WO1989007271A1

WO1989007271A1 - Immunoassays using monoclonal antibodies directed against natural binding proteins

Info

Publication number: WO1989007271A1
Application number: PCT/US1989/000342
Authority: WO
Inventors: Charles Walter Benner; Michael Terry Largen
Original assignee: E.I. Du Pont De Nemours And Company
Priority date: 1988-01-28
Filing date: 1989-01-27
Publication date: 1989-08-10
Also published as: EP0404783A4; GB9015666D0; JPH03503566A; GB2233091A; EP0404783A1; GB2233091B

Abstract

Monoclonal antibodies or immunoreactive monoclonal antibody fragments specific for naturally occurring binding proteins are used to form a monoclonal antibody-binding protein complex by which to detect a ligand in a variety of immuoassay formats. These antibodies or fragments can be labeled or unlabeled and when they are unlabeled then the unlabeled antibody or fragment is reacted with a labeled antibody specific for the unlabeled antibody or fragment. This approach preserves the inherent affinity of the binding protein for the ligand.

Description

TITLE IMMUNOASSAYS USING MONOCLONAL ANTIBODIES DIRECTED AGAINST NATURAL BINDING PROTEINS FIELD OF THE INVENTION This invention relates to immunoassays using monoclonal antibodies or immunoreactive monoclonal antibody fragments to non-immune natural binding proteins and a new method of labeling binding proteins.

BACKGROUND OF THE INVENTION Immunoassays for antigens are usually based on the binding of specific antibody to an antigen which might be present in a specimen and the subsequent identification of the antigen-antibody complex by means of a defined detection system. The performance characteristics of these systems depend to a great extent on the binding characteristics of the reactants utilized in the assay. It has been appreciated that other reactants such as non-immune natural binding proteins possess binding characteristics which make them more desirable to use in an assay format than monoclonal antibodies. Naturally occurring ligand-selective binding proteins, such as folate binding protein and intrinsic factor, are present in many biological systems and serve to transport or receive low molecular weight biomolecules by binding to them selectively and with affinity constants comparable to those of antibodies.

Commercial radioimmunoassay (hereinafter RIA) kits are available for folate and use folate binding protein as one of the reactants because it binds with the same affinity at pH 9.3 to both folic acid and the primary folic acid metabolite found in serum, 5-methyltetrahydrofolic acid (5MTHF) . This dual binding is important because the analyte of clinical interest is the primary folic acid metabolite, 5MTHF. Unfortunately, the instability of 5MTHF acid makes it difficult to use as a calibrator in assay formats. Since folic acid is stable, it is often used as a calibrator, thus allowing manufacturers to take advantage of folate binding protein's ability to bind both compounds at pH 9.3. 0 Furthermore, folate binding protein is commercially available which enables manufacturers to avoid the difficult task of generating an antibody of sufficiently high binding affinity to two or more structurally different compounds. 5 Similarly, B-12 RIAs employ intrinsic factor because it binds to all four major B-12 metabolites found in serum. The ready availability of intrinsic factor which has high affinity for all four major B-12 metabolites makes it easier to construct a commercial

₂₀ RIA kit for B-12. Indeed, many naturally occurring binding proteins have affinities greater than the affinities possessed by antibodies. Another advantage to working with binding proteins is that binding proteins will not recognize the bridging group of

_₅ hapten conjugates commonly used in enzyme immunoassays. None of the commercial RIAs utilize monoclonal antibodies and binding protein in a single assay format, these assays only use binding protein and radiolabeled ligand. Moreover, to date, no

₃₀ commercial enzyme immunoassays employing non-immune naturally occurring binding proteins are available.

There is an enzyme-linked competitive immunoasεay which has been described for folate using unlabeled folate binding protein (FBP) and folate

_₅ substituted glucose-6-phosphate dehydrogenaεe in two different assay formats. One format utilizes solubilized FBP as discussed by L. G. Bachas et al.. Homogeneous Enzyme-Linked Competitive Binding Assay for the Rapid Determination of Folate in Vitamin ₅ Tablets, Analytical Chemistry, Vol. 58, No. 4, pp.

956-961 (1986) . The other format employs immobilized FBP as discussed by L.G. Bachas et al. in Cooperative Interaction of Immobilized Folate Binding Protein with Enzyme-Folate Conjugates: An Enzyme-Linked Assay for ₀ Folate, Analytical Chemistry, Vol. 56, No. 9, pp.

1723-1726 (1984) . These assays have a limited range and the assay utilizing immobilized FBP exhibits a rather unique biphasic (hooked) dose response curve which makes analyte concentrations difficult to ₅ determine in that assay range.

