RU2329303C2 - Monoclonal antibody immunoreactive with nucleocapsid (core) protein of c-4g5 hepatitis virus, method of diagnostics of hepatitis c viral (hcv) infection, and combination of monoclonal antibodies for its implementation - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: obtaining monoclonal antibodies immunoreactive with the nucleocapsid protein of the hepatitis virus C-4G5, and the development of a method of diagnostics based on the double layer version of immunoenzymometric assay for the detection of the nucleocapsid (core protein) of the hepatitis C virus using a combination of linear and conformational core protein epitope monoclonal antibodies 27, 1F9, and 4G5: MCA 1F9: linear epitope with amino-acid residues (AAR) 81-85, MCA 27, and 4G5: conformational epitopes at site 1-150 of the core protein AAR. The combination of monoclonal antibodies 27, 1F9, and 4G5, immunoreactive with the hepatitis C virus nucleocapsid protein may be used both for the capture (capture antibodies) and for the detection (detection antibodies) of the core protein. The double layer variant of the immunoenzymometric assay developed for the detection of the nucleocapsid of the hepatitis C virus using a combination of monoclonal antibodies including MCA 4G5 has high sensitivity and allows to detect core proteins in blood of both asymptomatic HCV-infected donors and patients suffering from acute and chronic hepatitis C. The advantages of the immunoenzymometric assay of the core protein may be useful for determining the viral load during IF therapy.

EFFECT: allows to diagnose hepatitis C with high probability at early stages of disease and is efficient for detection of HCV of at least three genotypes most actual for Russia.

3 cl, 3 tbl, 4 ex

Description

The invention relates to the field of biotechnology and virology, and relates to the diagnosis of hepatitis C using a sandwich variant enzyme-linked immunosorbent assay based on a combination of monoclonal antibodies specifically reactive with hepatitis C virus core protein (HCV), including 4G5 monoclonal antibodies produced by cultured mouse cultured mouse Mus mus . Musculus.

The specific diagnosis of viral hepatitis C is an urgent medical problem. Currently, an enzyme-linked immunosorbent assay (ELISA) based on the detection of antibodies to viral proteins is the primary method of primary routine diagnosis of viral hepatitis C. However, this test alone is not enough, since specific antibodies are not detected in the early stages after infection with the virus in the so-called phase of the seronegative “window”, which lasts up to several months, as well as in immunodeficiency states. Precise detection of hepatitis C virus and monitoring of confirmed hepatitis C require the determination of direct markers of HCV: RNA and virus-specific proteins. For the quantitative detection of HCV RNA in blood plasma, including quantitative assessment, the method of polymerase chain reaction (PCR), which is highly sensitive, is currently used, but its widespread use in practical healthcare is limited. In recent years, enzyme immunoassay methods have been actively developed to detect the HCV nucleocapsid protein (core protein) in blood serum, which can serve as a definite alternative to PCR. Abroad, several commercial test systems have been proposed for the detection of HCV core protein in blood serum, but in our country they are expensive and inaccessible. There are no domestic test systems for detecting HCV core protein.

A known method for the diagnosis of HCV infection is a sandwich variant enzyme-linked immunosorbent assay using monoclonal antibodies (MCA) to core protein to detect core protein of HCV circulating in the serum and plasma of HCV-infected donors and patients with hepatitis C (Seme K., Poljak M., Babic DZ et al. The role of core antigen detection in management of hepatitis C: a critical review // J. of Clin. Virol. - 2005. - V.32. - P.92-101). In the HCV Core antigen ELISA Test System (Ortho Clinical Diagnostics, Raritan, NJ), 5F11 and 5E3 monoclonal antibodies were used in the ELISA sandwich for capture and detection of core protein, respectively. The inability to detect core protein in the presence of anti-HCV antibodies was one of the significant drawbacks of the first generation test systems, which led to the appearance of second generation diagnosticums. The Total HCV Core antigen ELISA Test System or trak-C (Ortho Clinical Diagnostics) test system is based on the use of four MCAs: two for capture and two for detection of core antigen (MCA C11-3, C11-7 and C11-10 , C11-14, respectively). These monoclonal antibodies and test systems designed on their basis can be selected as the closest analogue to the present invention.

