KR900008014B1 - Pepticle antibodies and their use in detecting oncogone products - Google Patents

Abstract

An antibody is claimed which binds selectively to a characteristic marker epitope of an oncogene prod., which epitope is not present in the corresp. proto-oncogene prod. Pref. the oncogene prod. is an activated form of P21 protein and the epitope encompasses position 12. Also claimed is a method for detecting a polypeptide or an oncogene prod. In a celluar sample of a patient, the polypeptide having a characteristic marker epitope which is not exposed in the undenatured polypeptide and the oncogene prod. having a characteristic marker epitope which is not present in the proto- oncogene prod. and which is not exposed in the undenatured protein.

Description

Method for producing peptide antibody

1 is a diagram showing the amino acid sequence of the p21 protein.

FIG. 2 is an autoradiogram showing an SDS-PAGE analysis of an immune complex.

3 is an autoradiogram showing the SDS-PAGE analysis of two p21 proteins.

4 shows an autoradiogram showing an SDS-PAGE analysis of an immune complex.

5 shows a plot of GTP binding to p21 protein.

6 is a photograph of cells transformed with V-ki-ras p21.

The present invention relates to a method for producing a peptide antibody for the detection of oncogene products.

More particularly, the present invention relates to the fields of immunology and cancer diagnosis and therapeutic applications, wherein the antibody has specificity for mutant oncogenes, not natural proto-oncogenes, and immunochemistry made from the antibodies. Drugs, and diagnostics and treatments using immunochemicals.

Carcinogenic genes are cytoplasmic genes that contribute to oncogenic transformation of cells (Weinberg, R., Sctentific American, 249: 126-142 (1983)). These are modified forms of normal cytoplasmic genes and have been detected in a wide range of human tumors and tumor cell lines. Oncogenic genes were detected by transfecting DNA of human tumors or tumor cell lines on a cell line known as NIH / 3 T3 cells, and the cells expressed by taking the oncogene from the transfected DNA expressed in morphologically modified and dense lesions. Begins to grow. Cells obtained from these lesions can form tumors when grown in mice. Lane, MA, Sainten, A. and Cooper, GM, Proc Natl Acad Sci USA, 78: 5185-5189 (1981): and Shilo B. and Weinberg, RA, Nature, 289: 607-609 (1981).

The p21 protein encoded by a group of ras-proto-carcinogens, when activated through somatic mutations, results in oncogenic transformation of certain cells in culture, which has caused various human cancers.

The frequency of activation of the ras gene in human tumors can be over 20%. From the structural comparison of the ras oncogenes, it was found that they are distinguished from their non-transgenic normal genes by a single mutation that modifies the amino acid sequence of the p21 protein, the gene product, at position 12 or position 61. Parada, LF, et al., Nature, 297: 474-479 (1982); Santos, E., et al., Nature, 298: 343-347 (1982); Yuasa, Y. et al., Nature, 303: 775-779 (1982). The most frequently observed mutation that activates the p21 protein as a carcinogenic gene transforms the amino acid residue at position 12 of the protein into one of the other residues. Marshall, C.J. Et al., Cancer Surveys, 3: 183-214 (1984).

The p21 protein is known to specifically bind GTP and hydrolyze GTP to GDP and inorganic phosphates. Shih, T.Y. Et al., Nature, 287: 686-691 (1980), McGrath, J.P. Et al., Nature, 310: 664-649 (1984) and Swelt, R. W. et al., Nature, 311: 273-275 (1984). In the form of a p21 protein containing a threonine residue at position 59, the phosphate migrates to a threonine residue by an autophosphorylation reaction. McGrath et al., Supra, Gibbs, J.B. Et al., P.N.A.S. USA, 81: 2674-2678 (1984) and Shih T.Y. Et al., Nature (London) 287: 686-691 (1980). The role of these actions in the transformation potential of activated p21 is not yet known. However, it has been speculated that some of the p21 protein near position 12 may be involved in the interaction of p21 protein with GTP. Gay, N.T. Et al., Nature, 301: 262-264 (1983) and Weirenga, R.K. Nature, 302: 842-844 (1983). It has been found that polyclonal serum and monoclonal antibodies that modulate various p21 determinants allow GTP binding (Furth, M. et al., J. Virol., 43: 294-304 (1982)). Monoclonal antibody Y13-259 does not distinguish between normal p21 protein and activated p21 protein.

European Patent Publication No. 108,564 to Cline et al. Describes the use of probes, such as antibodies, to detect expression products of the c-onc gene, including the ras carcinogen, in diagnosing the presence of malignancies. . This publication does not distinguish between wild-type and mutant of polypeptides. U.S. Patent Application No. 369,517 (filed April 1, 1982, by T. Papas et al., Titled invention: Expression of retroviral myc gene in human tumor cells) detects human tumor cells. A cloned recombinant DNA probe labeled with 32p containing the myc oncogene sequence of avian myelocytomatosis virus strain (MC29) is used. Various blood cells have been found to produce significant amounts of RNA homologous to the myc gene sequence of MC29.

Peptide segments that closely correspond to the amino acid sequences of the region of interest of the protein under study can be chemically synthesized, and the peptides can be combined with carrier proteins and introduced into the appropriate host to yield antibodies. For example, Alexander et al. Describe two leukocyte proteins (Thy-1) that differ only in one amino acid and its specific antiserum, derived from a simply chemically synthesized peptide leading to a sequence modification region. (Nature, 306: 697-699 (1983)). Document due to Tamura, T. There, a method for the pp60 _src immunoprecipitated using antibodies and of generating antibodies to the synthetic oligopeptide corresponding to the primary structure of pp60 _src have been described [see: Cell, 34: 587 -596 (1983)].

Lerner et al. Describe a method for using a peptide as a vaccine at a position where the peptide corresponds to an amino acid sequence estimated from the nucleotide sequence of a protein such as hepatitis B surface antigen. Proc. Natl. Acad Sci USA, 78: 3403-3407 (1981), Walter, G. et al., P.N.A.S. USA, 77: 5197-5200 (1980) and Lerner, R. A., Nature, 299: 592-596 (1982). Regarding the p21 protein, PCT WO / 84/01389, published April 12, 1984 by Weinberg et al, describes the difference between oncogenes of DNA isolated from human bladder cancer cells and their corresponding proto-oncogenes. have. As a form of experimental serum reagents, antibodies are described that are specific for p21 proteins expressed from proto-carcinogens or oncogenic genes that can be used to detect carcinogenesis. Antibodies may also be generated using chemically synthesized peptide fragments corresponding to amino acids of the critical region of the normal or modified peptide sequence under study.

