RU2276672C2 - Antibody raised to hldf, method for it preparing (variants), peptide showing antigenic and nucleic acid-hydrolyzing property and method for diagnosis of human cell anaplastic state - Google Patents

Abstract

FIELD: biotechnology, peptides.

SUBSTANCE: invention relates to a method for preparing antibodies raised to human leukocyte differentiation factor (HLDF) or to HLDF fragment (31-38) representing peptide of the following structure: Arg-Arg-Trp-His-Arg-Leu-Glu-Lys possessing with antigenic and nucleic acids-hydrolyzing properties, and for diagnostic aims also. Antibodies are prepared from rabbit plasma blood immunized with three injections of antigens wherein synthetic HLDF factor or conjugate is used as antigens. Diagnosis of anaplastic state of human cells is carried out by using solutions of antibodies to HLDF factor or HLDF fragment (31-38) in the concentration 0.0013 mg/ml as biological markers. Invention provides carrying out the differential diagnosis of tumors and normal organs and effective detecting initial stages in cell differentiation disturbances.

EFFECT: improved preparing method of antibody, improved method for diagnosis.

6 cl, 21 dwg, 1 tbl

Description

The invention relates to the field of biochemistry, to the production of biologically active substances, methods for their preparation, namely, to the production of substances having antigenic and immunospecific properties, and to medicine, and in particular to methods for diagnosing the anaplastic state of human cells, in particular, with oncological diseases.

The problem of creating effective methods for diagnosing human oncological diseases, especially in the early stages of the disease, is very relevant.

The statistics of oncological diseases shows that with early diagnosis of the processes of transformation of a healthy cell into anaplastic one, the most effective treatment of human cancer is possible both by therapeutic methods known to specialists working in this field, as well as by surgery. To diagnose the stage of cancer, it is very important to conduct studies of human cells using sensitive tests that capture even the earliest minor precancerous changes in the cell structure, which can lead to the further development of intense proliferative activity of these cells, that is, to the development of cancer.

Currently known methods for diagnosing cancer are based on the use of an immunohistochemical reaction of cell structures when exposed to external biological markers on the cell, causing, for example, the appearance of new structures in the cell, for example, antigens.

Currently known modern methods for the diagnosis of tumors are based on an immunohistochemical reaction in order to identify antigens specific for a given tumor type. In prostate cancer, a reaction is carried out to study the expression of prostate specific antigen (PSA) p53, bcl-2, p27kipl, AR and MIB-1 in tumor tissue cells. (Bai XZ., Masters J.R., O'Donoghue N. et al. Prognostic markers in clinically localized prostate cancer. Int .. J. Oncol (Greece) 1999, v. 14, No. 4, p. 785-791).

A known method of immunohistochemical diagnosis of the anaplastic state of a human cell, comprising the following stages:

- obtaining a test sample of human tissue in the form of postoperative material or a piece of tissue after a puncture biopsy,

- preparation of tissue sections for immunohistochemical analysis,

- immunohistochemical analysis of tissue sections, treatment of tissue sections with biological markers that cause the formation of stained precipitates in the structures of the section cells,

- a visual assessment of the results of the analysis by the nature of the location and concentration of stained precipitates to identify tissue sites with anaplastic state of cells (Bhargava V., Kell DL, van de Rijn M., Wamke RA Bcl-2 Immunoreactivity in Breast Carcinoma Correlates with Hormone Receptor Positivity. American Journal of Pathology 1994, v. 145, N3, p. 535-540).

Moreover, solutions containing antibodies to certain antigens expressed in cell structures are used as markers. A known method of immunohistochemical analysis of a section of tissue of the human prostate gland and a diagnostic method based on it. (Myers R. B., Grizzle W. E. Changes in biomarker expression in the development of prostatic adenocarcinoma. Biotech. Histochem. 199T, v. 72, N2, p. 86-95).

As biological markers, solutions are used that identify factors that inhibit the development of apoptosis in human cells, for example, anti-sera of mouse monoclonal antibodies to protein compounds of the Bcl-2 group (DAK.O Corporation, USA) (hereinafter Bcl-2), which are capable of visualizing the appearance of stained precipitates - antigens in the cytoplasm of gland epithelial cells. This method is quite effective for the diagnosis of high-, moderate- and low-differentiated adenocarcinomas (high-, moderate - and low-) in the prostate gland.

Currently, the most widely used immunohistochemical markers are immune solutions to factors p53 and bcl-2. However, the use of these markers has certain limitations; Thus, with the help of these markers, only pronounced types of tumors (advanced stage prostatic adenocarcinomas) are most clearly detected.

When studying the expression of bcl-2 antigen in normal tissue of the human prostate gland or in benign adenoma by immunohistochemical methods, a positive reaction is detected, as a rule, in the basal cell layer of ducts and acini and is not found in cells lining the gland. This marker allows you to identify pre-tumor processes - prostatic intraepithelial neoplasia of low and high gradations, while immunoreactivity is observed both in the cells of the basal layer and in the glandular epithelium. A limitation in the widespread use of this method is the fact that specific staining in prostatic intraepithelial neoplasia can be detected only in 22% of cases, while in 78% of cases immunohistochemical staining is observed, which is characteristic of a process not associated with the formation of a tumor, or characteristic of benign tumors - adenomas.

The p53 marker is also not intended to identify pre-tumor processes, but serves as a prognostic factor in various forms of therapy with adenocarcinomas.

In this regard, it is necessary to search for new, more universal tumor and precancerous biological markers that reveal the initial stages of anaplastic processes in tissues.

Specialists working in the field of bioorganic chemistry and microbiology, there are known methods for producing antibodies, both monoclonal and polyclonal, for example, using cultured cells with myeloma, hybridomas.

Known methods for producing antibodies by isolating them from the blood of animals immunized with a solution or suspension containing an antigen that initiates the appearance of specific antibodies to it in the blood plasma.

The methods include the steps of producing an antigen, immunizing an animal, obtaining blood serum of an immunized animal, and isolating antibodies from blood serum.

Such methods for producing antibodies are carried out mainly using standard immunization methods known to specialists in this field, using standard methods of control and purification of sera. The differences are due to the differences in the substances used in the methods as antigens.

The present invention was tasked with the development of new more sensitive biological markers and methods for their preparation and a new method for diagnosing the anaplastic state of a human cell based on the use of new biological markers with a more directed and more differentiated effect on human cell structures, and in particular on structures, containing human leukocyte differentiation factor, the content and localization of which in the cell depends on the degree of its differentiation and.