The essence of the invention consists in the creation of monoclonal antibodies immunoreactive with the hepatitis C virus 4G5 nucleocapsid protein, and the development of a diagnostic method based on a sandwich variant of the enzyme immunoassay for the detection of hepatitis C virus nucleocapsid (core protein) using a combination of monoclonal antibodies 27, 1F9 and 4G5 directed to linear and to conformation-dependent epitopes of the core protein. Moreover, the combination of monoclonal antibodies 27, 1F9 and 4G5 immunoreactive with the hepatitis C virus nucleocapsid protein is used both for capture (capture antibodies) and for detection (detecting antibodies) of the core protein.

The essential features of the invention that match the features of the prototype include the following.

1. The monoclonal antibodies that make up the sandwich were obtained by hybridoma technology when immunizing BALB / c mice with recombinant HCV nucleocapsid proteins and specifically interact with the HCV nucleocapsid protein. In a certain combination, they, as part of a sandwich variant of enzyme-linked immunosorbent assay, detect a nucleocapsid protein in the sera of HCV-positive individuals.

2. "Capture" (capture) monoclonal antibodies are adsorbed (applied) to the wells of 96-well plates of polystyrene or other polymeric materials with high sorption ability.

3. Contacting adsorbed on the solid phase of MCA with serum or blood plasma samples, as a result of which, with a positive interaction, an antigen-antibody complex is formed.

4. Detection of a complex of monoclonal antibodies with core antigen by known immunological methods, for example, enzyme immunoassay, radioisotope, chemiluminescent, etc. This is done by applying detecting monoclonal antibodies to a core protein labeled with an enzyme, a radioisotope, and the like.

5. The detection of core protein is a quantitative method. The concentration of core protein in serum samples is estimated by a calibration curve with a recombinant core protein.

The essential features of the invention that differ from the features of the prototype include the following.

1. The use of a combination of MKA 27, 1F9 and 4G5 directed to other epitopes of the HCV core protein: MKA 1F9 to a linear epitope with amino acid residues (amino acid residues) 81-85, MKA 27 and 4G5 to conformation-dependent epitopes on plot 1-150 a.o. core protein (E.A. Rechkina, G.F. Denisova, O.V. Masalova, L.F. Lideman, D.A. Denisov, E.I. Leonova, R.I. Ataullakhanov, S.V. Guryanova, A. A. Kushch. Mapping of antigenic determinants of hepatitis C virus proteins using phage display technology // Molecular Biology, 2006, v.40, No. 2, p.357-368), while MCA as part of the test prototype systems directed to linear epitopes of core protein: a.o. 41-60 (MKA 5F11), 21-40 (MKA 5E3), 100-120 (MKA S11-3), 120-130 (MKA S11-7), 21-40 (MKA S11-10) and 41-50 ( MKA C11-14) (Orito E., Mizokavi M., Tanaka T. et al. Quantification of serum hepatitis C virus core protein level in patients chronically infected with different hepatitis C genotypes. // Gut. - 1996. - V.39 .- P.876-880; Aoyagi K., Ohue C., Iida K. et al. Development of a simple and highly sensitive enzyme immunoassay for hepatitis C virus core antigen. // J Clin Microbiol - 1999. - V.37 . - P.1802-1808).

2. This combination of MCAs is used both as “capturing” antibodies and as detecting enzyme-labeled conjugates, while in the above test systems “capturing” and detecting MCAs are directed to different epitopes of the core protein.

Strain 4G5 producing monoclonal antibodies reacting with the HCV core protein was obtained by hybridization of cultured sp2 / 0 mouse myeloma cells with spleen cells of BALB / c mice immunized with a recombinant HCV core protein with a sequence of 1-150 a.a., corresponding to the genotype (subtype) 1b. Hybrid cells are cultured on a selective GAT medium, clones producing monoclonal antibodies to the HCV core protein are detected. By serial passages, the cells of the most productive 4G5 clone are introduced into mass culture. The strain of hybrid cultured mouse Mus. Musculus 4G5 is stored in the collection of transplantable cell lines at the Research Institute of Virology named after D.I. Ivanovsky RAMS under the number 8/1/1 and is characterized by the following features:

1. Morphological properties: 4G5 cell culture consists of rounded cells of various sizes with large nuclei that are weakly attached to the substrate.