Hand, H. et al. Describe a method for generating monoclonal antibodies using synthetic peptides reflecting amino acid sites 10-17 of the Hu-ras _T ²⁴ gene product. PNAS USA, 81: 5227-5231 (1984). The antibody was shown to react with the ras gene product p21, but showed no specificity to the activating p21 (modified at site 12) protein than the normal p21 protein.

In a publication published on Sep. 4, 1983 by Gallick, GE, et al., The temperature sensitivity of murine sarcoma virus in rats by microinjection into the cytoplasm of purified IgG from antisera produced against synthetic V-mos peptides. Transformation is inhibited in cell lines infected with mutant lines. Transformation of cells is induced by p85 _gag-mos fusion protein. Antibodies showed no specificity for activating (mutant) oncogene products.

Cancer Letter Vol. 9 describes the NIH RFP Scheme on the study of monoclonal antibodies against oncogene products of avian and mammalian retroviruses.

One difficulty in preparing antibodies that have specificity to single amino acid differences between p21 proteins is the synthesis of proteins that do not allow the antibody to bind to the specific amino acid epitopic site of interest. Another difficulty with the antibody regimens described above is that it is not possible to predict whether a given peptide fragment will be effective as an immunogen for the production of the required antibody. Thus there is a need in the art for methods of obtaining modified immunogenic proteins with peptide immunogens effective for proper antibody production and exposed epitopes to which antibodies can bind so that a single amino acid difference can be detected. In addition, it is necessary to determine the mechanism of p21 protein activation in order to develop effective therapies to prevent carcinogenic effects.

In accordance with the present invention, chemically synthesized peptides of constant amino acid sequence are used to prepare antibodies having specificity for single amino acid differences between proteins. These antibodies are characterized by their selective binding to characteristic marker epitopes of the oncogene product, wherein the epitope is not present in the corresponding proto-oncogene product. Preferably, the oncogene product is an activated form of p21 protein and the epitope surrounds site 12 thereof. "Epitope" in the text refers to the binding site of the oncogene product that recognizes the antibody.

In particular, the present invention provides an inhibitor that selectively binds a oncogene product to a region of the oncogene product containing a characteristic marker epitope of the oncogene product that is not present in the corresponding proto- oncogene product, and has a direct effect on this binding. And contacting with a carcinogen product to inhibit the oncogene product from binding to the cytoplasmic components necessary for carcinogenic action of the oncogene product. In a preferred embodiment, the cytoplasmic component is GTP and the inhibitor is an antibody and the oncogene product is the activated form of the p21 protein and the epitope surrounds site 12 thereof. Inhibitors that are compositions of matter also belong to the present invention.

Another embodiment of the present invention provides a method of contacting at least one cancer cell with a composition consisting of an antibody that selectively binds to a characteristic marker epitope of the oncogene product that at least partially causes cancer that is not present in the corresponding proto-oncogene product. The present invention relates to a method of treating cancer. One way to contact the cells is by injecting the antibody-containing composition into the cells.

FIG. 1 is a schematic showing the amino acid sequence of the p21 protein surrounding region 12 marked with X (site 12 of the protein corresponds to region 8 of the peptide). Peptides of amino acids 5 to 17 with cysteine residues inserted between sites 16 and 17 are used as immunogens to prepare the antibodies of the examples according to the invention.

Figure 2 shows the immunocomplex of the V-ki-ras p21 protein containing affinity purified anti-p21 serum directed against gly, ser or val at site 12 and serine residues expressed by E. coli. Autoradiogram showing SDS-PAGE analysis. Shown on the left represents the control.

FIG. 3 is an autoradiogram showing SDS-PAGE analysis of two p21 proteins with ser and arg amino acid substituents at site 12 from transformed mammalian cells immunoprecipitated with anti-P21 _ser antibodies. The two shown on the left in each group are the controls and the one on the right is the molecular weight standard. The second labeled pre-immune shown on the right is immunoprecipitated rabbit serum prior to injection.

4 shows E. coli expressing V-ki-ras p21 with monoclonal antibody Y13-259 (denoted as anti-p21 MAB in the figure), which is known to have failed to inhibit GTP hydrolysis by the anti-p21 _ser , or p21 protein Autoradiogram showing SDS-PAGE analysis of immune complex labeled with gamma labeled GTP of (E. coli). Marked A was immunoprecipitated prior to phosphorylation and marked B was immunoprecipitated after phosphorylation.

FIG. 5 binds to p21 protein as a percentage of control (without antibody) relative to the amount of anti-p21 _ser antibody added to immunoglobulin in non-immunized normal rabbits, or V-ki-ras p21 immunoprecipitated with Y13-259 A plot of GTP is shown.

6 shows a photograph of cells transformed with V-ki-ras p21 injected with anti-p21 _ser antibody (FIGS. 6a to 6c) or immunoglobulin (FIGS. 6d to 6f) of an immunized normal rabbit as a control. Indicates. Figures 6a and 6d represent 0 hours of injection, 6b and 6e represent 24 hours of injection, and 6c and 6f represent 36 hours of injection. 6A-6C show the progress of cells from the transformed (carcinogenic) state to the untransformed (noncarcinogenic, normal) state.

In one particular embodiment according to the present invention the detection of the product of the RAS oncogene in human cells is due to the ability of a particular antibody to distinguish proteins that differ by a single amino acid. Antibodies that can distinguish between normal products of the ras gene (p21 protein) and mutant activation forms through single amino acid differences at sites 12 or 61 include immunofluorescence, immunoperoxidase staining, immunoprecipitation, ELISA or Western blotting. Can be used to detect ras oncogene products by standard techniques such as

As used herein, the term "epitope" refers to the binding site of a carcinogenic product that recognizes an antibody of the invention.

The term "antibody" refers to both polyclonal and monoclonal antibodies. The term also includes whole immunoglobulins and antigen binding fragments thereof. Polyclonal antibodies are directed to host animals, such as rabbits, rats, sheep, mice, etc., to produce peptides or peptide fragments encoded by oncogenes containing amino acid (s) that distinguish mutant proteins from native proteins. Can be created by injection The serum is extracted from the host animal and screened to obtain polyclonal antibodies specific for the peptide immunogen. Monoclonal antibodies can be produced, for example, by immunizing mice with the peptides described above. Mice are inoculated intraperitoneally with an immunized amount of the peptide and then boosted with an analogous amount of the immunological peptide. Spleens are collected from the immunized mice a few days after the last second inoculation and from which cell suspensions are prepared for fusion.