U.S. Patent No. 4,271,140 (hereinafter '140) and PCT/GB85/00120 disclose the use of binding proteins and antibodies in a single assay format. The '140 reference describes the use of chemically odi- _Q fied binding proteins in an immunoassay which employs a complex of the structure A^*BL(^B*^L*)n^Al wherein BL is a binding ligand which is covalently bound to Ai. PCT International Application Number PCT/GB85/00120, which was published on October 10, 5 1985, describes the use of monoclonal antibodies capable of recognizing a complex of a small molecule and a binding protein against the small molecule wherein the monoclonal antibody is not an antibody against the small molecule or the binding protein. _Q Thus, these antibodies do not recognize binding protein unless it has bound ligand.

None of the references nor the commercial radioimmunoassay kits teach the instant invention which utilizes monoclonal antibodies or immunoreactive monoclonal antibody fragments specific for natural binding proteins in a single assay format. The binding protein is indirectly labeled via the attachment of a labeled monoclonal antibody specific for the binding protein. This eliminates the need for - direct chemical modification of the binding protein which can alter the binding protein's ability to bind ligand. Thus, monoclonal antibody-binding protein complex provides an alternative to existing binding protein labeling techniques which rely on chemical

₁₀ modification to prepare specifically labeled binding proteins suitable for use in immunoassays. This also provides an improvement over existing immunoassays by preserving the binding capability of the binding protein.

15. SUMMARY OF THE INVENTION Monoclonal antibodies or immunoreactive monoclonal antibody fragments specific for naturally occurring binding proteins are used to form a monoclonal antibody-binding protein complex by which

20 to detect a ligand in a variety of immunoassay formats. These antibodies or fragments can be labeled or unlabeled and when they are unlabeled then the unlabeled antibody or fragment is reacted with a labeled antibody specific for the unlabeled antibody

25 or fragment. This approach preserves the inherent affinity of the binding protein for the ligand. DETAILED DESCRIPTION OF THE INVENTION Techniques for preparing monoclonal antibodies of the present invention are well known and

3₀ have been cited in a wide variety of publications, the following of which are incorporated by reference: Kohler and Milstein, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity'⁷, Nature, 256: 495-497 (1975), Pereira et al., Infection

₃5 and Immunity, Vol. 29, No.2, pp. 724-732 (Aug. 1980), Oi et al., Immunoglobulin producing cell lines, pp. 351-371 in B. Mishell and S. Schiigi (ed.). Selected methods in cellular immunology, . H. Freeman Co., San Francisco (1980) and Galfre et al.. Preparation of Monoclonal Antibodies: Strategies and Procedures, Methods in Enzymology 73: 1-46 (1981). The term monoclonal antibody as used throughout embraces both intact monoclonal antibodies as well as immunoreactive monoclonal antibody fragments.

Monoclonal antibodies which bind with naturally occurring binding proteins such as those binding proteins illustrated in Table 1 below, and others can be produced and isolated using any of the above techniques for producing monoclonal antibodies. These antibodies can be purified and labeled using well known purification and protein conjugation techniques.

Table 1

Analyte Class Examples Specific Analyte

Vitamins Folate binding protein Folate, 5MTHF Intrinsic factor B12 and metabolites Riboflavin binding protein Riboflavin R protein B12 and metabolites

Hormones Cortisol binding globulin Cortisol Thyroxine binding globulin Thyroxine (T₄. , Triidothyxonine (T₃)

Androgen binding protein Androgens Glucocorticoid receptor Glucocorticoids

Drugs Cyclophilin Cyclosporin

Penicillin binding proteins Beta lactams

Amino Acids Perlplasmic binding proteins Most amino acids (Bacteria)

Cellular Receptors Epidermal Growth Factor (EGF) receptor EGF Interleu ln (I ) 2 receptor IL-2 Table 1 (continued)

Carcinogens Methylcholanthrene binding protein Met-hylcholanthrei-e Benzopyrene binding protein Benzopyrene

Allosteric Proteins cAHP protein kinase cAHP

Bihydrofolate reductase Ket_hotrexat_t

Carbohydrates Perlplasmic binding proteins Many sugars

(Bacteria.)

Leetins Many complex carbohydrates

.The monoclonal antibodies may be labeled with a component of a reporter system as defined below or with a member of a specific binding pair to which a component of a reporter system is attached.

Specific binding pairs may be of the immune or non-immune type. Immune specific binding pairs are exemplified by antigen-antibody systems or hapten- anti-hapten systems. There can be mentioned fluorescein/anti-fluorescein, dinitrophenyl/anti- dinitrophenyl, biotin/anti-biotin,peptide/anti-peptide and the like. The antibody member of the specific binding pair may be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, i.e., a hapten, it may be covalently coupled to a carrier protein to render it immunogenic.

Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies, for example, biotin-avidin, protein A-IgG and protein G-IgG.

