenue, Framingham, Massachusetts 01701 (508)872-3113. The standard was lysate from HBP cells (American Type Culture Collection, Rockville, Maryland. )

One hundred microliters of biotin labeled Cb antibody (10 μg/ml) was added to each well of a streptavidin coated plate. The plate was covered and incubated at room temperature with shaking (approx. 150 RPM) for 30 minutes.

Following the incubation the plate was washed three time with wash buffer PBS/Tween. Fifty microliters of FITC labeled Vβ conjugate (diluted to 250 ng/ml) was added to each well of the plate. Fifty microliters of HPB cell lysate diluted in standards diluent was then added to each well. The plate was then covered and incubated at room temperature with shaking (150 RPM) for 3 hours. The plate was then washed 3 times with wash buffer. One hundred microliters of anti-FITC-HRF conjugate diluted 1:80 in conjugate diluent was then added to each well. The plate was incubated at room temperature with shaking (150 RPM) for 30 minutes. Following this incubation the plate was washed 3 times in wash buffer PBS/Tween. One hundred microliters of OPD (O-phenylenediamine, a chromogen) substrate solution was added to each well . The plate was incubated at room temperature for 30 minutes. Following the incubation 50 μl of 2M H₂S0₄ Stop Solution was added to stop the reaction. The plate was read at 490 nm.

Example 8: CD3 Assay

Biotin labeled anti-CD3 antibody was purchased from Gen-Trak, Inc., Plymouth Meeting, PA 19462. FITC labeled rabbit anti-CD3 antibody, Code #A452 Lot #102 was purchased from DAKO A/S Produktionsvej 42, DK-2600 Glostrop, Denmark, 45 44 92 0044. EDTA whole blood lysate was the source for the standard.

One hundred microliters of biotin labeled anti-CD3 antibody solution in conjugate diluent (2 μg/ml) was added to each well of a streptavidin coated plate. The plate was covered and incubated at room temperature with shaking (approx. 150 RPM) for 30 minutes. Following the incubation the plate was washed three times with wash buffer PBS/Tween. Fifty microliters of FITC labeled anti-CD3 conjugate diluted to 850 ng/ml in conjugate diluent was added to each well of the plate. Fifty microliters of while blood lysate diluted in standards diluent was then added to each well. The plate was then incubated at room temperature with shaking (150 RPM) for 2 hours. The plate was then washed 3 times with wash buffer PBS/Tween. One hundred microliters of anti-FITC-HRP conjugate diluted to 823 ng/ml in conjugate diluent was then added to each well. The plate was incubated at room temperature with shaking (150 RPM) for 30 minutes. Following this incubation the plate was washed 3 times in wash buffer. One hundred microliters of TMB working substrate solution was added to each well . The plate was incubated at room temperature for 30 minutes. Following the incubation 100 μ l of 0.18M H₂S0₄ Stop Solution was added to stop the reaction. The plate was read at 450 nm. Example 9: DNA Amptamers

Biotin labeled primer sequence (0.5 μM, final concentration) , FITC labeled primer sequence (0.1 μM, final concentration) and dilutions of 1:10, 1:100, and 1:1000 of a DNA amptamer library in PBS containing 10 mM MgCl₂ were mixed together. The reagents were preincubated for 30 minutes at room temperature. One hundred microliters of the above mixture was added to each well of a streptavidin coated microtiter plate. The plate was then incubated for 1 hour at room temperature. Following the incubation the plate was washed three times with PBS. One hundred microliters of rabbit anti-FITC-HRP diluted 1:50 in conjugate diluent was then added to each well . The plate was incubated at room temperature with shaking (150 RPM) for 30 minutes. Following this incubation the plate was washed 3 times in wash buffer PBS/Tween. One hundred microliters of OPD Substrate solution was added to each well and the plate was incubated at room temperature for 30 minutes. Fifty microliters of 2M H₂S0₄ Stop Solution was added to each well to stop the reaction. The plate was read at 490 nm.

Claims

What is claimed is:

1. A kit comprising:

(a) a universal solid phase; and

(b) a universal conjugate.

2. The kit of claim 1 wherein the universal solid phase comprises a streptavidin coated microtiter plate.

3. The kit of claim 1 wherein the universal conjugate comprises an anti-fluorescein antibody labeled with a detectable marker.

4. The kit of claim 1 further comprising a working substrate solution capable of reacting with the universal conjugate to produce a detectable signal.