Hybridomas can be prepared from tumor partners in splenocytes and rats using kohler, B. and Milstein, C. general somatic hybridization techniques (Nature (1975) 256: 495-497). For example, a rat myeloma cell line, available from the Salk Institute of the Cell Home Care Center (San Diego, CA, USA), can be used for hybridization. Basically, the technique involves fusing tumor cells and splenocytes using a fusogen such as polyethylene glycol. After fusion the cells are separated from the fusion medium and grown in a selective growth medium such as HAT medium to remove unhybridized parental cells. In some cases, hybridomas can be expanded, and the supernatant can be tested by conventional immunoassay procedures (eg radioimmunoassay, enzyme immunoassay or fluorescence immunoassay) using an immunoreagent such as an antigen. Positive clones can be characterized to determine whether they meet the antibody criteria of the present invention.

Hybridomas that produce the antibodies described above can be grown in vitro or in vivo using known processes. Monoclonal antibodies can be separated from the culture medium or body fluid, if desired, by conventional immunoglobulin purification processes such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration.

As used herein, the term “partially denatured” protein has a characteristic marker epitope that has not been exposed to a characteristic non-denatured protein and is partially denatured or sufficiently relaxed to expose the epitope region to the antibody of the invention but at the same time solution Refers to a protein that remains intact and hardly inhibits antigen-antibody binding. Examples of protein alteration reagents that can be used for this purpose include urea, deoxycholate, guanidine hydrochloride, sodium dodecyl sulfate, and the like.

The binding of the antibody to the protein can be enhanced if a sufficiently high concentration of affinity purified antibody is used. Affinity tablets are well known in the art when the antigenic peptide binds to a carrier other than that used for immunoassay and the antibody is stagnated through the carrier.

The poptide used to obtain an antibody against p21 serologically useful as an immunogen comprises a variable amino acid at site 12, a sufficient amount of side residues defining the characteristic marker epitope to which the antibody selectively binds, and the second position at the C-terminus of the peptide. It consists of cysteine residues in. The peptide may be contacted with a carrier protein such as keyhole limpet hemocyanin or bovine serum albumin through cysteine residues before being injected into the host.

The peptide fragment should have enough amino acid residues to define that it is an epitope of the protein fragment to be detected, but not so large as to have a constant conformation different from that of the protein to be detected. If the peptide fragment is too short, the fragment turns out to be inappropriate for other proteins, which may be physically buried in the immune carrier protein. The second cysteine residue at the C-terminus is used to link the carrier protein to the peptide fragment. Figure 1 depicts one fragment useful for the p21 protein.

Carcinogen products that can be used herein are structurally modified (mutated) oncogene products other than proto-carcinogen products. Examples of such oncogene products include cytoplasmic gene products consisting of ras oncogene products (P21 _CH-ras and P21 _CK-ras ), myc oncogene products (P58 _C-myc ), sis oncogene products (PDGF B-chain), etc. There is this. Preferably the oncogene product in the body is an activated form of p21 protein. The term "activation" as used in a protein product refers to the replacement of DNA, which in turn alters the protein to cause oncogenic transformation.

Peptide antiserum against p21 protein representing an antibody according to the invention can be prepared as follows. The normal amino acid glycine found at site 12 of the native p21 protein is replaced at the same position with each possible amino acid resulting from a single base change of the codon, corresponding to amino acid sites 5 to 17 of the p21 RAS gene product. Synthetic peptides are prepared. These amino acids are serine, arginine, cysteine, valine, alanine and aspartic acid. In FIG. 1 the amino acid sequence for the relevant portion of the p21 protein at sites 5 to 17 is described. The peptide produced is a tetradecapeptide which should contain an additional cysteine residue second in the C-terminal serine terminus coupled to the carrier protein. The result of each amino acid change at site 12 is indicated in the right column. The peptides can be synthesized by solid phase synthesis known in the art described by Merrifield in Example 1 using an automated SAM peptide synthesizer and a manual solid phase synthesis device, "peptider". Liquid hydrogen fluoride is used to decompose the peptide from the resin used in the synthesis and purify by column chromatography. After synthesis, peptides are covalently coupled to carrier proteins such as clam hemocyanin (KLH) or bovine serum albumin (BSA) via cysteine residues at the C-terminus and then injected into rabbits.

Antibody titers are determined using an ELISA protocol, where an Imulon plate is coated with each peptide covalently coupled to a carrier protein used for the immunopeptide and another carrier protein to allow screening of the antibody to be performed. Let's do it. The process of isolating site 12-specific antibodies from nonspecific antibodies is performed by passing the serum on an affinity column to which the peptide BSA complex is covalently attached. In the case of antisera produced against a peptide having serine at site 12, the peptide used in the affinity column has glycine at the same site. As determined by ELISA, the antibody that failed to bind to the affinity column described above has specificity for serine at site 12.

The ability of the resulting anti-peptide antibody to distinguish amino acid substitutions at site 12 of the total protein is tested using immunoprecipitation under partial alteration conditions of the p21 protein. Thus, the antibody and protein are mixed and incubated for 0.5 to 1 hour at pH 7.5 to 8.0 in the presence of protein denatured buffer containing SDS and / or deoxyclate. More generally, protein alteration conditions are known, but for the purposes of the present invention an SDS concentration of about 0.05 to 0.10% by weight at a physiological PH, such as at a pH of about 7.5 to 9, preferably 8 and / or Or about 0.5 to 1 hour using a deoxycholate concentration of about 0.1 to 1% by weight, preferably 0.5 to 1% by weight. These conditions depend on the specific carcinogenic product used and the purity of the antibody.

After immunoprecipitation, immune complexes are collected on Protein A Sepharose, washed and analyzed by SDS-PAGE.

In the present specification, the antibody may be used as an immunochemical for therapeutic and diagnostic purposes. As a therapeutic antibody attached to a carrier can be inserted into a tumor and reverse its malignant state.