The HLDF differentiation factor (SEQ ID N0: 1) was originally isolated from the culture medium of HL-60 cells of human promyelocytic leukemia treated with retinoic acid (IAKostanyan, MV Astapova, EV Starovoytova, SM Dranitsyna, VM Lipkin - A new human leukemia cell 8.2 kDa differentiation factor: isolation and primary structure. (1994) FEBS Lett., v.356, n.2-3, pp327-329).

It has been shown that the HLDF factor causes differentiation of the initial cell line along the granulocytic pathway and stops cell proliferation. During the study, it was determined that as the processes of differentiation and apoptosis develop in cells, a significant increase in the level of HLDF factor is observed, mainly in the near-membrane peripheral region.

The HLDF factor is a glycosylated protein consisting of 54 amino acid residues (see the List of sequences attached to this application), which has pronounced nonspecific nuclease properties. In the course of the study, an 8-membered factor fragment (amino acid residues 31-38) was identified that was able to hydrolyze nucleic acids. This factor site is highly immunogenic.

The problem was solved by creating a new antibody with immunospecificity to the human leukocyte differentiation factor (hereinafter - HLDF) or the HLDF fragment (31-38), characterized by the following features:

- physico-chemical properties of the antibodies correspond to the physicochemical properties of antibodies to immunoglobulins of class G (hereinafter - IgG):

a) the specificity of the interaction of antibodies with protein A, confirmed by affinity chromatography;

b) the magnitude of the electrophoretic mobility of antibodies in a polyacrylamide gel in the presence of sodium dodecyl sulfate is confirmed by electrophoregram data;

c) the specificity of the interaction of antibodies with secondary antibodies to class G immunoglobulins is confirmed by the data of antibody studies by enzyme immunoassay (ELISA);

- the specificity of interaction with the HLDF factor, determined by enzyme-linked immunosorbent assay (ELISA) and immuno-transfer using HLDF factor and its fragment;

- the ability, when interacting with the HLDF factor or the HLDF fragment (31-38), to cause staining of cell structures-precipitates.

Moreover, the antibody according to the invention is polyclonal.

The problem was also solved by obtaining a new peptide with antigenic and NK-hydrolyzing properties, which is a fragment of the HLDF factor (31-38) (SEQ W N0: 2), by the method of solid-phase peptide synthesis by sequential peptide chain extension and having the following characteristics:

- structural formula

Arg-Arg-Trp-His-Arg-Leu-Lys-Glu;

- molecular weight 1180 ± 2 Yes, determined using MALDI spectrometry;

- the amino acid composition determined by amino acid analysis and confirmed by determining the N-terminal amino acid sequence on a solid-phase sequencer Applicol Diosistem 470A, with the following ratio of amino acid residues in one molecule of the peptide:

Arginine (Arg) - 3, Tryptophan (Tgr) - 1, Histidine (His) - 1, Leucine (Leu) - 1, Glutamic acid (Glu) -1, Lysine (Lys) - 1;

- isoelectric point 11.82, determined in a solution of pH 7.0;

- pH stability in the pH range from 2 to 9;

- thermal stability in the dry state in the temperature range from 0 to 56 ° C, thermal stability in an aqueous solution in the temperature range from 0 to 37 ° C for 24 hours;

- 99.2% homogeneity determined by high performance liquid chromatography;

- solubility in water, physiological saline and in buffer solutions, pH from 2 to 11;

- cytotoxicity, determined by the ability of the peptide to cause apoptotic cell death of promyelocytic leukemia HL-60.

The problem is also solved by the development of methods for producing these antibodies from rabbit blood plasma, including the steps of producing an antigen, immunizing a rabbit with an antigen, obtaining blood serum of an immunized animal, and isolating antibodies from blood serum, characterized in that in one method according to the invention, the rabbit is immunized by three times injection with a dose of antigen of about 0.07 mg / kg body weight, and synthetic factor HLDF is used as antigen, and in another method according to the invention, imm rabbit ization is carried out by injecting three-fold in the first dose of the antigen of about 0.150 mg / kg body weight, in the second and third time - antigen dose of about 0.035 mg / kg and used as the antigen peptide conjugate according to the new invention.

The problem was also solved by the development of a new method for the diagnosis of the anaplastic state of human cells, including the following stages:

- obtaining a test sample of human tissue, fixing his condition, dehydration and processing of a tissue sample to obtain a slice in paraffin,

- preparation of a paraffin section of tissue for immunohistochemical analysis,

- immunohistochemical analysis of a tissue section, treatment of the section with biological markers that cause the formation of stained precipitates in the structures of the section cells,

- visual assessment of the results of the analysis by the nature of the location and concentration of stained precipitates to identify tissue sites with anaplastic state of cells, characterized in that the biological markers use a solution of antibodies according to the invention in PBS with a concentration of 0.0013 mg / ml.

The invention is further explained in the description of specific, not limiting the present invention, embodiments of the invention and the accompanying drawings, in which:

Figure 1 is a chromatogram of the blood serum of immunized rabbits on Protein-A-Sepharose.

Figure 2 is an electrophoregram of the molecular weight distribution of antibodies according to the invention, obtained by the Laemmly method in a 12% polyacrylamide gel in the presence of sodium dodecyl sulfite (SDS-PAGE).

Figure 3 - electrophoregrams 3A, 3b, 3C, illustrating the NK-hydrolyzing activity of the peptide according to the invention.

4 is an electrophoregram 4a, 4b, illustrating the study of the specificity of the interaction of the antibodies according to the invention with the differentiation factor HLDF using the method of immunoprotection.

Figure 5 - image of a tissue section of a normal prostate gland obtained by a diagnostic method using a solution of antibodies to proteins of the Bcl-2 group.

6 is an image of a tissue section of a normal prostate gland obtained by the diagnostic method according to the invention.

Fig.7 is an image of a section of prostate tissue with a diagnosis of benign prostatic hyperplasia, obtained by the diagnostic method using a solution of antibodies to proteins of the Bcl-2 group.

Fig. 8 is an image of a tissue section of benign prostatic hyperplasia obtained by the diagnostic method of the invention.

Fig.9 is an image of a section of prostate tissue with a diagnosis of precancerous disease (low grade prostatic intraepithelial neoplasia (PIN)) obtained by the diagnostic method according to the invention.