A variety of methods are available to covalently label monoclonal antibodies with members of specific binding pairs. Methods are selected based upon the nature of the member of the specific binding pair, the type of linkage desired and the tolerance of

the antibody to various conjugation chemistries. Biotin may be covalently coupled to monoclonal antibodies by utilizing commercially available active derivatives. Some of these are biotin-N-hydroxy- succinimide which binds to amine groups on proteins; biotin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which both bind to sulfhydryl groups. Fluorescein can be coupled to protein amine groups using fluorescein isothiocyanate. Other standard methods of conjugation may be employed to couple monoclonal antibodies to a member of a specific binding pair including dialdehyde, carbodiimide coupling, homobifunctional crosεlinking, and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another. Carbodiimide coupling is facilitated by using the commercially available reagent, l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDAC) .

Homobifunctional crosslinkers, including the bifunctional imidoesters and bifunctional N-hydroxy- succinimide esters, are commercially available and are employed for coupling amine groups on one substance to amine groups on another. Heterobifunctional crosslinkers are reagents which possess different functional groups. The most common commercially available heterobifunctional crosslinkers have an amine reactive N-hydroxysuccinimide ester as one functional group and a sulfhydryl reactive group as the second functional group. The most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens. One of the functional groups may be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups. In order to facilitate signal detection, either the monoclonal antibodies or a member of a specific binding pair is labeled with a component of a reporter system. The term reporter system refers to the reporter selected and any means of linking the reporter to the monoclonal antibody or to a member of a specific binding pair which in turn is attached to the monoclonal antibody. Thus, a reporter may be linked directly or indirectly, covalently or non-covalently to the monoclonal antibodies or to a member of a specific binding pair. Reporters include, but are not limited to, radioactive isotopes, enzymes, metal sols of metals or metal compounds such as metal oxides, metal hydroxides and metal salts or polymer nuclei coated with a metal or metal compound, particles which alter the permittivity, conductivity or magnetic permeability of a surface, fluorogenic, chemiluminescent, or electrochemical materials. Two commonly used radioisotopes are 125χ _and 3JJ. Standard radioactive isotopic labeling procedures include the chlora ine T, lactoperoxidase and Bolton-Hunter methods for 125χ _an(j reductive methylation for ³H.

Enzymes which are also used as reporters for immunoassays include, but are not limited to, horseradish peroxidase, alkaline phosphatase, BETA-galactosidase, glucose oxidase, luciferase, BETA-lactamase, urease and lysozyme. Labeling with enzymes is facilitated by using dialdehyde, carbodiimide coupling, homobifunctional crosslinkers and heterobifunctional crosslinkers as described above for labeling monoclonal antibodies with members of specific binding pairs. The labeling method chosen depends on the functional groups available on the enzyme and the material to be labeled, and the tolerance of both to the conjugation conditions. The labeling method used in the present invention may be one of, but not limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistry 8, 871 (1971), Avrameas and Ternynck, Immunochemistry 8, 1175 (1971), Ishikawa et al., J. Immunoassay 4(3): 209-327 (1983) and Jablonski, Anal. Biochem. 148: 199 (1985). Labeling may be accomplished by indirect methods such as using spacers or other members of specific binding pairs. An example of this is the detection of biotinylated antibodies with unlabeled εtreptavidin and biotinylated enzyme, with the unlabeled streptavidin and biotinylated enzyme being added either secjuentially or simultaneously. Detection of enzyme activity may be facilitated by measuring chromogenic, magnetic, fluorogenic, chemiluminescent or electrochemical changes or by any other methods commonly known in the art.

Also within the scope of this invention there can be mentioned anti-antibodies specific to the monoclonal antibodies wherein the anti-antibodies are used to generate the signal and can be labeled using any of the above-mentioned labels and conjugation methods.

Monoclonal antibodies to naturally occurring binding proteins which fall within the scope of this invention include but are not limited to the following functionalities: 1) antibodies which recognize binding protein and do not interfere with binding of ligand by the binding protein; and

2) antibodies which recognize binding protein only when ligand is not bound. With respect to antibodies having functionality 2 as described above, these antibodies may recognize the active site or may recognize a distinct site which is altered following binding of ligand.

Important to the practice of this invention are judicious screening experiments that allow for the discrimination of hybrido a clones which produce monoclonal antibodies against binding proteinε having the foregoing functionalitieε as discuεsed below. See, E. A. Pierce et al., A Radiometric Immunosorbent Assay for the Detection of Anti-Hormone-Binding _ Protein Antibodies, Analytical Biochemistry, 153: 67-74 1986) and D. 0. Morgan, Plate Binding Assay for Monoclonal Anti-receptor antibodies. Endocrinology, 116(3): 1224-1226 (1985) which are hereby incorporated by reference. In a preferred embodiment, the invention can be practiced with monoclonal antibodies having the functionality of recognizing binding protein and not interfering with binding of ligand by the binding protein. Monoclonals of this type can be prepared by immunizing appropriate animals, such as mice or rats, with purified or partially purified binding protein. Purified binding protein is not required and, in fact, it is not even necessary to know the identity of the binding protein due to the nature of the monoclonal antibody screening procedure as described below.

