Monoclonal Antibody Against HCV Core Antigen, Hybridoma Cell Line and Application

Abstract

The present invention discloses an anti-HCV core antigen monoclonal antibody, a hybridoma cell line and their applications. Specifically, a hybridoma cell line screened against the common conserved sequence of the core proteins of different genotypes of HCV, and the antibody secreted by it is used in the detection of HCV antigens, which can detect hepatitis C viruses of different genotypes, has good inclusiveness, and only two antibodies are needed to achieve the above technical effects. This can not only reduce the production cost of HCV antigen detection kits, but also reduce the production difficulty and improve the production efficiency.

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody against HCV core antigen, a hybridoma cell line and their applications, belonging to the technical fields of molecular biology and infection immunology. Background Art

[0002] Hepatitis C virus (HCV) is the pathogenic factor of hepatitis C and is mainly transmitted through blood transfusion. Chronic HCV infection often leads to liver cirrhosis, and some of these patients may develop hepatocellular carcinoma. Currently, there are mainly three common HCV detection methods:

[0003] (1) HCV antibody detection, which detects HCV antibodies in the serum of patients;

[0004] (2) HCV antigen detection, which detects HCV core antigen in the serum of patients;

[0005] (3) HCV-RNA detection, which determines the presence of the virus by qualitatively or quantitatively detecting HCV RNA.

[0006] HCV-RNA detection is mainly used for the selection and efficacy monitoring of antiviral treatment, but it requires strict environmental control, relatively expensive equipment, the testing personnel need to be professionally trained and obtain corresponding qualifications, and has high requirements for the quality control of samples.

[0007] HCV antibody detection is the most commonly used detection method at present, but its fatal drawback is the existence of a "window period", that is, there is a time period of 40 - 70 days between HCV infection and the production of HCV antibodies. The human body has been infected and is infectious during this period, but the antibody detection reagent cannot detect it at this time. This stage is called the window period before seroconversion after infection (Perseroconversion Window Phase, PWP). The existence of the window period is the main cause of blood transfusion infection. In addition, HCV patients who have been cured or have recovered spontaneously may still have a relatively high concentration of antibodies in their bodies and can be detected. Therefore, antibody detection cannot distinguish between current infection and past infection.

[0008] HCV antigen detection can advance the window period by about 50 days on average and reduce the risk of HCV infection during the window period. Within 1 - 2 days after the appearance of HCV nucleic acid, HCV core antigen will appear in the infected person's body, and there is a certain correlation with the level of HCV nucleic acid, which can be used as a marker for HCV detection. However, there are many genotypes of HCV, and there are significant differences in the amino acid sequences of core antigens among different genotypes, and most of these differences are located in the strong epitope regions. Usually, monoclonal antibodies are obtained by immunizing with the core antigen of the main genotype, and a combination of multiple monoclonal antibodies with different epitopes is used for detection to reduce the missed detection caused by different genotypes, but this will increase the cost and reduce the production efficiency.

[0009] In addition, since patients will produce antibodies about a period of time (average 49 days) after being infected with hepatitis C virus, forming antigen-antibody immune complexes, which leads to a decrease in the free antigen, thus greatly reducing the detection rate of HCV antigen. Summary of the Invention

[0010] The object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide anti-HCV core antigen monoclonal antibodies, hybridoma cell lines and their applications.

[0011] To achieve the above object, the present invention adopts the following technical solutions:

[0012] 1. HCV core antigen, whose amino acid sequence is selected from:

[0013] (A) C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1;

[0014] (B) C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.2.

[0015] 2. A hybridoma cell line 3C2 secreting anti-aforementioned HCV core antigen monoclonal antibody, whose taxonomic name is Hybridoma cell line 3C2, was deposited at the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024355.

[0016] 3. A hybridoma cell line 5E1 secreting anti-aforementioned HCV core antigen monoclonal antibody, whose taxonomic name is Hybridoma cell line 5E1, was deposited at the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024356.

[0017] 4. Anti-HCV core antigen monoclonal antibody, which is secreted by the aforementioned hybridoma cell line and is specific to polypeptides selected from the following amino acid sequences:

[0018] (A) C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1;

[0019] (B) C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.2.

[0020] As one of the preferred technical solutions, the anti-HCV core antigen monoclonal antibody is monoclonal antibody 3C2, and the sequences of the three complementarity-determining regions of its heavy chain variable region are respectively:

[0021] Heavy chain CDR1: GFSLTSYG, as shown in SEQ ID NO.18;

[0022] Heavy chain CDR2: IWAGGTT, as shown in SEQ ID NO.19;

[0023] Heavy chain CDR3: ARERNGMDY, as shown in SEQ ID NO.20;

[0024] The sequences of the three complementarity determining regions of the light chain variable region are respectively:

[0025] Light chain CDR1: SSITY, as shown in SEQ ID NO.21;

[0026] Light chain CDR2: DTS;

[0027] Light chain CDR3: QQWRSDPPT, as shown in SEQ ID NO.22.

[0028] As one of the further preferred technical solutions, the gene encoding the aforementioned heavy chain variable region has the following nucleotide sequence:

[0029] Heavy chain CDR1: GGATTTAGCCTGACCTCCTACGGC, as shown in SEQ ID NO.23;

[0030] Heavy chain CDR2: ATCTGGGCTGGCGGAACCACC, as shown in SEQ ID NO.24;

[0031] Heavy chain CDR3: GCCAGGGAGAGGAATGGCATGGATTAT, as shown in SEQ ID NO.25;

[0032] Light chain CDR1: TCCTCCATCACCTAT, as shown in SEQ ID NO.26;

[0033] Light chain CDR2: GATACCTCC;

[0034] Light chain CDR3: CAGCAGTGGCGGAGCGACCCTCCTACC, as shown in SEQ ID NO.27.