5. The kit of claim 1 further comprising a means of derivatizing a first and a second derivatized reagent.

6. The kit of claim 1 further comprising a means for derivatizing a first derivatized reagent with biotin NHS ester.

7. The kit of claim 5 wherein the first derivatized reagent is selected from a group consisting of antibodies, haptens, peptides, proteins and nucleic acid probes.

8. The kit of claim 1 further comprising a reagent for derivatizing a second derivatized reagent in a sample with fluorescein isothiocyanate.

S . The kit of claim 7 wherein the second derivatized reagent is selected from a group consisting of antibodies, haptens, peptides, proteins and nucleic acid probes.

10. The kit of claim 1 further comprising a fluorescein isothiocyanate derivatized reagent capable of binding to a hapten, peptide, protein or antibody.

11. A kit comprising: (a) a universal solid phase onto which a first derivatized reagent may be immobilized; and

(b) a universal conjugate capable of detecting a second derivatized reagent.

12. A kit comprising a universal streptavidin coated plate and a universal anti-fluorescein isothiocyanate conjugate.

13. The kit of claim 11 further comprising a protein labelled with biotin-NHS ester and fluorescein isothiocyanate.

14. A kit for detecting a selected antigen comprising:

(a) a streptavidin coated surface;

(b) a first reagent for derivatizing a first antibody specific for said selected antigen with biotin NHS ester, the biotinylated antibody being capable of:

(i) binding to the streptavidin coated surface so that the biotinylated antibody is immobilized; and

(ii) binding the antigen in a sample placed in contact with the streptavidin coated surface; (c) a second reagent for derivatizing a second antibody specific for the selected antigen with isothiocyanate fluorescein, the isothiocyanate fluorescein derivatized antibody being capable of binding to the bound antigen; and (d) a third reagent containing anti- fluorescein isothiocyanate antibody capable of binding to the isothiocyanate fluorescein labeled antibody bound to the bound antigen.

15. The kit of claim 14 wherein the selected analyte comprises a cytokine.

16. The kit of claim 15 wherein the cytokine comprises IL-4.

17. A kit for detecting antibodies to a selected receptor comprising;

(a) a streptavidin coated surface; (b) a first reagent for derivatizing a selected receptor with biotin NHS ester, the biotinylated receptor being capable of :

(i) binding to the streptavidin coated surface so that the biotinylated receptor is immobilized; and (ii) binding the antibody to the immobilized receptor in a sample placed in contact with the streptavidin coated surface;

(c) a second reagent for derivatizing a second antibody specific for the selected receptor with isothiocyanate fluorescein, the isothiocyanate fluorescein labeled antibody being capable of binding to the immobilized receptor; and

(d) a third reagent containing anti- fluorescein isothiocyanate antibodies capable of detecting the isothiocyanate fluorescein derivatized antibody bound to the immobilized receptor.

18. The kit of claim 17 wherein the receptor comprises CD4.

19. A method for measuring a selected component in a sample comprising: (a) providing a universal solid phase for immobilizing a first derivatized reagent and a universal conjugate;

(b) attaching a derivatized moiety to a first derivatized reagent wherein the label facilitates immobilization of the first derivatized reagent to the universal solid phase;

(c) binding the first derivatized reagent to the universal solid phase so that the first derivatized reagent is immobilized;

(d) contacting a sample with the universal solid phase containing the immobilized first derivatized reagent so that a selected analyte component may bind to the immobilized first derivatized reagent; (e) derivatizing a second derivatized reagent with a derivative moiety capable of reacting with the universal conjugate to produce a detectable signal;

(f) contacting the second derivatized reagent with the universal solid phase so that the second derivatized reagent may bind to the selected component bound to the immobilized first derivatized reagent;

(g) contacting the universal conjugate to the universal solid phase containing the bound component; and

(h) detecting the bound component .

20. The method of claim 19 wherein the components being measured are selected from a group consisting of proteins, receptors, antigens, antibodies, viral proteins and nucleic acids .

21. The method of claim 19 wherein the universal solid phase in the provided kit comprises a streptavidin coated microtiter plate.

22. The method of claim 19 wherein the first derivatized reagent is labeled with biotin NHS ester.

23. The method of claim 19 wherein the first derivatized reagent is selected from a group consisting of antibodies, haptens, proteins and nucleic acid probes.

24. The method of claim 19 wherein the universal conjugate comprises fluorescein antibody labeled with a detectable marker.

25. The method of claim 20 wherein the second derivatized reagent is derivatized with fluorescein isothiocyanate.

26. The method of claim 19 wherein the selected component is detected by adding a reagent that reacts with the universal solid phase.

27. The method of claim 19 wherein the selected component is detected by adding a reagent that reacts with the universal conjugate.