2. Cultural properties: RPMI-1640 medium containing 15-20% fetal calf serum (ETS), 4.5 g / l glucose, 3 mM glutamine, 0.2 u / ml insulin, 50 was used as a growth medium for cultivation mcg / ml gentamicin. The nature of the growth is a stationary suspension, the removal method is shaking. The passivation frequency is 2-3 days. Sowing dose of 200 thousand cells in 1 ml. The sieving ratio is 1: 2-1: 3. The introduction of strain cells at a concentration of 10 ⁷ / ml into the abdominal cavity of mice pre-sensitized for 10-14 days by injection of 0.3-0.5 ml pristan causes them to form ascites tumors after 10-16 days.

3. Cryopreservation of hybridoma. 24 hours before freezing, the hybridoma is subcultured 1: 2. Cells precipitated by centrifugation (1000 rpm, 10 min) are suspended in a cryoprotective medium (90% ETS, 10% DMSO) and poured into ampoules at a concentration of 3-5 mln / ml, 1-1.8 ml each. Store 24 hours at -70 ° C, then transferred to liquid nitrogen.

4. Characterization of immunoglobulins. MAB produced by hybridoma belong to the IgG class, subclass - IgG1.

As samples of antibodies using a culture fluid generated during the cultivation of a hybridoma, or ascites fluid.

Example 1. The activity and specificity of monoclonal antibodies was determined in the solid-phase version of the enzyme immunoassay. Recombinant core protein 1-150 a.a. was adsorbed onto the solid phase. at a concentration of 1 μg / ml, as well as synthetic peptides of various lengths (from 12 to 40 a.o.), overlapping the core protein sequence, at a concentration of 10 μg / ml in 0.05 M carbonate-bicarbonate buffer, pH 9.5 overnight at room temperature. Then washed the wells of the plates (PBS + 0.1% tween-20) and layered culture liquids or ascites of clone 4G5 in serial 5-fold dilutions in blocking solution (1% casein in PBS-tween-20) and incubated for 1 hour at 37 ° C. After washing, goat antibodies were applied against mouse immunoglobulins labeled with peroxidase (DAKO, Denmark). The enzymatic activity of bound peroxidase was detected using a tetramethylbenzidine substrate (TMB). The optical density (OD) of the samples was measured in a two-wave mode — at a wavelength of 450 nm (measurement) and 620 nm (reference). The last dilution was taken as the MCA titer, the OD of which exceeded the negative control OD (MCA for non-structural HCV proteins) 2.5 times.

With recombinant protein 1-150 a.a. the culture fluid of clone 4G5 interacts with a titer of 1: 256-1: 512, ascites fluid interacts with a titer of 1:10 ⁷ . Reactions with peptides are not fixed. This indicates the conformationally dependent nature of the epitope recognized by 4G5 MAB: the amino acid residues forming the epitope are spatially aligned, but are spaced apart in the primary protein structure. Epitope mapping data for MCA 4G5, carried out using phage display technology, confirmed this conclusion. The key motif of amino acid residues that is part of the 4G5 epitope: QSPSRLxD and KQxKExxxE a.o. (E.A. Rechkina, G.F. Denisova, O.V. Masalova, L.F. Lideman, D.A. Denisov, E.I. Leonova, R.I. Ataullakhanov, S.V. Guryanova, A .A. Kushch. Mapping of antigenic determinants of hepatitis C virus proteins using phage display technology // Molecular Biology, 2006, v.40, No. 2, p.357-368).

The invention provides a method for the diagnosis of hepatitis C using a sandwich ELISA using a combination of monoclonal antibodies directed to different epitopes of the HCV core protein.

The development of this method for the diagnosis of hepatitis C was preceded by a study of the immunochemical properties of 7 original MCAs and their epitope mapping. Monoclonal antibodies were obtained from mouse ascites liquids and purified by Protein A - Sepharose CL-4B column affinity chromatography (Pharmacia, Sweden) according to the method recommended by the company. Conjugation of MCA with horseradish peroxidase was carried out by the periodic method. The protein concentration in the purified immunoglobulins (Ig) from the MCA, Ig isotypes was determined, their specific activity was measured in the indirect variant of the solid-phase ELISA during sorption on the solid phase of recombinant core proteins. The study of the epitope specificity of MCA was performed using peptide screening, reciprocal competitive analysis and phage display technology (Masalova O.V., Abdulmejidova A.G., Atanadze S.N., Lakina E.I., Semiletov Yu.A., Burkov A.N., Ulanova T.I., Novikov V.V., Pimenov V.K., Fields G., Khudyakov Yu.E., Kushch A.A. Characterization of the panel of monoclonal antibodies and epitope mapping of hepatitis C virus proteins // Reports of the Academy of Sciences, 2002, Vol. 383, No. 4, pp. 455-550; E.A. Rechkina, G.F. Denisova, O.V. Masalova, L.F. Lideman, D.A. Denisov , E.I. Leonova, R.I. Ataullah Anov, SV Guryanova, AA Kushch. Mapping of antigenic determinants of hepatitis C virus proteins using phage display technology // Molecular Biology, 2006, v.40, No. 2, p.357-368).