Animals are immunized using conventional immunization procedures. This usually includes primary immunization with binding protein in complete Freund's adjuvant (CFA) and one or more booster immunizations utilizing binding protein emulsified in incomplete Freund's adjuvant or binding protein in phosphate-buffered saline. Following a booster immunization given one to four days preceding fusion, lymphocytes from lymph nodes or spleen are fused with a suitable non-immunoglobulin secreting myeloma according to well established procedures such as that of Kohler and Milstein referenced above. Following ten days or more of incubation in medium containing a mixture of hypoxanthine, a inopterin and thymidine (HAT) , supernatants from individual hybrid clones secreting monoclonal antibodies are screened in a solid state radioimmunosorbent assay (RISA, see Figure 1 below) . This assay consists of rabbit or goat anti-mouse IgG antibodies coated onto the wells of eight or twelve well break apart well strips. Supernatants containing antibodies from hybrid clones to be screened are placed individually into the wells and allowed to incubate for one hour at 37^βC. The wells are then washed and a solution containing pure or partially pure binding protein (BP) , e.g. folate binding protein (FBP) , is added to each well. Following incubation for an additional hour at 37°C, the well is washed extensively. Those supernatants containing antibody with sufficiently high affinity for the binding protein will now have indirectly bound the binding protein to the well. By adding radiolabeled binding ligand, e.g. folate, and incubating, those wells containing antibodies to the binding protein which do not interfere with the binding protein's ability to bind folate are detected. 1) Surface-anti IgG ψ Hybridoma Supernatant 2) Surface-anti IgG o Hybridoma Supernatant

1 BP

3) Surface-anti IgG o Hybridoma Supernatant o BP ψ Radiolabeled ligand (RL)

4) Surface-anti IgG o Hybridoma Supernatant o BP o RL Figure 1 In addition to the above-described RISA, a direct ELISA, enzyme-linked immunosorbent assay, (Figure 2) can be performed in which the purified binding protein is bound to polystyrene plates. This is a standard ELISA hybridoma screening assay. In the direct assay hybridoma supernatants presumably containing antibodies to binding protein are added to the wells, incubated, and following subsequent washing a second antibody directed against mouse antibodies (to which an enzyme or radioactive label is chemically attached) . Following incubation and washing substrate is added and antibody bound to the wells is determined.

1) Surface-BP Hybridoma Supernatant

2) Surface-BP o Hybridoma Supernatant

J, anti IgG-enzyme-labeled

3) Surface-BP o Hybridoma Supernatant o Anti IgG- enzyme labeled Figure 2

An indirect ELISA (Figure 3) can also be performed in which ligand (hapten) is chemically coupled to a carrier protein such as BSA and this hapten-carrier is bound to polystyrene plates. In this indirect screening assay, binding protein is added to the well which contains ligand conjugated to carrier protein to which the binding protein can bind by binding to ligand. Following incubation and washing hybridoma supernatants are added and if the appropriate type antibody with specificity for the binding protein is present the monoclonal antibody will be bound and can be detected as described for the direct assay. 1) Surface-analyte carrier - BP

2) Surface-analyte carrier o BP Hybridoma Supernatant

3) Surface-analyte carrier o BP o Hybridoma

Supernatant

Anti IgG-enzyme labeled

4) Surface-analyte carrier o BP o Hybridoma o Anti

Supernatant IgG- enzyme labeled Figure 3

The preferred class of antibodies, which bind to binding protein irrespective of whether ligand is bound and which do not interfere with subsequent binding of ligand antibodies, are expected to produce a positive result in the RISA as well as in the direct and indirect ELISA assays. Antibodies presumed to bind binding protein only when ligand is not bound would be expected to be positive in the direct ELISA and RISA and would be expected to be negative in the indirect ELISA. Based upon these primary screening results antibodies can be further characterized as to their abilities to react with binding protein under the various conditions of analyte binding.

Having generated monoclonal antibodies with the requisite functionalities, they can then be used with the appropriate binding protein to measure the ligands of interest. A variety of assay formats can be used to practice the invention. Some exemplary formats include, but are not limited to, sandwich assays and affinity column-mediated immunoassay (ACMIA) as discussed in U.S. Patent 4,551,426 in which one can utilize monoclonal antibodies which do not interfere with binding of ligand to binding protein.

Application to an ACMIA type assay would involve mixing a solution of monoclonal antibody-binding protein complex with a solution that contains ligand recognized by the binding protein followed by a brief period of incubation to bind the ligand to a predetermined amount. A solid surface which has ligand or ligand analog attached is added, and again the mixture is incubated. The complex that had not captured analyte will bind to immobilized ligand or ligand analog, i.e., something that acts like the ligand but is not the ligand. After separating the immobilized and aqueous phases, either supernatant or immobilized phase is measured for the label's activity.