Figure 10 - image of a section of prostate tissue with a diagnosis of high-grade prostatic intraepithelial neoplasia (PIN), obtained by the diagnostic method using a solution of antibodies to proteins of the Bcl-2 group.

11 is an image of a tissue section with a diagnosis of low-grade prostatic intraepithelial neoplasia (PIN) obtained by a diagnostic method using a solution of antibodies to proteins of the Bcl-2 group.

Fig. 12 is a slice image of the same prostate tissue with a high degree of PIN diagnosis as in Fig. 10, but obtained by the diagnostic method according to the invention.

Fig - image of a section of tissue of the human endometrium with a diagnosis of simple hyperplasia, obtained by the diagnostic method according to the invention.

Fig - image of a tissue section of the human endometrium with a diagnosis of adenomatous endometrial hyperplasia, obtained by the diagnostic method according to the invention.

Fig - image of a slice of prostate tissue with a diagnosis of differentiated adenocarcinoma obtained by the diagnostic method using a solution of antibodies to proteins of the Bcl-2 group.

Fig. 16 is a sectional image of prostate tissue with a diagnosis of differentiated adenocarcinoma obtained by the diagnostic method according to the invention.

Fig - image of a section of prostate tissue with a diagnosis of low-grade adenocarcinoma when processed by androgen receptors (left side of the figure), on the right side - moderately differentiated adenocarcinoma with the distribution of androgen receptors in individual nuclei of anaplastic cells.

Fig is an image of a section of prostate tissue with a diagnosis of low-grade adenocarcinoma obtained by the diagnostic method using a solution of antibodies to proteins of the Bcl-2 group.

Fig. 19 is an image of a section of prostate tissue diagnosed with a low-grade adenocarcinoma obtained by the diagnostic method according to the invention.

Fig - image tissue section of the glandular epithelium of the colon with a diagnosis of differentiated adenocarcinoma, obtained by the diagnostic method according to the invention.

Fig - image tissue section of smooth muscle cells of the uterus with a diagnosis of leiomyosarcoma, obtained by the diagnostic method according to the invention.

The invention may be practiced by the methods described below.

The preparation of a peptide according to the invention having the structural formula Arg-Arg-Trp-His-Arg-Leu-Glu-Lys can be carried out by any methods known to those skilled in the art, for example, by solid phase peptide synthesis using a tert-butyloxycarbonyl scheme according to the BOC / Bzl methodology (Peptide Synthesis, 2nd edn., Pierce Chem. Co., Rickford IL), (Rodionov IL, Baru MB and Ivanov VT A Swellographic approach to monitoring continuous flow solid phase peptide synthesis, Peptide Res., 1992, v.5. No.2, pp119-125).

The synthesis is carried out automatically on a peptide synthesizer. A copolymer of polystyrene and divinylbenzene with 4-hydroxylmethylacetamide methyl (PAM) anchor group BOC-ARG (Tos) -PAM RESIN (100-200 mesh, 0.47 mmol / g. Advanced Chem. Tech., USA Cat. No.SR 5605) in an amount of 250 μmol.

For temporary protection of the α-amino groups of amino acids, a blocking tert-butyloxycarbonyl (BOC) moiety is used. To carry out the condensation reaction, four-fold excesses of reagents are used: pentafluorophenyl ether of the BOC amino acid and hydroxybenzotriazole.

The amino acids Arginine, Tryptophan, Histidine, Leucine, Glutamic acid are used as starting products. Solid lysine, available in the form of drugs on the market, for example, preparations of the company Reanal, in the amount of 0.564 g of each amino acid for each synthesis cycle.

The terminal carboxyl group of the C-terminal amino acid of the synthesized peptide is covalently linked to a carrier.

The stepwise extension of the peptide chain from the N-terminus is carried out by sequentially cleaving the blocking group from the α-amino group and linking the carboxy group of the subsequent amino acid to it. The completeness of the reactions is monitored using a qualitative and quantitative ninhydrin test using swellographic monitoring (Kaiser E., Colescott R. L., Bossinger C. D., Cook P. I., 1970, Anal. Biochem. 34, p. P. 595-598).

The resulting peptidyl polymer is cleaved from the carrier with the simultaneous removal of blocking groups by the action of liquid hydrogen fluoride (19 ml per 700-1000 mg of peptidyl polymer) in the presence of meta-cresol (1 ml) and cysteine (500 mg) at a temperature of minus 10 ° С to 0 in two hours.

The peptide is chromatographed by gel filtration on a column of Sephadex G-15 resin (26 × 500 mm) in 1-N-acetic acid. The structure of the peptide is then analyzed by amino acid composition by well-known specialists working in the field of peptides, methods of amino acid analysis and MALDI spectrometry.

The peptide is purified to homogeneity (99.2%) by reverse phase high performance liquid chromatography on a NUCLEOSIL C18 column on an ALTEX device, USA, or by other well-known specialists working in this field by chromatography methods.

The yield of the final product is about 56% dry matter relative to the total amount of starting amino acids.

Then the peptide is subjected to analysis to determine the physico-chemical properties.

Thus, a peptide having the structural formula

Arg-Arg-Trp-His-Arg-Leu-Lys-Glu,

and the following properties:

- molecular weight -1180 ± Yes;

- amino acid composition with the following ratio of amino acid residues in one molecule of the peptide:

Arg-3, Tgr-1, His-1, Leu-1, Glu-1, Lys-1;

- isoelectric point 11.82;

- solubility in water, saline and in buffer solutions, pH from 2 to 11;

- pH stability in the pH range from 2 to 9;

- thermal stability in the dry state in the temperature range from 0 to 56 ° C, thermal stability in an aqueous solution in the temperature range from 0 to 37 ° C for 24 hours;

- homogeneity of 99.2%.

An analysis of the obtained peptide for immunogenicity, carried out by immunizing rabbits with a peptide conjugate with ovalbumin chicken eggs or cochlear hemocyanin (KLH) showed that the peptide has high immunogenicity, initiating the appearance of antibodies in rabbit serum. The antibody titer in the resulting serum was 1: 1000.

The resulting peptide was examined to determine its NK-hydrolyzing activity.

For this purpose, the peptide at a concentration of equal to or more than 10 ^-6 M was incubated with nucleic acids in a slightly acidic medium in 0.05 M Na-acetate buffer, pH 4.5, for 1 hour at a temperature of 37 ° C. The reaction products were analyzed by electrophoresis in 1% agarose gel in 0.04 M Tris-Acetate buffer, pH 8.0, with 0.002 M EDTA, followed by staining with ethidium bromide solution.