Antibodies that can distinguish RAS oncogenes from their normal genes by single amino acid differences in the gene product (p21 protein) can be used for the diagnostic detection of malignant cells in various clinical situations. For example, the presence of RAS oncogenes may have prognostic significance in determining the course of treatment for certain types of cancer. In another situation, the detection of RAS oncogenes in malignant cells may be a useful clue for the monitoring of disease processes and their early discovery by facilitating the detection of these cells in many normal cells. The primary diagnostic use of anti-p21 antibodies is that they can be used as immunohistochemicals or as serum markers for the detection of any kind of cancer, including c-ras mutants.

Chemically important derivatives of the antibodies according to the invention, which are of critical importance, are labeled (e.g., radiolabeled, enzyme-labeled, or fluorochrome-labeled) which provide a means for identifying immune complexes comprising labeled antibodies. ) Derivatives. Whether an antibody can act in a therapeutic form such as an immunotoxin depends on what protein the antigen is.

The label used to prepare the labeling portion of the antibody includes residues such as enzymes and directly detectable residues such as fluorochromes and radiolabels, which must be reacted or induced to be detected. Examples of such labels include ³² P, ¹²⁵ I, ³ H, ¹⁴ C, fluorescence and derivatives thereof, rhodamine and derivatives thereof, dansyl, umbelliferon, luciferin, 2, 3-dihydrophthalazinedione, horseradish Peroxidase, alkaline phosphatase, β-galactosidase, lysozyme, and glucose-6-phosphate dehydrogenase. The above label is attached to the antibody by a known method. For example, coupling agents such as dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized benzidines and the like can be used to attach the above fluorescence, chemiluminescence and enzyme labels to the antibody.

Antibodies and labeled antibodies can be used in a variety of immunoassay processes to detect the presence of cancer in the human body or to monitor the condition of cancer in patients already suffering from cancer. When used to monitor the condition of cancer, a quantitative immunoassay process should be used. In such monitoring, tests are performed periodically to compare the results to determine if the tumor size of the host has increased or decreased. Common test techniques that can be used include direct and indirect tests. Direct testing is the incubation of tissue samples or cells from a host with one or more labeled antibodies in vivo and in vitro. If the sample contains cancer cells, the labeled antibody will bind to these cells. After washing the tissue or cells to remove unbound labeled antibody, the presence of the labeled immune complex is read from the tissue sample. In indirect studies, tissue or cell samples are incubated with one or more unlabeled antibodies. The sample is then treated with a labeled antibody (e.g., labeled anti-rabbit antibody) against the antibody and washed and the presence of the labeled tricomponent complex is read.

In an in vitro test for detecting cancer in a bodily fluid sample, the sample can be incubated with an antibody against an unlabeled protein, preferably an antibody immobilized on a solid support. Samples are incubated with other antibodies labeled before, during or after incubation. After washing, the presence of the labeled complex is read for the sample. Both antibodies used may be against p21 (different epitopes) or the second antibody may be against the first antibody.

The antibodies can also be used in competitive RIA formats.

For diagnostic purposes the antibody is typically distributed in kit form. These kits typically comprise antibodies in appropriate containers or antibodies in altered or unlabeled form, alteration buffers, culture reagents and washes if necessary, labeled anti-rabbit antibodies if the kit is for indirect testing, and substrates according to the nature of the label. Or inducing agent. Human cancer antigen controls and indicators may also be included. The antibodies in the kit can also be affinity purified.

The high specificity of antibodies to specific oncogenic products, along with their ability to modulate the biochemical action of oncogenic products, allows these antibodies to study the structure of oncogene products and their role in in vitro and in vivo tumorigenesis. Makes it a very useful probe. Potentially antibodies can be used to inhibit the action of oncogene products in complexes and to explore the interaction of oncogene products with other cytoplasmic components in in vitro systems.

The antibody can also be used for cancer treatment. For example, these and other suitable pharmaceutical compositions or medicaments may be used as inhibitors in the process of inhibiting the binding of the cellular components necessary for carcinogenicity of the oncogene product to the oncogene product. In this process, the oncogene product as described above is contacted with an inhibitor capable of inhibiting the binding of the oncogene product to the cytoplasmic component. The specificity inhibitor used depends, for example, on the oncogene product and the cytoplasmic components. If the oncogene product is a mutant of the proto-oncogene product, the inhibitor will selectively bind to a characteristic marker epitope of the oncogene product, which is not present in the corresponding proto-oncogene product. Inhibitors should also exert a direct effect on the binding of cellular components to oncogene products. This direct effect may be that the epitope comprises at least a portion of the site where the cytosolic component binds to the oncogene product, or the inhibitor alters binding by disturbing the three-component structure of the oncogene product by binding or acting at a certain distance from the epitope. have. The present invention is not bound by any particular theory of how inhibitors act on binding.

The cytoplasmic components required for the function of the oncogene product may be, for example, proteins, lipids, carbohydrates, nucleic acids, metabolites such as nucleotides (eg ATP, CTP, GTP, UTP) and the like. The specific components of the cells required for the function of the oncogene product depend on the product. For example, in the case of an activated p21 protein, the cytoplasmic component is GTP, which binds to the activated p21 protein and makes it a carcinogen. Different proteins have specificity for the same or different cytoplasmic components.

Preferably the inhibitor is an antibody, which may be an affinity purified polyclonal antibody, and the oncogene product is an activating p21 protein with a characteristic marker epitope surrounding region 12 of the protein.

Antibodies can be used, in particular, in the treatment of cancer patients, wherein the process selectively binds one or more cancer cells to a characteristic marker epitope of a carcinogenic product that at least partially causes cancer, which is not present in the corresponding proto-oncogene product. It is made to contact with the composition which consists of antibodies. The antibody may be attached to a carrier molecule that allows the antibody to pass freely through the cell membrane without the need for contact or infusion with the cells by microinjection. Carrier molecules for this purpose may be hydrophobic or similar or perhaps identical to the B-chain of lysine, which pulls the active A-chain of lysine through the cell membrane. Preferably the oncogene product of the process is an activated form of p21 protein with a marker epitope that surrounds region 12 of the protein.

The invention is illustrated by the following examples in which all ratios are by weight and temperatures are in degrees Celsius unless otherwise noted.

Example 1

A. Peptide Synthesis

SAM peptide synthesizer (Biosearch Corp., Calif.), Wherein the tetradecapeptides shown at the bottom of FIG. ) And "peptides" (Peninsula Laboratories), a passive solid phase synthesis instrument.