An example of a sandwich assay using monoclonal antibodies which recognize binding protein and do not interefere with binding of ligand by binding protein is illustrated in Figure 4 below.

Yl = anti-FBP monoclonal antibody which recognizes BP and does not interfere with binding of ligand;

Y2 = anti-FBP monoclonal antibody which recognizes BP when ligand is not bound;

1) Surface-Yi * FBP * FA + Surface-Yχ * FBP

Y2-label

2) Surface-Yi * FBP * FA + Surface-Yχ * FBP * Y -label

Figure 4

This invention provides an important alternative to chemical modification means of labeling

binding protein because chemical modification can reduce the binding protein's ability to recognize and bind ligand. Increased dynamic range of the assay is also obtained by carefully controlling the ratio of labeled antibody to binding protein as illustrated in Examples 1 and 2. Signal generation is proportional to this ratio. The binding protein concentration in an assay can be held constant at a value optimized for analyte capture efficiency and the required signal is

10 optimized by titration of the antibody conjugate as is illustrated in Example 3. Increased signal and sensitivity are also obtained by employing monoclonal antibody conjugates which recognize different epitopes on the binding protein. Furthermore, two or more

15 labels can be attached to one binding protein by using selectively labeled monoclonal antibodies which recognize the identical or different epitopes on the binding protein.

Use of monoclonal antibodies allows use of _₀ heterogeneous, impure preparations of binding protein in both the immunization procedure and assay. Clones are selected that satisfy the criteria of screening experiments one to three set forth above.

The following examples are intended to

25 illustrate the invention and should not be construed as limitations thereon.

EXAMPLE 1

Folic Acid (FA) Assay Using Preformed FBP Anti-FBP-Alkaline Phosphatase Complex 0 and Human Serum Samples

Reagents:

A. Anti-FBP-Alkaline Phosphatase Conjugate: 1. Sulfhydryl modification of alkaline phosphatase. To 11.2 g, 8.0 E-8 moles of alkaline 5 phosphatase (AP) (Boehringer Mannheim EIA grade) was added 8.4 E-4 g, 4.8 E-6 moles, of S-acetylsuccinic anhydride (Sigma) in 20 μl of dimethylforma ide (DMF) . The reaction was magnetically stirred at room temperature for one hour followed by addition of 40 μl of an aqueous, pH 7.0, 1.45 molar solution of hydroxylamine (Aldrich) . The solution was magnetically stirred for 0.5 hour at room temperature and purified by size exclusion chromatography using a 1.5 x 30 cm column packed with Sephadex G-25 (Pharmacia) eluted with 100 mM sodium phosphate, 1.0 mM EDTA, pH 6.5. The fractions containing the sulfhydryl modified alkaline phosphatase were pooled and the concentration was determined by ultraviolet spectroscopy using an extinction coefficient at 280 nm of 1.0 mL/(mg cm). Final concentration of chemically modified alkaline phosphatase was 1.2 mg/mL.

2. Maleimidyl modification of anti-FBP monoclonal antibody. To 1.0 mg, 6.7 E-9 moles, of anti-FBP monoclonal antibody in 1.9 L phosphate buffer saline (PBS), pH 7.4, was added 8.0 molar equivalents, 1.8 E-5 g of succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxylate (Pierce Chemical Co.) in 6.7 μl> DMF. The reaction solution was magnetically stirred for 1.5 hours at room temperature and purified by size exclusion chromatography using a 1.5 x 30 cm column packed with Sephadex G-25 (Pharmacia) eluted with 100 mM sodium phosphate, pH 6.5. The fractions containing the chemically modified anti-FBP monoclonal were pooled and the protein concentration was determined using an extinction coefficient at 280 nm of 1.4 mL/(mg cm). The volume recovered was 8.2 mL, concentration of the chemically modified antibody was 0.09 mg/mL.

3. 1.6 mL of the chemically modified alkaline phosphatase solution and 8.2 mL of the modified anti-FBP monoclonal antibody solution were combined and allowed to stand at 4^βC for one hour. 13 uL of a 10 mM solution of 2-mercaptoethylamine hydrochloride in 100 mM sodium phosphate was added. The solution was stored at 4^βC for one hour, concentrated by ultrafiltration and dialyzed against 10 mM Tris, 1.0 mM MgCl2, 0.1 mM ZnCl2« The conjugate was diluted 1:2 with an aqueous buffer solution composed of 10 mM Tris, 10% mannitol, 5% fraction V BSA, 150 mM NaCl, 1.0 mM MgCl2, 0.1 mM ZnCl2 0.3% 2-chloroacetamide, 0.2% sodium azide, 0.05% thimerisol, 0.05% Tween-20, pH 8.1, and stored at 4°C. Approximate concentration of conjugate — 0.1 mg/mL.