The results of the study are presented on electrophoregrams Figa, 3b, 3c, where Fig.3a illustrates the hydrolyzing activity of the peptide against DNA of plasmid pSp65, Fig.3b - against DNA of phage λ, Fig.3c - against ribosomal RNA, while on the tracks 2 (Figs. 3a, 3b, 3c) shows the forms of nucleic acids incubated under the same conditions, but without peptide, in lanes 3 (Figs. 3a, 3b, 3c), the nucleic acids before incubation, in the initial state.

Electrophoregrams show that the original nucleic acid degrades under the action of the peptide (lanes 1 in FIGS. 3a, 3b, 3c).

The results showed that the peptide at concentrations of 10 ^-6 M or more is able to hydrolyze the molecules of plasmid DNA, phage λ DNA, and RNA under acidic conditions (pH 4.5 or less), which is indirect evidence of the participation of HLDF differentiation factor in apoptosis, part which is a peptide.

The cytotoxicity of the peptide was studied by its ability to cause apoptotic death of HL-60 cells by exposure to a solution of the peptide in PBS with a peptide concentration in PBS of 10 ^-6 M, 10 ^-7 M, 10 ^-8 M, on HL-60 cells after incubation for 4 hours at a temperature of 37 ° C.

As control, HL-60 cells treated with PBS were used. For comparison, the results obtained by exposing the HL-60 cells to the well-known apoptosis inducer actinomycin D in the same PBS solution are presented.

The cytotoxicity of the peptide was evaluated by apoptotic death of HL-60 cells compared with the control calculated by the formula

where N _A is the proportion of cells killed by apoptosis,

to _{A exp} is the number of apoptotic cells in an HL-60 culture exposed to a peptide or actinomycin D,

to _{A const} is the number of apoptotic cells in the control experiment.

The results of the analysis are presented in the table.

Cytotoxicity of the peptide when exposed to HL-60 cells N _A ,% The effect of the peptide in concentration The effects of actinomycin D 10 ^-6 M 10 ^-7 M 10 ^-8 M 10 ^-6 M 385 ± 10 395 ± 3 239 ± 5 280 ± 5

It is clear from the table that the peptide at concentrations of 10 ^-6 M and 10 ^-7 M exhibits a stronger cytotoxic effect than the known inducer of apoptosis actinomycin D at a concentration of 10 ^-6 .

The results of a study of the cytotoxic and NK-hydrolyzing activity of the peptide suggested the involvement of the HLDF differentiation factor in apoptosis as a nuclease.

From the foregoing, it is clear to every person skilled in the art that the obtained peptide has pharmaceutical purity, does not need further purification, can be obtained as a solid, and can be used as solutions in cases of its use in scientific research or in methods and technological processes using its antigenic properties, as well as its NK-hydrolyzing properties.

Obtaining antibodies to human leukocyte factor HLDF and / or to a fragment of HLDF (31-38) can be carried out by methods known to specialists working in this field, using standard methods at various stages.

Obtaining these antibodies, according to the invention, from the blood plasma of an immunized rabbit and includes the following steps: obtaining an antigen, immunizing a rabbit, obtaining blood serum of an immunized animal, and isolating antibodies from blood serum.

The stage of obtaining the antigen can consist of various operations, for example, synthesis of the necessary antigen, or its acquisition, preparation of antigen solutions for subsequent injections, performed by standard methods known to those skilled in the art.

Immunization of a Chinchillan rabbit weighing about 2.5 kg is carried out according to the standard method by injecting a specific dose of antigen subcutaneously by injection at 5-6 points along the spinal column with a volume of 1 / 5-1 / 6 of the volume of the solution containing the antigen.

In this case, the antigen is administered in the form of a solution of antigen in PBS of standard concentration and composition in a volume of 1 ml emulsified in Freund’s adjuvant; moreover, Freund’s complete adjuvant is used for the first injection, incomplete adjuvant is used in subsequent injections.

Blood sampling is carried out from the marginal ear vein of a rabbit in a volume of up to 50 ml once. To form a clot and obtain serum, the blood is incubated for 30 min at 37 ° C, then for 12 hours at 4 ° C, and then centrifuged for 10 min at 4 ° C at a speed of 10,000 rpm.

The resulting blood serum was diluted 4 times with buffer, pH 8, containing TRIS-HCl to a concentration of 0.1 M and applied to a Protein A-Sepharose CL-4B column (Pharmacia Fine Chemicals, Sweden), equilibrated with the same buffer. The elution of antibodies from the sorbent is carried out with a buffer, pH3, containing 0.1 M Gly-HCl. The eluate is titrated with a solution of 1M TRIS to a neutral pH.

The antibodies are then purified from the antigen on K.LH-Sepharose synthesized according to the standard procedure using BrCN-activated Sepharose (Pharmacia Fine Chemicals, Sweden). Antibodies that did not bind to immobilized K.LH were concentrated by rechromatography on Protein A-Sepharose.

In accordance with one method of the invention, HLDF cell differentiation factor is used as an antigen to produce antibodies, which are synthesized using the solid phase synthesis method or isolated from the culture medium of trans-retinoic acid-treated HL-60 cells by a known method (FEBS Letter, 1994 , Vol. No. 12) or using a solid phase peptide synthesis method from commercially available amino acids, for example, Reanal.

According to the present invention, in this method for producing antibodies, the rabbit is immunized with the antigen - factor HLDF - three times by injection with an antigen dose of about 0.070 mg / kg body weight, then blood sampling is carried out, blood serum is obtained by the above-described methods and antibodies are isolated.

The concentration of antibodies in the resulting 50% aqueous solution of glycerol is 1.5 mg / ml, the working titer of antibodies is 1: 1000.

In another method according to the invention, the HLDF factor fragment (31-38) of the peptide according to the invention obtained from the previously described method of peptide solid-phase synthesis is used as an antigen to immunize a rabbit.

For use in the specified method, two types of conjugates of the indicated peptide with various high molecular weight carriers were synthesized: cochlear hemocyanin (KLH) and ovalbumin from chicken eggs.

The synthesis of conjugates was carried out according to standard methods known to specialists in this field. The synthesis was carried out using water-soluble 1-ethyl-3- (3-dimethylaminopropanol) -carbodiimide-hydrochloride (EDAC, Bio-Rad, USA) by forming a covalent peptide bond between the hapten carboxyl group and the amino groups of the carrier according to the following procedure.