Example 2. An experimental test of the ability of 7 original MCAs to core protein in various combinations to detect core protein in blood plasma samples of HCV-positive blood donors.

Antigenic activity of core protein was determined using a sandwich ELISA in plasma samples of blood donors, among which 17 samples contained antibodies to HCV and HCV RNA, 20 samples did not contain these markers (negative control). HCV RNA was determined by polymerase chain reaction (PNR), the presence and spectrum of antibodies to HCV proteins were detected using screening and confirmatory test kits of Diagnostic Systems (Nizhny Novgorod).

Sample preparation for core protein analysis. Polyethylene glycol (PEG) 4000 (Serva, Germany) was added to plasma samples to a final PEG concentration of 4%. A precipitate formed overnight or at least 3 hours at 4 ° C, then the mixture was centrifuged at 12,000 rpm for 30 minutes at 4 ° C. The pellet was resuspended in 0.1 M phosphate-buffered saline, pH 7.4 (PBS) so that the final volume was 1/15 of the original volume of the plasma sample. Then, tween-80 was added to the samples to a final concentration of 0.5% in order to remove the lipid membrane of virions and surface proteins.

MCA for core protein was sorbed into the wells of 96-well Nunc Maxisorp panels (Denmark) at a concentration of 5 μg / ml (50 μl) in a PBS solution overnight at room temperature, washed (PBS + 0.1% Tween-20) and blocked non-specific binding sites with 5% skimmed milk powder on PBS for one hour at 37 ° C. Then, prepared samples (50 μl) were applied and incubated for 2 h at 37 ° С with constant shaking. At the same time, dilutions of the positive control, recombinant core protein, were introduced. After washing for 1 h (37 ° C), conjugates — MCAs — were added to the core protein labeled with horseradish peroxidase in blocking solution (1% casein in PBS-tween-20). The enzymatic activity of bound peroxidase was detected using a tetramethylbenzidine substrate (TMB). The optical density (OD) of the samples was measured in a two-wave mode — at a wavelength of 450 nm (measurement) and 620 nm (reference). Samples were considered positive if their OD exceeded critical. The latter was calculated as the average OD of negative controls (donor plasma not containing anti-HCV) plus two standard deviation values. The amount of core protein in the samples was estimated by a calibration curve with a recombinant core protein, for which a linear dependence of OD on concentration was shown in the range of 0.1-15 ng / ml.

Studies of samples in ELISA showed that of the 25 variants of various combinations of the tested monoclonal antibodies, the combinations consisting of MCA 27, 1F9, 4G5 and 6D1 have the highest sensitivity (table 1). A comparative analysis showed that the combination of MCA 27, 1F9, and 4G5 (in equal proportions) during sorption on the solid phase and in the form of conjugates more effectively reveals core protein in most samples that gave negative results when using individual MCAs. Thus, the use of a combination of MCA data made it possible to achieve a sensitivity of 88% and to detect core protein in 15 of the 17 samples studied. For example, in comparison with the MCA variant 27-27, core protein was additionally detected in 3 samples, and in 3 more the intensity of the positive signal was significantly increased. The sensitivity of detection of recombinant core protein was 300 pg / ml, which, in terms of the initial plasma volume, taking into account 15-fold concentration, was 20 pg / ml. The absence of core protein in 2 samples in the presence of HCV RNA may indicate a low concentration of the virus in the blood. Plasma of HCV-negative donors did not reveal nonspecific interactions with MCAs 27.1F9 and 4G5 (specificity 100%).

Example 3. Detection of HCV core protein in serum and plasma samples of individuals infected with HCV of various genotypes.