B. Bovine folate binding protein (FBP) in 120 mM Na Phosphate, 30 mM NaCl (Biochemicals Inc.), specific activity = 18 icrogram of folate binding ability per mL.

C. FBP anti-FBP-AP complex was prepared by combining equal volumes of FBP diluted 1:1000 with 50 mM sodium tetraborate, 150 mM NaCl, 1.0 mM MgCl2, 0.1 mM ZnCl2, 0.1% rabbit IgG (Sigma, used as a stabilizer), pH 9.3, and anti-FBP-AP conjugate diluted 1:200 with the same buffer.

D. Crθ2-IgG-FA reagent, 10 mg/mL in PBS, was prepared by coating chromium dioxide particles with covalently bound folic acid rabbit IgG (IgG-FA) conjugate. (See U.S. Patent 4,661,408, issued April 21, 1987 to Lau et al., hereby incorporated by reference.) The IgG-FA conjugate was prepared as discussed below using standard methods known in the art for attaching carboxylic acid functionalized compounds to proteins containing amine groups. The rabbit IgG (Sigma) was used as an inert carrier protein and not for its i munological properties. E. Human serum samples obtained from Massachusetts General Hospital which were supplied with clinically determined values for folate.

F. Wash buffer consisting of 10 mM Tris, 0.05% Tween-20, 150 mM NaCl, 0.3%

2-chloroacetamide, pH 7.0.

G. 2.4 M DEA buffer, pH 9.1.

H. 15 mM 4-methylumbelliferyl phosphate (MUP) in 0.1 N NaOH. I. 0.5 M EDTA, pH 8.9.

J. 50 mM sodium tetraborate, 150 mM NaCl, 1.0 mM

MgCl2, 0:1 mM ZnCl₂, pH 9.3. Assay Procedure:

1. 100 μL of a normal human serum sample was placed in a 12 x 75 mm polypropylene test tube and

500 μl> of reagent J was added. The sample was heated at 100 ^~ C for 15 minutes, then cooled to room temperatur .

2. 100 μL of complex (reagent C) was added followed by addition of 25 μL of reagent D.. The reaction solution was vortexed and placed in a 37^βC bath for 21 minutes. The solid phase was magnetically separated at room temperature and the supernatant was discarded. The solid phase was washed three times with 500 μL of reagent F.

3. 250 μL of reagent G was added to the solid phase, the solution was heated to 37^βC and 50 μL of reagent H was added. The solution was vortexed and incubated for 4 minutes at 37'C. 300 μL of reagent I was added, the solid phase was separated at room temperature and the fluorescent intensity of this solution was determined after diluting 1:5 with reagent I. The results appear in Table 2. 19

Table 2

0

Utilization of the FBP anti-FBP-AP complex in an immunoassay for the detection of folic acid in human serum samples has provided positive correlation with clinically determined values. The complex binds both ₅ folic acid in solution and folic acid covalently attached to a solid surface. Under the prescribed experimental conditions there is a competition of the complex for soluble and immobilized folic acid. Assay signal is inversely proportional to the concentration o °f sample 5-methyltetrahydrofolic acid.

EXAMPLE 2 Folic Acid Assay Using Preformed FBP Anti-FBP-AP

Complex and Folic Acid Standard Solutions

Reagents: 5 1. The reagents were identical to those in Example 1 with one noted exception. Standard solutions of folic acid were used in place of human serum samples. The standard solutions were prepared by diluting folic acid dihydrate to 20, 10, 5, 1 ng/mL with PBS, 5% human serum albumin (HSA) , 0.3%

2-chloroacetamide, pH 7.4. The diluent was used as a

0 ng/mL standard.

Procedure:

1. As outlined in Example 1. The results appear in Table 3.

The data in Tables 2 and 3 show an inverse relationship between folic acid concentration and signal strength. The magnitude of the fluorescent intensity differs in the two examples due to dissimilarities of solution matrices.

EXAMPLE 3 Signal Variation as a Function of Anti-FBP-AP Conjugate Titer

Reagents:

A. Anti-FBP-AP conjugate: See reagent A, Example 1.

B. Bovine folate binding protein: See reagent B, Example 1.

C. FBP anti-FBP complex: Three FBP anti-FBP-AP solutions were prepared using different

FBP anti-FBP-AP conjugate εtoichiometric ratios. Equal volumes of FBP diluted 1:3222 and anti-FBP-AP conjugate diluted 1:200, 1:400 and 1:800 were combined to make three solutions of complex with decreasing titer of conjugate. Dilutions were made using 50 mM sodium tetraborate, 150 mM NaCl, 1.0 mM MgCl , 0.1 mM ZnCl2_/ 10% mannitol, 0.3% 2-chloroacetamide, 0.1% triton X-100, 5% BSA, pH 9.3.