A solution of 4 mg of EDAC in 100 μl of water is added dropwise to a solution of 2 mg of the indicated peptide in water at 4 ° C. with stirring. Then, a solution of 6 mg KLH in 200 μl of a TBS standard composition of 0.15 M NaCl, 0.05 M Tris-HCl and a pH of 7.5-8 was dropwise added to the reaction mixture. The reaction mixture was incubated for 4 hours at room temperature with stirring.

The reaction products are then dialyzed against a TBS solution. A ready-to-use peptide conjugate is stored at minus 20 ° C in a frozen state.

The rabbit is immunized with the conjugate of the indicated peptide by three injections: the first time with an antibody dose of about 0.150 mg / kg body weight, the second and third times with an antibody dose of about 0.035 mg / kg body weight.

Then, by the previously described methods, the blood serum of the immunized animal is obtained and antibodies are isolated.

The concentration of antibodies in the resulting 50% aqueous solution of glycerol is 1.3 mg / ml, the working titer of antibodies is 1: 1000.

The antibodies obtained by the indicated two methods according to the invention are examined to determine their physicochemical properties and to reveal their specificity, for example, by affinity chromatography methods, SDS-PAGE electrophoresis, enzyme-linked immunosorbent assay (ELISA), immuno-transfer method.

The research results showed the following.

Antibodies were obtained whose physicochemical properties correspond to the physicochemical properties of IgG antibodies:

a) the specificity of the interaction of antibodies with protein A is confirmed by the results of affinity chromatography (Figure 1), which shows that the antibody fractions are eluted from the Proteain A-Sepharose column only in buffer B containing 0.1 M Gly-HCl, pH 3.0 ;

b) the level of electrophoretic mobility in 12% SDS page-SDS is confirmed by the data of the electrophoregram shown in Fig.26, where the bands of columns "a" and "d" correspond to standard molecular masses of protein markers 97.4; 66.2; 55.0; 42.7; 40.0; 31.0; 21.5; 14.4 KDa, and the protein bands of columns “b” and “c” correspond to the molecular weights of antibodies according to the invention: column “c” obtained by the first method, according to the invention, using HLDF as antigen, column “b” obtained by the second the method according to the invention, using a fragment of the HLDF factor (31-38) - the peptide according to the invention as an antigen;

c) the specificity of interaction with secondary goat antibodies to class G immunoglobulins is confirmed by enzyme-linked immunosorbent assay (ELISA).

The specificity of the interaction of the obtained antibodies with the HLDF factor was determined by ELISA and immuno-transfer

The results of the study of antibodies using the method of immunoprotection are presented in Figure 4 (a, b). Fig. 4a is an electrophoregram of a synthetic differentiation factor. Fig. 4b is an image of a nitrocelluloid membrane onto which the proteins shown in Fig. 4a and treated with the antibody solution of the invention in 1: 1000 dilution were transferred using the standard method of immunoprotection. (Harlow E., Lane D. Antibodies (A Laboratory Manual). Cold Spring Harbor; N.-Y .: Cold Spring Harbor Press, 1988).

A clear image of the precipitate was obtained only with the HLDF protein, which indicates the high specificity of the antibodies obtained. The ability, when interacting with an HLDF factor or an HLDF fragment (31-38), to cause the formation of colored precipitates in human cells is confirmed by the use of antibodies as biological markers in the method for diagnosing the anaplastic state of a human cell according to the invention.

A method for diagnosing the anaplastic state of a human cell according to the invention comprises the following steps:

a) obtaining a test sample of human tissue, for example, by standard methods from postoperative histological material or puncture biopsy material (Ekman H., Hedberg K., Persson P. Cytological versus histological examination of needle biopsy speciments in the diagnosis of prostate cancer. Br. J .Urol. 1967, v. 39, p. 544) and preparing it by the standard method for subsequent research, for example, consisting in the fact that tissue samples not exceeding 0.5 × 0.5 × 0.2 cm are fixed in neutral 10% formalin solution in distilled water for 24 hours, then carry out their dehydrate cation in ethanol sequentially in solutions of 70%, 80%, 90% concentration and then three times in a solution of 100% alcohol with exposure in each solution for 4 hours at room temperature. Then the samples are kept in 3 shifts of xylene, 4 hours each, immersed in 2 shifts of liquid paraffin with a temperature of 60 ° C for 12 hours. After that, tissue samples are placed in blocks and embedded in paraffin, and 5 μm thick sections are cut from the block at room temperature;

b) the preparation of a paraffin section for immunohistochemical analysis, for example, by the standard method (Bonhoff H. Neuroendocrine cells in benign and malignant prostate tissue. Morphogenesis, proliferation and androgen receptor status. Prostate Supplement, 1998, v. 8, pp18-22), which consists in that paraffin sections are placed on glass coated with poly-L-lysine (preparation of SIGMA, USA, P 0425), then the sections are dewaxed by immersing them in 3 xylene shifts, and then in 3 shifts of 100% ethanol, then placed sequentially in ethanol solutions 96%, 90%, 80%, 70%, 50% concentration for 5 min each, and then in a buffer first phosphate buffer saline pH 7.4 in deionized water - PBS standard composition: 0.01 M - phosphate buffer containing 0.138 M NaCk, pH - 7.4.

Sections were then microwaved according to a known method (Loda M., Fogt F., French FS, Posner M., Cukor B., Aretz HT, Alsaign N. Androgen receptor immunohistochemistry on paraffin-embedded tissue. Mod. Pathol. J. , 1994, v.7, pp388-391) in a 10 M citrate buffer (pH 6.0) in a 750 W furnace power mode, sequentially bringing the solution to a boil and cooling it to room temperature according to the scheme: boiling for 5 min , cooling for 2 min at room temperature, boiling for 5 min.

Then the glass with sections in the same buffer solution is cooled for 15-10 minutes at room temperature.

To inhibit the endogenous peroxidase activity of tissue cells after cooling, the sections are removed from the solution and incubated for 5 minutes. in a 3% hydrogen peroxide solution.

Previously prepared sections were washed with a PBS solution of the previously indicated composition;

c) immunohistochemical analysis of a tissue section, treatment of the section with biological markers that cause the formation of stained precipitates in the cell structures.