Solid phase synthesis methods used herein are described in Merrifield, RB, Adv. Enzymol. Relat, Areas Mol. Biol., 32: 221-296 (1969) and “The chemistry of Polypeptides” (PGkatsoyannis). ed.), pp. 336-361, Plenum, New York (1973). The experimental part of this technique is described in the following literature (Stewart. J.M. et al., "Solid Phase Peptide Synthesis", Freeman, San Francisco, California (1969)).

The synthesis is carried out using a commercially available N-tert-butyloxycarbonyl-L-serine (O-belzyl ether) covalently attached to polystyrene resin by benzyl ester as C-terminal amino acid. The synthesis deprotects the protecting group from the α-amino group with trifluoroacetic acid, neutralizes the trifluoroacetate salt with diisopropylethylamine and then converts the N-tert-butyloxycarbonyl-protected amino acid to Iterative circulation method of coupling to bodyimide. All amino acid derivatives and reagents used are commercially available.

Mass synthesis of the resin is necessary during synthesis, ie after each reaction (deprotection, neutralization and coupling). The resin is washed with suitable organic solvents such as dichloromethane, dioxane and dimethylformamide.

Cysteine is not naturally produced at the 16 or 17 sites from the amino terminus of the p21 protein, but is inserted between the sites described above to allow subsequent attachment of the peptide necessary for immunity to the protein carrier.

Peptides are degraded from the resin by liquid hydrogen fluoride added with a small amount of anisole. The degraded peptide is extracted from the resin using 2N aqueous acetic acid and lyophilized. 1 g of the dried peptide-resin yields 339 mg of crude peptide.

For purification, the peptide (100 mg) is treated with dithiothreitol (30 mg) in 5 ml of 2N acetic acid solution and chromatographed with 2N acetic acid on an LH Sephadex column with a fill volume of 150 ml. Fractions containing large amounts of peptide are combined and lyophilized. A total of 56 mg of peptide is recovered.

Homogeneity of the peptide is studied from the amino acid composition of the hydrolyzate of the peptide by reverse phase HPLC.

Conjugation of these peptides to clam hemocyanin (KLH) or bovine serum albumin (BSA) is via a sulfohydryl group of cysteine residues. N-maleimido-6-amino caproyl ester of 1-hydroxy-2-nitrobenzene-4-sulfonic acid sodium salt, which is a heterodifunctional crosslinker, is prepared by the following process.

1 molar equivalent (2.24 g) of 4-hydroxy-4-nitrobenzene sulfonic acid sodium salt (HNSA) in 1 molar equivalent (2.06 g) of dicyclohexyl carbodiimide in 25 ml of dimethylformamide (DMF) and 1 Molar equivalents (2.10 g) of N-maleimido-6-aminocaproic acid are mixed overnight at room temperature. A white precipitate of dicyclohexyl urea is formed. The precipitate is filtered off and 300 ml of diethyl ether is added to the mother liquor. After about 10 minutes to 4 hours a gum-like solid precipitated out of the mother liquor. The solid was found to contain 58% active HNSA ester and 42% free HNSA.

The analysis is made by dissolving a small amount of precipitate in phosphate buffer at pH 7.0 and measuring the absorbance at 406 mm, giving an unreacted amount of free HNSA that is a contaminant in the HNSA ester formulation. Additional very small amounts of strong strong bases (eg 5N NaOH) immediately hydrolyze the esters formed and perform secondary reads therefrom. The primary yield is subtracted from the secondary yield to obtain the amount of ester in the original material. The solid is then dissolved in DMF, left on an LH20 Sephadex column and eluted with DMF to separate the ester from the contaminating free HNSA. Purification is monitored by thin layer chromatography using chloroform, acetone and acetic acid (6: 3: 1 volume / volume) as eluent. The product was identified as mal-sac HNSA ester by its reactivity with amines. The yield of pure esters was estimated at approximately 30% of theory, and the purified material consisted of 99% esters.

The obtained ester is completely dissolved in water and stable for several hours in the condition that no nucleophile is added. When placed in 1N ammonia the ester yields the corresponding amide, some of which is hydrolyzed to free acid. Purified esters have been proven to be stable for a long time on dry storage.

Approximately 0.5 mg of purified mal-sac HNSA ester is dissolved in 1 ml of distilled water. 10 μl of the solution is diluted at pH 7.0 with 1 ml of 10 mM phosphate buffer. Absorbance at 406 nm is used to calculate the concentration of free HNSA as described above. When 50 μl of 4.8 N sodium hydroxide solution is added to the diluted ester and mixed, the absorbance of the solution at 406 nm increases significantly, indicating that the hydroxide nucleophile rapidly hydrolyzes the ester to the acid and free HNSA anion components. .

The difference between the post-base and the initial free HNSA concentration indicates the concentration of the ester. From the actual concentration of the ester and the glycoprotein amino group, one can calculate the amount of ester to be added to the protein solution to obtain the desired degree of substitution.

당 1.0×10^-2몰의 총아민 농도(대략 59리신/BSA분자)를 수득한다. 분말 형태의 상기 제조원 mal-sac HNSA 에스테르(97.7% 순도)의 계산량(11mg, 2.35×10^-5몰)을 2.0ml의 BSA 용액에 용해시킨다. 반응을 실온에서 수행한다. 10μl의 분취량을 용액으로부터 적당한 시간 간격으로 분리하여 각기 1.0ml의 0.01M 포스페이트 완충액으로 pH 7.0에서 희석시킨다. 각기 희석시킨 분취량의 스팩트럼은 Hewlett-Packard) 분광광도계를 사용하여 기록하고 406nm에서의 흡수도를 기록한다. 이어서 총 50μl의 4.8N NaOH를 각 분취량에 가하여 각 분취량을 혼합시키고 이의 스펙트럼을 재차 기록하고 406nm에서의 흡수도를 측정한다. 결과는 표 1에 기재되어 있다.The purified HNSA ester is then reacted with BSA as follows (the reaction with KLH is similar to this process): 22 mg (20 μmol) total and BSA (molecular weight 66, 296) are dissolved in 2.0 ml 0.1 M phosphate buffer at pH 7.5. Let

A total amine concentration of about 1.0 × 10 ⁻² moles of sugar (approximately 59 lysine / BSA molecules) is obtained. A calculated amount (11 mg, 2.35 × 10 ⁻⁵ mol) of the above-mentioned mal-sac HNSA ester (97.7% purity) in powder form is dissolved in 2.0 ml of BSA solution. The reaction is carried out at room temperature. Aliquots of 10 μl are separated from the solution at appropriate time intervals and diluted at pH 7.0 with 1.0 ml of 0.01 M phosphate buffer, respectively. Aliquots of each diluted aliquot were recorded using a Hewlett-Packard spectrophotometer and the absorbance at 406 nm. A total of 50 μl of 4.8 N NaOH is then added to each aliquot to mix each aliquot and record its spectrum again and measure the absorbance at 406 nm. The results are shown in Table 1.