HCV is known to have high genetic variation. Genotypes 1 (subtype 1b) and 3 (subtype 3a) are most common in Russia, HCV of genotype 2 is less common. Original MCAs were obtained by immunizing mice with recombinant proteins synthesized in accordance with the amino acid sequence of the core protein of genotype 1b. It was important to find out the possibility of using the developed method for the diagnosis of hepatitis C to detect HCV of different genotypes.

We studied serum and plasma from 57 HCV-positive individuals, among whom were patients with acute and chronic hepatitis C (OGS and CHC) and infected blood donors. Samples were genotyped by PCR using primers from the core protein region. Sample preparation and ELISA were carried out according to the methods described in example 2. In the sandwich used a combination of MCA 27, 1F9 and 4G5.

The detection of core protein in sera with genotypes 1 (1a and 1b) and 3 (3a) was similar and reached 89% (table 2). In samples with genotype 2 (2a, 2b, and 2c), core protein was detected less frequently (in 69% of cases); this indicator, however, did not significantly differ from that for genotypes 1 and 3 (p> 0.05). Thus, the developed method for the diagnosis of hepatitis C effectively detected HCV of at least three genotypes that are most relevant for Russia. The detection of core protein of genotypes 2 and 3 is probably due to the presence of MCA 1F9 in the sandwich: these antibodies, as determined using phage display technology, recognize a portion of the amino acid sequence (81-84 a.a.), conserved in all known HCV genotypes (E.A. Rechkina, G.F. Denisova, O.V. Masalova, L.F. Lideman, D.A. Denisov, E.I. Leonova, R.I. Ataullakhanov, S.V. Guryanova, A .A. Kushch. Mapping of antigenic determinants of hepatitis C virus proteins using phage display technology // Molecular Biology, 2006, v.40, No. 2, p.357-368).

Example 4. Using the developed method for the diagnosis of the early stages of acute hepatitis C.

When infected with hepatitis C virus, a long (up to several months) phase of the seronegative “window” is characteristic; during this period, only direct HCV markers - RNA and virus-specific proteins can be detected. The purpose of the work is to identify HCV core protein in patients with acute hepatitis in the seronegative period.

We studied serum samples from 4 patients with symptoms of acute hepatitis (jaundice, hepatosplenomegaly, an increase in alanine aminotransferase (ALT) by more than 10 times compared to the norm). At the initial screening of sera, antibodies to HCV, markers of other viral hepatitis, and other etiological causes were not found, and therefore, this patient was diagnosed with acute hepatitis of unknown etiology. Next, dynamic monitoring of these patients was carried out.

Sample preparation and sandwich ELISA were performed as described in example 2, using a combination of MCA 27, 1F9 and 4G5. RNA was determined by PCR. The presence and spectrum of antibodies to HCV proteins was detected using screening and confirmatory test kits from Diagnostic Systems (Nizhny Novgorod).

In 2 patients (No. 1 and No. 2) in the absence of anti-HCV antibodies, both core protein and HCV RNA were detected (table 3). Thus, these patients were diagnosed with acute hepatitis C. Subsequently, the diagnosis was confirmed in these patients - HCV seroconversion was noted. In 2 patients (No. 3 and No. 4), neither core protein nor HCV RNA was detected in the samples; dynamic observation of these patients also did not reveal any of the markers of hepatitis C, and therefore the diagnosis of acute hepatitis of unknown etiology remained unchanged.

Thus, the developed sandwich variant of enzyme-linked immunosorbent assay for the detection of hepatitis C virus nucleocapsid using a combination of monoclonal antibodies, including 4G5 MCA, is highly sensitive and allows detecting core protein in the blood of both asymptomatic HCV-infected donors and patients with acute and chronic hepatitis C. A significant coincidence (p <0.05) of the results of determining core protein by this method and HCV RNA by PCR was shown. The proposed method allows you to diagnose with high probability hepatitis C already in the early stages of the disease, namely: at a time when specific antibodies have not yet been detected, and is effective for detecting HCV of at least three genotypes that are most relevant for Russia. The benefits of quantifying core protein may be useful for determining viral load during interferon therapy. Putting the method into practice will increase the reliability of the diagnosis of hepatitis C and the viral safety of blood transfusions.