D. Cr0₂-IgG-FA: 0.25 mg/mL, see reagent D, Example 1.

E. Standard solutions of folic acid prepared in PBS, 2.5% BSA and 0.3% 2-chloroacetamide to final concentrations of 10 and 0 ng/mL.

F. Wash buffer: See reagent F, Example 1.

G. 2.4 M DEA, pH 9.0.

H. 200 mM para-nitrophenyl phosphate, 0.1 N NaOH. I. 150 mM phosphate buffer, pH 7.8. Assay Procedure:

1. 50 μL of the 0 and 10 ng/mL folic acid solutions were added to polypropylene test tubes in triplicate.

2. 100 μL of complex solution were added to the folic acid standards, the solutions were vortexed and incubated at 37"C for five minutes.

3. 25 μL of reagent D were added and the solutionε were incubated at 37°C for an additional 6 minuteε 40 εecondε.

4. 325 μL of reagent F were added, the εolid phase was magnetically separated at room temperature and the supernatant was discarded. The solid phase was washed twice with 500 μL of reagent F.

5. 559 μL of reagent G were added to each tube, the solutions were brought to 37°C followed by addition of 21 μL of reagent H. The samples were incubated for 10 minutes at 37°C and the reactions were quenched by addition of 600 μL of reagent I.

6. The solid phase was magnetically separated at room temperature. The absorbance of each εolution at 405 nm was determined. The results appear in Table 4. Table 4

Aεεay Signal Variation with Anti-FBP-AP Titer

Dilution of Abεorbance 404 nm favg,

Anti-FBP-AP Dilution of FBP [FAI-P ng/mL 10 ng/mL

1:200 1:3222 3.10 0.54

1:400 1:3222 2.00 0.42

1:800 1:3222 1.09 0.28

Example 3 demonstrates signal generation is proportional to the binding protein/antibody conjugate ratio. The signal desired over a given assay range can be optimized by titration of the antibody conjugate.

Claims

1. A method of labeling a non-immune naturally occurring binding protein comprising reacting the protein with at leaεt one first monoclonal antibody which may be labeled or unlabeled and which is specific for the binding protein and thereafter if the first antibody is unlabeled, reacting said first unlabeled antibody with a labeled antibody to the firεt antibody.

2. A method according to claim 1 wherein said binding proteins are binding proteins to analytes selected from the group consisting of vitamins, hormones, drugs, amino acids, cellular receptors, carcinogens, allosteric proteinε, and carbohydrates.

3. A method according to claim 1 wherein the monoclonal antibodies are selected from the group consisting of antibodies that recognize binding protein and do not interfere with binding of ligand by the binding protein and antibodies that preferentially recognize binding protein when no ligand is bound.

4. A method according to claim 1 wherein the monoclonal antibodies are labeled with a component of a reporter system selected from the group consisting of radioactive isotopes, enzymes, stable free radicalε, metal sols, magnetic, fluoroge^"nic, chemiluminescent and electrochemical materials.

5. A method according to claim 1 wherein the labeled antibodies have attached thereto a first member of an immune binding pair or a non-immune binding pair wherein said first member is reacted with a second member of the binding pair and further wherein said second member is labeled with a component of a reporter syεtem.

6. A method according to claim 5 where the component of the reporter system is selected from the group consiεting of radioactive isotopes, enzymes, εtable free radicalε, metal sols, magnetic, fluorogenic, chemiluminescent and electrochemical materials.

7. An immunoassay to detect or quantitate a ligand which binds to a non-immune, naturally occurring binding protein compriεing: a) incubating a εample εuspected or known to contain the ligand with a naturally occurring binding protein for that ligand, and b) determining the extent of reaction between the ligand and binding protein, characterized in that the binding protein is reacted with at least one monoclonal antibody which may be labeled or unlabeled and which is specific for the binding protein and when the antibody iε unlabeled then reacting εaid unlabeled monoclonal antibody with a labeled antibody to the unlabeled antibody, the extent of reaction between the ligand and binding protein iε determined by meaεuring the amount of bound or unbound monoclonal antibody.

8. An i munσaεsay according to claim 7 wherein said binding protein is a binding protein to an analyte selected from the group consisting of vitamins, hormones, drugε, amino acidε, cellular receptorε, carcinogens, allosteric proteins, and carbohydrates.

9. An immunoaεεay according to claim 7 wherein the monoclonal antibody or monoclonal antibodieε are selected from the group conεiεting of antibodieε that recognize binding protein and do not interfere with binding of ligand by the binding protein and antibodieε that preferentially recognize binding protein when no ligand is bound.

10. An immunoassay according to claim 7 wherein the monoclonal antibody or monoclonal antibodies may be labeled with a component of a reporter syεte εelected from the group conεiεting of radioactive iεotopes, enzymes, stable free radicals, metal sols, magnetic, fluorogenic, chemiluminescent and electrochemical materials.