TABLE 1

From the absorbance at 406 nm before and after base addition, the concentration of the residual ester and the ratio of the reaction ester are measured for the reaction mixture. The results show that the reaction rate is nearly straight over 15 minutes.

When the reaction time is 15 minutes, the reaction mixture is placed in a pp10 desalted Sephadex G-25 column (pharmacia, Inc.) equilibrated with 0.1 M phosphate buffer at pH 6.0 to stop the reaction. It was found that 2.6 × 10 ⁻³ mol / l of ester reacted and thus 25.9% of 59 epsilon-amino groups of BSA were probably substituted. The product thus contains 16 mal-sac groups per molecule.

The first reaction product mal-sac-BAS (or mal-sac-KLH) is separated by adding the reaction mixture to a pp10 desalted Sephadex G-25 column equilibrated with 0.1 M phosphate buffer at pH 6.0. The column is eluted in 1.0 ml fractions using 0.1 M phosphate buffer. After column elution the absorption spectrum is monitored and peak fractions containing mal-sac BSA are collected. 18 mg of cysteine-containing tetradecapeptide is synthesized as described above and the combined mixture is stirred overnight at room temperature. Conjugates are mass dialyzed and lyophilized against distilled water and, in some cases, changes in amino acid composition are analyzed.

B. Preparation of Antisera

New Zealand Baeksan rabbits are immunized with 1/2 mg of KLH derivative of peptides by peripheral lymph node injection in Freund's complete adjuvant and 2 weeks later subcutaneously injected with 1/2 mg of the substance in Freund's incomplete adjuvant. Subcutaneous injection of an additional 1/2 mg of the material is performed every three weeks. One month later, three intravenous doses of 1/2 mg each are given for 4 days and the serum samples used in this study are taken 11 days later.

C. ELISA test for the determination of antibody titers

96-well Imulon I plates are coated overnight at 4 ° C. with 50 μl (per well) of 250 μg / ml solution of the appropriate BSA-peptide conjugate. After washing the wells with PBS-Tween, dilutions of the antiserum to be tested are added and incubated for 2 to 3 hours at room temperature. After further washing with PBS-Tween, both anti-rabbit IgG (peroxidase conjugates) were added at a dilution of 1/1000 and incubated for 1 hour at room temperature. It is then further washed with PBS-Tween and peroxidase substrate is added (ABTS, Sigma) and read after 30 minutes.

D. Affinity Purification of Antibodies

Isolation of site 12-specific antibodies from non-specific antibodies is accomplished by passing serum on a Reacti-Gel affinity column to which the peptide BSA conjugate is covalently attached. In the case of antisera produced against a peptide having serine at site 12, the peptide used in the affinity column has glycine at that site. As determined by ELISA, antibodies that failed to bind the affinity column described above have specificity for serine at site 12.

The effluent from the column is passed through a secondary column to which the peptide with serine at site 12 is covalently attached. Antibodies that bind this column are eluted with 50 mM glycine.HCl at pH 2.5, dialyzed against 1/20 × phosphate buffered saline (PBS) and concentrated by centrifugation under vacuum to give a final concentrate of 10 mg / ml in PBS. To obtain.

E. Immunoprecipitation of p21 Protein by Antibodies

The ability of anti-peptide antiserum to immunoprecipitate p21 protein is shown in Figure 2, which is an autoradiogram of an SDS-PAGE gel.

In this example, the antibody is used to immunoprecipitate a form of p21 protein known to contain serine at site 12. The protein is E. Coli, which expresses the V-Ki-ras p21 protein [which is produced using plasmid pINIII-ras prepared by M. Inouge of SUNY of Stony Brook, New York, where transcription is lac Directed by a promoter—see Masui, Y. et al., In Experimental Manipulation of Gene Expression, M. Inouge, ed., Academic Press, NY, pp 15-32 (1983). Expression of the protein is induced by treating the culture with 2 mM isopropylthiogalactoside (IPTG). The trimo pellet is resuspended in 50 mM glucose, 25 mM Tris.HCl, 10 mM EDTA, 2 mg / ml lysozyme, and 0.01% aprotinin at pH 8.0 and then incubated at 4 ° C. for 30 minutes. Triton × -100 is added to the 1% concentrate for 5 minutes. After 15,000 × g spin for 10 minutes, the supernatant is spun for another 2 hours at 150,000 × g to obtain clear lysate. 100 μl of clear cell lysate samples are incubated for 30 minutes at 37 ° C. with 40 μCi ³² p gamma-labeled guanosine triphosphate (GTP). 0.2 μg of control monoclonal antibody Y13-259, 10 μl of anti-p21 ^ser , 10 μl of anti-p21 ^val , or 10 μl of anti-p21 ^gly (the latter serum is shown in FIG. X as shown in the figure is generated against peptides that are valine or glycine, respectively).

The culture is carried out in the presence of a partially altered buffer consisting of 0.05% SDS, 2 mM dithiothreitol, 0.5% NP 40, 50 mM Tris.HCl and 120 mM NaCl, pH 8.0. After 1 hour the immune complexes are collected on 10 μl of protein A Sepharose beads pre-coated with affinity-economyd sheep anti-rat IgG in reaction with Y13-259 and murine IgG. Immune complexes are washed with 0.5% NP 40, 1M LiCl, 50mM Tris HCl, and 120mM NaCl, pH 8.0 and analyzed by SDS-PAGE. The obtained gel was dried on a piece of paper and then placed on a piece of film to obtain a second figure by a standard autoradiograph process, which shows the analysis result. The control on the left is the monoclonal antibody Y13-259, which is known to immunoprecipitate this specific form of p21 protein (obtained by Mark Furth of the Sloan-kettering Institute, NYNew York First Ave.). Furth , M. et al., J. Virol., 43: 294-304 (1982). FIG. 2 shows that it is produced against serine-containing peptides of the only three tested sera that react with this form of p21 protein. Thus, antibodies can distinguish single amino acid changes in proteins.