Table 1
Efficiency of detecting core protein in virions in RNA-positive donor plasmas using various combinations of monoclonal antibodies Option No. Sandwich Composition - MCA Conjugate Efficiency of detecting core protein in virions in RNA-positive donor plasmas one 27-27 12/17 * 71 ** 2 27-4G5 1/5 twenty 3 27-1F9 4/11 36 four 27-6D1 10/15 67 5 D4-27 5/9 56 6 D4-4G5 8/14 57 7 D4-1F9 3/11 27 8 D4-6D1 5/13 38 9 D4-1A12 4/10 40 10 1A12-27 2/10 twenty eleven 1A12-4G5 6/10 60 12 1A12-6D1 6/9 67 13 1A12-1A12 2/14 fourteen fourteen 1F9-27 6/12 fifty fifteen 1F9-1F9 10/14 71 16 1F9-6D1 10/14 71 17 6D1-27 6/13 46 eighteen 6D1-4G5 7/10 70 19 6D1-6D1 6/14 43 twenty 6D1-1A12 4/10 40 21 4G5-27 6/10 60 22 4G5-4G5 5/10 fifty 23 4G5-1F9 3/10 thirty 24 4G5-6D1 6/10 60 25 4G5-1A12 6/11 55 26 27, 1F9, 4G5 - 27, 1F9, 4G5 15/17 88 Note: * the number of core-positive plasmas / the number of investigated RNA-positive plasma donors; ** -% core-positive blood plasma

table 2
Comparative efficacy of detecting core protein in HCV-positive donors and patients infected with HCV of various genotypes HCV genotypes Serum core protein detection efficacy HCV-positive donors Patients OGS and CHC 1a not studied 2/3 1b 21/23 * 8/9 Total in genotype 1 31/35 (88.6%) 2a 1/2 6/9 2b not studied 1/1 2s 1/1 not studied Total in genotype 2 9/13 (69.2%) 3a 4/4 4/5 Total in genotype 3 8/9 (88.9%) Note: * number of core-positive sera / number of tested sera of this genotype

Table 3
The identification of HCV markers in patients with acute hepatitis in dynamics patient no. day from the onset of the disease / jaundice conc. core protein, pg / ml RNA HCV antibodies * ALT (μmol / min · L) Core NS3 NS4 NS5 one 14/10 139 + neg. screening 412 27/23 <20 - 0.8 ** 1,2 0.3 0 135 2 20/18 152 + neg. screening 1200 25/23 512 + 0.5 3,1 0 0 652 31/29 74 n / a 0.7 3.3 0 0 394 53/51 69 + 1,5 3.0 0 0 155 3 14/12 <20 - neg. screening 1050 35/33 <20 - 0 0 0 0 41 2.5 months <20 - 0 0 0 0 39 7 months <20 - 0 0 0 0 thirty four 13/3 <20 - neg. screening 1316 19/9 <20 - 0 0 0 0 700 26/16 <20 - 0 0 0 0 192 4 months <20 - 0 0 0 0 23 Notes: * - the determination of antibodies to HCV was carried out using screening and confirming test systems "Diagnostic systems" (Nizhny Novgorod); neg. screening - total antibodies to HCV were not detected, gender. screening - detected; ** - OD of samples with HCV antigens in ELISA; "-" - not found; n / a - not investigated.

Claims (3)

1. Monoclonal antibody 4G5 produced by a strain of hybrid cultured mouse Mus cells. Musculus 4G5 deposited in the collection of transplantable cell cultures of the Research Institute of Virology named after D.I. Ivanovsky RAMS under the number 8/1/1, immunoreactive with the protein of the nucleocapsid (core protein) of hepatitis C virus (HCV) and specific for the conformation-dependent antigenic determinant in the region of 1-150 amino acid residues of the HCV core protein. 2. A method for the diagnosis of HCV infection, including the detection of hepatitis C virus core protein in the sandwich version of an enzyme-linked immunosorbent assay using a combination of monoclonal antibodies immunoreactive with hepatitis C core protein, characterized in that the monoclonal antibodies 27, 1F9 used in combination and 4G5 are directed to linear and conformation-dependent epitopes (antigenic determinants) of the HCV core protein: MKA 1F9 - to a linear epitope with amino acid residues (a.a.) 81-85, MKA 27 and 4G5 - to conformation-dependent epitopes opam on a plot of 1-150 a.o. core protein. 3. The combination of monoclonal antibodies 27, 1F9 and 4G5, immunoreactive with the hepatitis C virus nucleocapsid protein, used to capture and detect core protein when implementing the method according to claim 2.