In an immunological analysis according to the standard method, serum of biological markers is applied to tissue sections, which are then incubated in a humid chamber for 12 hours at 4 ° C, then washed thoroughly in a PBS solution of the above composition. Then, tissue antigens are visualized, for example, by the vidin-biotin method using the universal peroxidase DAKO LSAB + kit (DAKO Corporation, USA), in which the biotinated secondary antibodies react with the corresponding molecules of peroxidase-conjugated streptavidin. (Giomo RA Comparison of the two immunoperoxidase methods based on the avidin-biotin interaction. Diagnostic Immunology, 1984, v. 2, p. 161).

The sections are washed and treated with biotinized solutions of anti-rabbit immunoglobulins in PBS buffer containing a protein carrier and 15 mM sodium azide. After 30 minutes of incubation, the sections were washed again and then incubated for 30 minutes in a solution of horseradish peroxidase labeled streptavidin in PBS buffer containing a protein carrier and preservative.

At the end of the reaction, the sections are washed with PBS and stained with substrate-chromogenic solution of DAB (3,3-diaminobenzidine). Then the sections are washed in running water for 10 minutes.

If necessary, a brighter staining of the background structures of the sections is stained with Mayer hematoxylin.

Then the sections are subjected to dehydration in ethyl alcohol, successively in solutions of 50%, 70%, 80%, 90% and 100% concentration, keeping in each solution for 1 min at room temperature, then in xylene, and then filled with Canadian balsam ;

d) a visual assessment of the results of the immunohistochemical analysis by the nature of the location and concentration of stained precipitates to identify tissue sites with anaplastic state of the cells.

The assessment is made by visual observation of the preparations under a light microscope at a magnification of 100x or 400x.

The results of the immunohistochemical reaction can be "positive" or "negative." A positive reaction is characterized by the appearance of brown staining in the test drug in the central part of the prostate glands or in the cytoplasm of glandular epithelial cells, which indicates the specificity of the immunohistochemical reaction. The absence of brown color in the epithelial cells of the prostate gland indicates errors in the immunohistochemical reaction.

A "positive" result of an immunohistochemical reaction, depending on the distribution of its brown insoluble products in the studied samples, allows us to draw conclusions about the state of the processes occurring in the cells of the studied sample.

According to the invention, in the method for diagnosing the anaplastic state of a human cell, antibody solutions to the differentiation factor or to the HLDF peptide (31-38) according to the invention are used as biological markers.

Solutions of antibodies to the differentiation factor at a concentration of 0.0013 mg / ml were prepared as previously described.

Solutions of antibodies to the HLDF peptide (31-38) at a concentration of 0.0013 mg / ml were prepared as previously described.

The diagnosis of the method according to the invention is further illustrated by examples of the diagnosis of the state of the glandular organs of a person.

1. Diagnosis of a normal prostate gland.

In the study of biopsy material obtained by transrectal biopsy, it is possible to obtain tissue samples from the central and peripheral zones of the prostate gland.

As an example, a comparative diagnosis of a tissue section of the prostate gland was carried out, in which it was assumed that a benign tumor was adenoma, according to the invention, using antibodies to the HLDF factor as an apoptosis marker, and the previously known method, using antibodies to proteins as apoptosis markers Bcl-2 groups (DAKO Corporation, USA). Immunohistochemical analysis was carried out according to the previously known method.

The results of the study are shown in Fig.5 and Fig.6.

The normal prostate gland in the test material contains small glandular structures of a rounded shape, in which, when stained with Mayer hematoxylin, two layers of cells are clearly distinguished concentrically one to another. The inner main layer is made up of cylindrical secretory epithelial cells, basal layer cells lying on the basal membrane and wedged between the bases of cylindrical epithelial cells are located on the periphery. The cells of the basal layer are smaller than the secretory epithelial cells, have scanty cytoplasm and a large, irregularly shaped nucleus.

When using antibodies to Bcl-2 as a marker for apoptosis, an immunohistochemical method in the normal prostate reveals a brown staining of the cytoplasm of the basal layer epithelial cells associated with the expression of Bcl-2 gene products in actively proliferating basal cells (Figure 5).

When conducting an immunohistochemical reaction with antibodies to the HLDF factor according to the invention in normal glands, it was for the first time possible to detect the distribution of brown products of the diaminobenzidine reaction (DAB-reaction) in the central part of the glands in the form of “honeycomb structures”, which are a layer of cell membranes that died due to apoptosis and not visible when stained by other methods (Fig.6).

The diagnostic method according to the invention allows to visualize such structures due to the distribution of proteins of the HLDF type in them along the periphery of the cytoplasm, which are detected using the method proposed according to the invention.

Thus, in contrast to the known markers, for example, antibodies to Bcl-2, which stain the cytoplasm of the basal layer epithelial cells in normal glands, the method according to the invention makes it possible to detect a normal process of apoptosis in gland cells by the presence of nuclear-free cell walls in the center of the glands in form of "honeycomb structures", as illustrated in Fig.6.

Conclusion: a normal prostate gland was diagnosed.

2. Diagnosis of suspected prostate adenoma.

Prostate tissue was diagnosed in which a benign cancer was suspected. Prostate adenoma (benign prostatic hyperplasia) is characterized by hyperplasia of the epithelial and stromal cells of the prostate, which leads to the formation of large nodes in the periurethral region of the prostate gland. At the same time, small or large cystoid formations are formed in the gland, consisting of microscopic nodal structures of proliferating gland cells and having two epithelial layers: an inner cylindrical (columnar) and an outer cubic (cuboidal or flattened) located on an intact basement membrane. These formations in the gland are grouped in the form of a large number of papillary protrusions and folds characteristic of an adenoma.

The results of immunohistochemical studies are presented in Fig.7 and 8.

When diagnosing adenomas using an immunohistochemical method with a known marker of antibodies to apoptosis Bcl-2, brown staining, as in the case of normal glands, is observed only in the cytoplasm of cells of the outer basal layer (Fig. 7).

The immunohistochemical reaction with antibodies to the HLDF factor according to the invention allows revealing brown honeycombs in the inner part of the papillary protrusions and folds of glandular structures (Fig. 8).

Conclusion: adenoma is diagnosed.

3. Diagnosis of precancerous conditions of glandular epithelium.

Two types of pathology are distinguished as precancerous processes in the prostate gland, one of them is low-grade prostatic intraepithelial neoplasia, characterized by an increase in the number of cells and a multilayered secretory glandular epithelium, which is accompanied by an increase in the size of the nuclei and a variety of nuclear forms. Moreover, the distribution of nuclear chromatin in the cells is normal. Small nucleoli are not often observed in the nuclei of the secretory epithelium. This type of precancerous condition has a benign course and does not require surgical intervention.