In another experiment on immunoprecipitation, mammalian cells transformed with Harvey sarcoma virus DNA (V-Ha ras) or kirsten sarcoma virus DNA (V-Ki-ras) are used as feed of p21 antigen. The former cell (Rat-2 fibroblast line named kp6) was prepared by Dr. Mike Kreigler of Fox-Chase Cancer Institue, PA 19111 Philadelphia Burholme Ave. 7701. The latter cells (K-balb) were obtained as ATCC CCL 163.3 (K-234) from the American Type Culture Collection of Rockville Parklawn Drive 12301, MD 20852-1776, and Kirsten rat sarcoma virus-transformed Balb / 3T3 nonproducer ( nonproducer). The latter culture is infected with an ecotropic strain of rat leukemia virus to increase the expression of V-Ki-ras p21.

A single total of these transformed cells was metabolized for 4 hours at 37 ° C. with 2 μCi of carrier-free inorganic phosphate per 50 mm plate, and the extract was treated with surfactant PBS + 1% Triton X-100, detergent 0.5% Prepared in 0.01% aprotinin, an oxycholate and protease inhibitor. An aliquot of the extract containing V-Ha-ras p21 (with arginine at site 12) or V-Ki-ras p21 (with serine at site 12) was 0.25 μg of Y13- as a control for 1 hour at room temperature. 259 monoclonal antibodies, 0.25 μg rat IgG, 5 μg preimmunized rabbit serum, or 5 μg affinity purified anti-p21^serAntisera [ie anti-p21 generated against peptide having serine at site 12 and isolated from site 12 nonspecific antibody using affinity chromatography as described above ser antibody, wherein the peptide used in the affinity column has glycine as the amino acid corresponding to site 12 of the p21 protein. The reaction is carried out using the partially altered conditions as described above, except that the amount of SDS used is 0.1% by weight, not 0.05% by weight. Immune complexes are collected on Protein A Sepharose, washed and analyzed by SDS-PAGE using standard autoradiograph processes as described in FIG. In Figure 3, the autoradiogram, the rightmost control is the molecular weight standard, the control on the left is rabbit serum before immunization, and the two left on the left are the monoclonal antibodies Y13-259 and Rat IgG, respectively, as controls. . Figure 3 demonstrates that serum produced against serine-containing peptides can immunoprecipitate p21 of Kitsten sarcoma virus with serine at site 12, but Harvey sarcoma virus p21 with arginine residues at the same site cannot immunoprecipitate. do. The same antiserum was found not to react with the p21 protein containing glycine at site 12. There was no immunoprecipitation in the absence of SDS or deoxyclate partial alteration.

F. Suppression of autophosphorylation of V-Ki-ras p21 by anti-p21 ^ser antibody

Anti-p21 ^ser antibodies have also been shown to inhibit the autophosphorylation of the V-Ki ras carcinogenic gene product in vitro experiments, suggesting that some of the p21 protein surrounding site 12 is involved in the interaction of p21 protein with GTP. Indicates.

The extract from E. coli expressing V-Ki-ras p21 was immunoprecipitated with the anti-p21 ^ser antibody or Y13-259 monoclonal antibody as described above, and the resulting immune complex was subjected to 10 μCi ³² p gamma. Incubate for 30 minutes at 37 ° C with labeled GTP. Immune complexes are collected on Protein A Sepharose, washed and analyzed by SDS-PAGE and then introduced into standard autoradiograph methods as described above. The results indicate that autophosphorylation does not occur in anti-p21 ^ser immune complexes. In contrast, E.Coli-producing p21 immunoprecipitated with Y13-259 monoclonal antibody was actively phosphorylated (see Figure 4a, which is an autoradiogram of both materials). The use of pre-labeled p21, which has been efficiently immunoprecipitated under conditions such as lack of autophosphorylation, inhibits enzymatic activity but fails in serum binding at p21.

A total of 25 μl of extracts from E.Coli expressing V-Ki-ras were subjected to 100 mM NaCl, 5 mM MgCl ₂ and 50 mM Tris. Primary adjustment with HCl (pH 8.0) and the resulting immune complexes are incubated for 30 minutes at 37 ° C. with 1010 μCi gamma-labeled GTP. It is then immunoprecipitated with anti-P21 _ser or Y13-259 monoclonal antibodies under the conditions described above. Immunoprecipitates were placed on SDS-PAGE and introduced into the autoradiograph as described above to obtain FIG. 4b.

Figure 4a shows that GTP-dependent autophosphorylation of E.Coli-producing p21 ^ser antibodies was completely eliminated after immunoprecipitation by anti-p21 ^ser antibodies. In contrast, the p21 protein is efficiently autophosphorylated after immunoprecipitation by monoclonal antibody Y13-259 in the control group, which has been found to fail to inhibit GTP hydrolysis by the p21 protein. However, if p21 is phosphorylated before immunoprecipitation (Fig. 4b), it can be seen that the two antibodies efficiently precipitate the labeled p21 protein.

Thus binding of anti-p21 ^ser to V-Ki-ras p21 results in inhibition of p21 autophosphorylation. The most likely explanation for this result is that the antibody competes with GTP for binding to the same site.

G. Inhibition of GTP Binding by Anti-p21 ^ser Immunoglobulins

In order to confirm the above results and determine whether the antibody inhibits binding of GTP rather than transferring phosphate to threonine at site 59, Applicants used a monoclonal antibody-linked immunosorbent assay for p21 protein. The ability of anti-p21 ^ser antibodies to directly block the binding of α-32pdGTP was tested.

V-Ki-ras p21 generated in E. Coli is immunoprecipitated with 2 μl monoclonal antibody Y13-259 coupled to Reactigel beads purchased from Pierce Chemical Co. The immune complexes are washed with 0.5% NP 40, 50 mM Tris. HCl (pH 8.0), 1M LiCl and 120 ml NaCl. The washed complexes were anti-p21 ^ser antibody or non-immune normal in 0.5% Triton X-100, 0.5% deoxyclate, 150 mM NaCl, 50 mM Tris.HCl, pH 7.5, 5 mM MgCl ₂ , 0.1 mM ATP and 2 mM DTT. Incubate for 1 hour at 4 ° C. under rabbit immunoglobulin addition. The immunocomplex was washed and 4-10 μCi of alpha-labeled ³² p dGTP in GTP binding buffer consisting of 100 mM NaCl, 20 mM Tris.HCl, pH 7.2, 5 mM MgCl ₂ , 1% Triton X-100 and 0.1 mM ATP. Resuspend for 30 minutes at 4 ° C. under the addition of 0.2-0.52 μM, 800 Ci / mmol). The immune complexes are then washed in GTP binding buffer and counted with a liquid scintillation counter.