11. An immunoassay according to claim 7 wherein the labeled antibody or antibodies have attached thereto a first member of an immune binding pair or a non-immune binding pair wherein εaid firεt member is reacted with a second member of the binding pair and further wherein said εecond member is labeled with a component of a reporter system.

12. An immunoasεay according to claim 11 wherein the component of the reporter system is εelected from the group conεisting of radioactive isotopes, enzymes, stable free radicals, metal sols, magnetic, fluorogenic, chemiluminescent and electrochemical materials.

13. An immunoasεay according to claim 7 wherein a) a first antibody, a sample suspected or known to contain the ligand and a naturally occurring binding protein for that ligand are incubated, and b) the extent of reaction between the ligand and binding protein is determined, characterized in that the binding protein is reacted with a second monoclonal antibody which may be labeled or unlabeled and which is specific for the binding protein and when the second antibody is unlabeled then reacting said unlabeled second antibody with a labeled antibody to the unlabeled second antibody, the extent of reaction between the ligand and binding protein is determined by measuring the amount of bound or unbound second monoclonal antibody.

14. An immunoasεay according to claim 13 wherein εaid aεεay iε conducted as a sequential

₅ forward aεεay or a εimultaneouε asεay.

15. An immunoasεay according to claim 13 wherein the first antibody is immobilized.

16. An immunoasεay according to claim 13 wherein εaid binding protein iε a binding protein to ₀ an analyte selected from the group consisting of vitamins, hormones, drugs, amino acids, cellular receptors, carcinogens, allosteric proteinε, and carbohydrateε.

17. An immunoasεay according to claim 13 ₅ wherein the monoclonal antibodieε are εelected from the group consiεting of antibodies that recognize binding protein and do not interfere with binding of ligand by the binding protein, and antibodies that preferentially recognize binding protein when no o ligand is bound.

18. An immunoassay according to claim 13 wherein the monoclonal antibodies are labeled with a component of a reporter system selected from the group consisting of immune binding pairs, non-immune binding ₅ pairs, radioactive iεotopeε, enzymeε, stable free radicalε, metal εolε, magnetic, fluorogenic, chemilumineεcent and electrochemical materials.

19. An immunoassay according to claim 13 wherein the labeled antibody or antibodies have ₀ attached thereto a first member of an immune binding pair or a non-immune binding pair wherein εaid firεt member iε reacted with a second member of the binding pair and further wherein said second member is labeled with a component of a reporter system. 5

20. An immunoasεay according to claim 19 wherein the component of the reporter εystem is selected from the group consisting of radioactive isotopeε, enzymeε, εtable free radicalε, metal sols, magnetic, fluorogenic, chemiluminescent and electrochemical materials.

21. An immunoassay according to claim 7 wherein: a) immobilized ligand or ligand analog, sample εuεpected or known to contain the ligand and a naturally occurring binding protein for that ligand are incubated, characterized in that the binding protein iε reacted with at least one monoclonal antibody which may be labeled or unlabeled and which is specific for the binding protein and when the monoclonal antibody is unlabeled then the unlabeled monoclonal antibody is reacted with a labeled antibody to the unlabeled monoclonal antibody, the extent of reaction between the ligand and binding protein iε determined by meaεuring the amount of bound or unbound monoclonal antibody.

22. An i munoaεεay according to claim 21 wherein εaid assay is conducted as a sequential forward assay or a simultaneous assay.

23. An immunoassay according to claim 21 wherein said binding protein is a binding protein to analytes selected from the group consiεting of vitaminε, hormones, drugs, amino acids, cellular receptors, carcinogens, allosteric proteins, and carbohydrates.

24. An immunoasεay according to claim 21 wherein the monoclonal antibodies are selected from the group consiεting of antibodieε that recognize binding protein and do not interfere with binding of ligand by the binding protein, and antibodies that preferentially recognize binding protein when no ligand is bound.

25. An immunoassay according to claim 21

_ wherein the monoclonal antibodies are labeled with a 5 component of a reporter system selected from the group consiεting of immune binding pairε, non-immune binding pairs, radioactive isotopes, enzymes, stable free radicals, metal sols, magnetic, fluorogenic, 0 chemiluminescent and electrochemical materials.

26. An immunoasεay according to claim 21 wherein the monoclonal antibodieε have attached thereto a firεt member of an immune binding pair or a non-immune binding pair wherein εaid firεt member iε 5 reacted with a εecond member of the binding pair and further wherein εaid second member iε labeled with a component of a reporter system.

27. An immunoassay according to claim 26 wherein the monoclonal antibodies are labeled with a component of a reporter syεtem selected from the group consisting of radioactive isotopes, enzymes, stable free radicals, metal sols, magnetic, fluorogenic, chemiluminescent and electrochemical materials.

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