Prostate tissue was diagnosed in which a precancerous disease was suspected.

Treatment of the preparations with a commercial marker of apoptosis, antibodies to Bcl-2, revealed a brown staining of the cytoplasm of the epithelial cells of the basal layer, as in prostate adenomas.

When treated with anti-HLDF antibodies according to the invention, brown honeycombs are clearly visible in the center of the glands against the background of the multilayer secretory epithelium (Figure 9), the formation of which is characteristic of normal glands and prostate adenomas.

Another common type of precancerous glandular epithelium disease is high-grade prostatic intraepithelial neoplasia. It is characterized by an increase in the number of cells and a multilayer distribution of the gland epithelium, the presence of large nucleoli and an increase in the amount of nuclear chromatin. High-grade prostatic intraepithelial neoplasia on conventional hemetoxylin-eosin sections is practically indistinguishable from highly differentiated adenocarcinomas and is often a sign that adenocarcinoma is located nearby in this prostate gland.

A study of the expression in epithelial cells of products belonging to the Bcl-2 family in such pre-tumor formations as high-grade prostatic intraepithelial neoplasia (PIN, PIN) revealed two different types of staining. In most cases, the expression of Bcl-2 was observed only in the cells of the basal glandular epithelium, and thus, the immunohistological picture did not differ from those described previously with low grade prostatic intraepithelial neoplasia and prostate adenoma (Figure 10). The second type of immunohistochemical reaction with antibodies to Bcl-2 with IDU was highly characterized by intensive staining of the cytoplasm of not only the basal cells of the glands, but also directly of the secretory cells lining the acini of the prostate gland (Figure 11).

Thus, the studies confirm the data available in the scientific literature that antibodies to Bcl-2 reveal a high degree of PIN in only a small number of cases, which significantly limits the use of these antibodies in clinical practice.

The treatment of the preparations with antibodies to the HLDF factor in high-grade prostatic intraepithelial neoplasia (Fig. 12) revealed the presence of a positive brown coloration of the central part of the glands, but the cell-like coloration was not described previously. With this type of pathology, two types of distribution of the products of the immunohistochemical reaction are observed:

1. General reduction in the size of "honeycomb" formations or their complete absence in the lumen of the gland.

2. The appearance of brown products of the diaminobenzidine reaction around the cytoplasm of glandular cells.

On Fig presents a picture of a positive immunohistochemical reaction with antibodies to the HLDF factor on a tissue section with the presumptive pre-tumor process of high-grade IDUs described earlier (Figure 10), where the nature of the immunohistochemical reaction with antibodies to Bcl-2 did not allow an accurate diagnosis. 12, the absence of pronounced hollow "honeycomb" formations in the center of the gland and the appearance of intense brown staining around the cytoplasm of the cells indicate the presence of a pre-tumor formation.

Conclusion: a high degree of prostatic intraepithelial neoplasia was diagnosed.

The method according to the invention allows to diagnose pre-tumor processes in other glandular tissues, in particular in the human endometrium. Endometrial hyperplasia is given much attention because of the ability of its individual species to turn into adenocarcinomas.

Endometrial hyperplasia is characterized by a violation of the architecture of the glands, the growth of epithelial cells and the peculiarities of the cytology of epithelial cells. Low grade hyperplasias includes simple hyperplasia and complex hyperplasia.

Simple hyperplasia is characterized by general changes in the structure of the endometrial glands, which have a histological structure of epithelial cells, similar to normally proliferating endometrium, but of different sizes and irregular shapes. This type of hyperplasia rarely goes into adenocarcinoma and can be considered a benign process.

When staining the endometrium with the alleged simple hyperplasia of antibodies to the HLDF factor in the center of the glands against the background of the layering of the secretory epithelium, brown honeycombs are clearly visible (Figure 13), reminiscent of the previously described similar formations in normal prostate glands and adenomas.

Complex adenomatous hyperplasia (Complex hyperplasia) is characterized by an increase in the number and size of the endometrial glands and the asymmetry of their shape.

In the lining gland of the epithelium, more layers of cells can be observed than with simple hyperplasia, but with simple hyperplasia the cells have smooth edges, are deprived of pronounced cytological atypia, which distinguishes them from endometrial adenocarcinomas.

However, this type of hyperplasia can develop into adenocarcinoma.

Staining of endometrial preparations with suspected adenomatous hyperplasia with an apoptosis marker for the HLDF factor revealed the presence of a positive brown staining of the cytoplasm of glandular epithelial cells (Fig. 14).

Conclusion: adenomatous endometrial hyperplasia (complex hyperplasia ofendometrium) was diagnosed.

4. Diagnosis of suspected adenocarcinoma.

The method according to the invention was used to diagnose prostate tissue in which an oncological disease was suspected.

Differentiated adenocarcinoma. This type of adenocarcinoma (differentiated adenocarcinoma) is characterized by polymorphic but differentiated glands. There is a lot of connective tissue in the tumor, cribriform structures and solid gland fields are formed in the glands.

Immunohistochemical studies of the expression in the epithelial cells of products related to Bcl-2 with antibodies to Bcl-2 revealed a pronounced staining of the cytoplasm of not only the basal cells of the glands, but also directly the secretory cells of the glandular epithelium of the prostate (Fig. 15).

When conducting an immunohistochemical reaction with polyclonal antibodies to the HLDF factor according to the invention, a positive immunohistochemical reaction was observed in prostate tissue with differentiated adenocarcinoma. The cytoplasm of anaplastic glandular cells was stained intensely brown, with no “honeycomb structures” in the center of the glands (Fig. 16).

Conclusion: differentiated adenocarcinoma was diagnosed.

Low-grade adenocarcinoma. With this type of oncological pathology in the prostate gland, anaplastic cancer cells are freely located in the stroma and practically do not form glandular structures, often there are tumors consisting of dedifferentiated structures that are sharply polymorphic and acquire a solid character. In such tumors, the staining of anaplastic cells for androgen receptors specific for the glandular epithelium of the prostate is practically not observed (Fig. 17), which indicates a pronounced anaplasia of the glandular cells, their loss of sensitivity to hormonal regulation and to the corresponding treatment with antiandrogen drugs.