The results are shown in FIG. 5, which is a graph showing the concentration function of each antibody as a percentage of GTP binding to p21 protein against various classes of antibodies versus control (no antibody added). The points in the data represent five means of determination for the control without the antibody.

After treatment with anti-p21 ^ser antibody, GTP binding to immunoprecipitated p21 protein was reduced five-fold, while no decrease was seen in the control non-immune antibody. These results indicate that the anti-p21 ^ser binding site on the p21 protein overlaps with the GTP binding site. Alternatively, it can be explained that the antibody binds at a certain distance from the GTP binding site to change GTP binding by disturbing the three-component structure of the protein. The first explanation is that (1) Furth et al. Found that polyclonal serum and monoclonal antibodies against various p21 determinants allow GTP binding (see Supra) and (2) high concentrations (10 mM) under mild altered conditions. This is even more possible due to Applicants' finding that GTP (rather than ATP) actually inhibits the binding of anti-p21 ^ser antibodies to the p21 protein.

The present invention is not limited to any description.

The results raise the question of how changes in the amino acid at site 12 activate the p21 protein oncogene. Blocking GTP binding by an antibody recognizing region 12 strongly suggests that the amino acid is involved in the interaction of p21 with GTP and that a change in interaction can directly contribute to the oncogenic potential of the protein.

Example 2

Anti-p21 serine was injected into normal rat kidney (NRK) cells transformed with Kirsten sarcoma virus purchased from ATCC of Accession No. ATCC CCL 163.3 (K-234) as described above with 5 mg of affinity purified antibody per ml of solution. Micro injection. Kirsten sarcoma virus transforms cells by producing p21 with serine at site 12. After 12 hours of injection, the injected cells appear to have changed morphologically and resemble normal, untransformed NRK cells. After 30 hours the cells are returned to their fully transformed state. Non-immune normal rabbit antibodies as a control show no effect.

In addition, injection of anti-p21 serine antibody into cells transformed with p21 having arginine at site 12 has no effect. This indicates that the anti-p21 serine antibody inhibits the transformed phenotype (ie the known biochemical properties of the protein in vivo) while not the non-immune normal antibody.

The micro-injection experiment described above is repeated. In the new experiment, the injected cells changed morphologically 24 hours after injection (Figure 6a shows cells at 0 hours of injection) (Figure 6b). After 36 hours of injection, the injected cells change even more so that they resemble NRK cells as they were without normal transformation (see Figure 6c). Figures 6d-6f show that normal non-immune rabbit antibodies as controls have no effect on the transformed cells (6d is 0 hours, 6e is 24 hours, 6f is 36 hours, respectively). Eventually the cell returns to its fully transformed state but they are still alive.

In summary, the present invention provides an anti-peptide antiserum (antibody) capable of distinguishing amino acid substituents located at a single amino acid site of a protein.

Thus, the antibody can be used for detecting the presence of oncogene products that cause cancer in the human body or for therapeutic applications of cancer. The antibody contains amino acids 5 to 17 of the normal p21 protein, except that glycine at site 12 is replaced with valine, serine, arginine, cysteine, aspartic acid or alanine and the cysteine residue is inserted in the second position at the C-terminus, It is prepared by synthesizing specific peptides that produce antibodies (eg anti-p21 antiserum). Other peptides do not necessarily produce such antibodies. The protein to be immunoprecipitated with the antibody is usually partially altered to expose the epitope of the protein. Antibodies that come into contact with the protein can be affinity purified to enhance their binding to the p21 protein.

Anti-p21 ^ser antibodies have been found to immunoprecipitate p21 protein with serine at site 12 to quantitatively inhibit its autophosphorylation. Antibodies also significantly reduce the ability of p21V-Ki-ras to bind GTP in monoclonal antibody immunoabsorbance testing. Thus, perhaps the anti-p21 ^ser binding site at site 12 contains part of the GTP binding site. These results strongly support the concept that G21 binding and hydrolysis of p21 plays a critical role in the activity of oncogenes. Thus, anti-p21 ^ser antibodies can serve as a powerful means for the study of p21 protein function in vitro and in vivo.

Claims (10)

An antibody that selectively binds to a characteristic marker epitope that is not present in the corresponding proto-oncogene product of the oncogene product. The antibody of claim 1, wherein the oncogene product is an activating form of the p21 protein and the epitope surrounds its 12 position. The antibody according to claim 2, wherein the amino acid at position 12 is valine, serine, arginine, cysteine, aspartic acid or alanine. 12. A peptide fragment useful as an immunogen, comprising a variable amino acid at position 12, an amount of side residues sufficient to define an epitope and a cysteine residue inserted at the C-terminus of the fragment at the second position. The peptide fragment of claim 4 which is bound to a carrier protein. The polypeptide or sample is treated with a protein alteration reagent that exposes the epitope and hardly inhibits antigen-antibody binding: selectively binding the polypeptide or sample to the epitope under conditions that allow binding of the antibody to the epitope. After incubation with the antibody, the presence of an epitope and an immunocomplex of the antibody in the culture is detected, thereby producing a polypeptide or a carcinogen product in the cytoplasmic sample of the subject, wherein A characteristic marker epitope that is not exposed, and the oncogene product has a characteristic marker epitope that is not present in the proto-oncogene product and is not exposed in an unaffected protein. The host animal is immunized with a peptide fragment consisting of a variable amino acid at position 12, a residue sufficient to define an epitope and a cysteine residue inserted at the C-terminus of the fragment at the second position, and a serum obtained to obtain an antibody from the serum obtained. Characterized in that the method for producing an antibody according to claim 2. A partially denatured protein having a characteristic marker epitope that is not exposed in a characteristic unaltered protein and sufficiently altered to expose the epitope. A method for producing a protein according to claim 8, wherein the unmodified protein is brought into contact with the altering reagent and under conditions that sufficiently expose the epitope but hardly inhibit antigen-antibody binding. Characterized by an antibody according to any one of claims 1 to 3 which binds to or is labeled with a detectable label, a modified buffer, and a labeled antibody which binds to said antibody if the antibody is not labeled. Kit.

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