The specific staining of such cells upon reaction with Bcl-2 apoptosis antibodies also indicates the intense proliferative activity of these anaplastic cells, which in content of this antigen resemble actively proliferating cells of the basal layer of the epithelium, corrected for pronounced anaplasia and invasive activity (Fig. 18).

Antibodies to the HLDF factor according to the invention make it possible to detect intense expression of the HLDF factor in the cytoplasm of anaplastic cells by intense brown color and thus allow the detection of low-grade adenocarcinomas (Fig. 19).

Conclusion: low-grade adenocarcinoma was diagnosed.

The method of detecting anaplastic processes in a human cell according to the invention is universal for the diagnosis of various types of tumors, while a positive immunohistochemical reaction is noted in the cytoplasm of anaplastic cells in the large intestine, sarcomas, etc.

For example, FIG. 20 shows an image of a positive immunohistochemical reaction to the HLDF factor in the cytoplasm of anaplastic differentiated adenocarcinoma cells in the glandular epithelium of the large intestine.

On Fig presents an image of a positive immunohistochemical reaction in the cytoplasm of cells of a malignant tumor of smooth muscle cells of the uterus - leiomyosarcoma (smooth muscle cells of uterus - leiomvosarcoma)

Analysis of the results of the application of the diagnostic method according to the invention allows to draw the following conclusions.

1. A method for diagnosing the anaplastic state of a human cell according to the invention using, as biological markers, antibodies to the HLDF factor or an HLDF fragment (31-38) compared to known diagnostic methods, allows differential diagnosis of malignant and benign tumors and normal organs and to identify the very initial stages cell differentiation disorders, which can be considered as precancerous processes.

2. Antibodies to the HLDF factor or HLDF fragment (31-38) according to the invention are more sensitive markers for the earliest precancerous changes in anaplastic cells compared to antibodies to proteins of the Bcl-2 family and can be used to detect early stages of anaplastic processes in cells human when conducting histological and puncture biopsy studies.

3. Antibodies to the HLDF factor or HLDF fragment (31-38) according to the invention can serve as a unique tool in scientific research in studying the molecular mechanisms of cell differentiation and proliferation.

The peptide according to the invention can be obtained by well-known specialists working in this field, chemical synthesis methods using widely known reagents and equipment, widely known control methods and devices, and can be used both for scientific and clinical purposes based on the application of its special properties such as immunogenicity and NK-hydrolyzing activity of the peptide.

Antibodies according to the invention can be obtained by the methods according to the invention using widely known methods and equipment and can be used both in scientific and in clinical purposes based on their special properties, such as immunospecificity.

The method for diagnosing the anaplastic state of a cell according to the invention can be used both in laboratory and clinical conditions, and can be carried out by specialists working in this field, without additional special training.

Claims (6)

1. Antibody to human leukocyte differentiation factor HLDF (SEQ ID NO: 1) or to a fragment of HLDF (31-38) (SEQ ID NO: 2), characterized by the following features: physicochemical properties correspond to the physicochemical properties of antibodies of the IgG class: a) the specificity of the interaction of antibodies with protein A is confirmed by affinity chromatography; b) the level of electrophoretic mobility in a polyacrylamide gel in the presence of sodium dodecyl sulfite is confirmed by electrophoregram data; c) the specificity of interaction with secondary antibodies to class G immunoglobulins is confirmed by data from antibody studies by enzyme immunoassay (ELISA); immunospecificity to the HLDF factor was determined by enzyme-linked immunosorbent assay (ELISA) and immuno-transfer using HLDF factor and its fragments; the ability when exposed to a human cell to cause the appearance of colored precipitates in the structures of the cell. 2. The antibody according to claim 1, characterized in that it is a polyclonal antibody. 3. A peptide with antigenic properties and NK-hydrolyzing activity, which is a fragment of the HLDF factor (31-38), obtained by the method of solid-phase peptide synthesis by sequential peptide chain extension and having the following characteristics: structural formula Arg-Arg-Trp-His-Arg-Leu-Lys-Glu (SEQ ID NO: 2); molecular weight 1180 ± 2 Yes, determined using MALDI spectrometry; amino acid composition determined using amino acid analysis and using MALDI spectrometry, with the following ratio of amino acid residues in one peptide molecule: Arginine (Arg) - 3, Tryptophan (Trp) - 1, Histidine (His) - 1, Leucine (Leu) - 1, Glutamic acid (Glu) - 1, Lysine (Lys) - 1; isoelectric point 11.82, determined in a solution of pH 7.0; pH stability in the pH range from 2 to 9; thermal stability in the dry state in the temperature range from 0 to 56 ° C; thermal stability in an aqueous solution in the temperature range from 0 to 37 ° C for 24 hours; 99.2% homogeneity determined by high performance liquid chromatography; solubility in water, physiological saline and in buffer solutions, pH from 2 to 11; cytotoxicity was determined by the ability of the peptide to cause apoptotic cell death of promyelocytic leukemia HL-60. 4. The method of producing antibodies according to claim 1 from rabbit blood plasma, comprising the steps of: obtaining an antigen; rabbit immunization; obtaining blood serum of an immunized animal; the selection of antibodies from blood serum, characterized in that the immunization is carried out by three injections with an antigen dose of about 0.07 mg / kg of body weight, and the synthetic differentiation factor HLDF (SEQ ID NO: 1) is used as antigen. 5. The method of producing antibodies according to claim 1 from rabbit blood plasma, comprising the steps of: obtaining an antigen; rabbit immunization; obtaining blood serum of an immunized animal; isolation of antibodies from blood serum, characterized in that the immunization is carried out by three injections, for the first time with a dose of antigen of about 0.150 mg / kg of weight, a second and third time with a dose of antigen of about 0.035 mg / kg, and the peptide conjugate according to claim 1 is used as an antigen. 3 with a high molecular weight carrier. 6. A method for diagnosing anaplastic state of a human cell, comprising the following stages: obtaining a test sample of human tissue, fixing his condition, dehydration and processing of a tissue sample to obtain a slice in paraffin; preparation of a paraffin section of tissue for immunohistochemical analysis; immunohistochemical analysis of a tissue section, treatment of the section with biological markers that cause the formation of stained precipitates in the structures of the section cells; visual assessment of the results of the analysis by the nature of the location and concentration of stained precipitates to identify tissue sites with the anaplastic state of the cells, characterized in that the antibody markers according to claim 1 in PBS with a concentration of 0.0013 mg / ml are used as biological markers.

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