[0035] As one of the further preferred technical solutions, the amino acid sequence of the heavy chain variable region is: QVHLKESGPGLVASSQSLSITCTVS GFSLTSYG LHWVRQPPGKGLEWLGV IWAGGTT NY NSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYC ARERNGMDYWGQGTSVTVSS, as shown in SEQ ID NO.28;

[0036] The amino acid sequence of the light chain variable region is:

[0037] QIVLTQSPAIMSASPGEKVTMTCSAS SSITY MHWYQQKSGTSPKRWIY DTS KLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYC QQWRSDPPT FGGGTKLEIK, as shown in SEQ ID NO.29.

[0038] As one of the preferred technical solutions, the anti-HCV core antigen monoclonal antibody is monoclonal antibody 5E1, and the sequences of the three complementarity-determining regions of its heavy chain variable region are respectively:

[0039] Heavy chain CDR1: GFSITSSYSC, as shown in SEQ ID NO.30;

[0040] Heavy chain CDR2: ICFEGSI, as shown in SEQ ID NO.31;

[0041] Heavy chain CDR3: SRERHWGSFAMDY, as shown in SEQ ID NO.32;

[0042] The sequences of the three complementarity-determining regions of the light chain variable region are respectively:

[0043] Light chain CDR1: KSLLHSNGITY, as shown in SEQ ID NO.33;

[0044] Light chain CDR2: QMS;

[0045] Light chain CDR3: AQNLELP, as shown in SEQ ID NO.34.

[0046] As one of the further preferred technical solutions, the gene encoding the aforementioned heavy chain variable region has the following nucleotide sequence:

[0047] Heavy chain CDR1: GGATTCTCCATCACCAGCAGCTATAGCTGC, as shown in SEQ ID NO.35;

[0048] Heavy chain CDR2: ATCTGTTTCGAGGGCAGCATC, as shown in SEQ ID NO.36;

[0049] Heavy chain CDR3: TCCAGGGAGAGGCACTGGGGCAGCTTCGCTATGGATTAT, as shown in SEQ ID NO.37;

[0050] Light chain CDR1: AAGTCCCTGCTGCACTCCAACGGCATCACCTAT, as shown in SEQ ID NO.38;

[0051] Light chain CDR2: CAGATGAGC;

[0052] Light chain CDR3: GCTCAGAATCTGGAGCTGCCT, as shown in SEQ ID NO.39.

[0053] As one of the further preferred technical solutions, the amino acid sequence of the heavy chain variable region is:

[0054] QIQLKESGPAVIKPSQSLSLTCKVS GFSITSSYSC WHWIRQPPGKGLEWMGR ICFEGSI FYSPSIKSRSTISRDTSLNKLFMQLSSVTREDTAMYYC SRERHWGSFAMDY WGQGTSVTVSS, as shown in SEQ ID NO.40;

[0055] The amino acid sequence of the light chain variable region is:

[0056] DIVMTQAAFSNPVTLGTSASISCRSS KSLLHSNGITY LYWYLQKPGQSPQLLIY QMS NLASGVPDRFSSSGSGADFTLRISRVEAEDVGVYYC AQNLELP PTFGGGTKLEIK, as shown in SEQ ID NO.41.

[0057] As one of the preferred technical solutions, the anti-HCV core antigen monoclonal antibody is monoclonal antibody 3C2, whose heavy chain nucleotide sequence is as shown in SEQ ID NO.3, and the amino acid sequence is as shown in SEQ ID NO.4; the light chain nucleotide sequence is as shown in SEQ ID NO.5, and the amino acid sequence is as shown in SEQ ID NO.6; monoclonal antibody 3C2 is specific to the polypeptide with the following amino acid sequence:

[0058] C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1.

[0059] As one of the preferred technical solutions, the anti-HCV core antigen monoclonal antibody is monoclonal antibody 5E1. Its heavy chain nucleotide sequence is as shown in SEQ ID NO.7, and the amino acid sequence is as shown in SEQ ID NO.8; the light chain nucleotide sequence is as shown in SEQ ID NO.9, and the amino acid sequence is as shown in SEQ ID NO.10; monoclonal antibody 5E1 is specific to the polypeptide with the following amino acid sequence:

[0060] C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.2.

[0061] 5. Application of the aforementioned monoclonal antibody in the preparation of an HCV detection kit.

[0062] Advantages of the present invention:

[0063] The hybridoma cell lines screened in the present invention for the common conserved sequences of the core proteins of different genotypes of HCV, and the antibodies secreted by them are applied to HCV antigen detection, which can detect HCV of different genotypes, has good inclusivity, and only two antibodies are required to achieve the above technical effects. It can not only reduce the production cost of the HCV antigen detection kit, but also reduce the production difficulty and improve the production efficiency. Description of the Drawings

[0064] Figure 1 It is the analysis of the HCV core antigen sequence.

[0065] Deposited Information

[0066] Classification name: Hybridoma cell line 3C2

[0067] Latin scientific name: Hybridoma cell line 3C2

[0068] Deposit number: CCTCC NO: C2024355

[0069] Name of the depository: China Center for Type Culture Collection (CCTCC)

[0070] Address of the depository: Wuhan University, Wuhan, China

[0071] Deposit date: October 15, 2024

[0072] Classification name: Hybridoma cell line 5E1

[0073] Latin scientific name: Hybridoma cell line 5E1

[0074] Deposit number: CCTCC NO: C2024356

[0075] Name of the depositary institution: China Center for Type Culture Collection (CCTCC)

[0076] Address of the depositary institution: Wuhan University, Wuhan, China

[0077] Date of deposit: October 15, 2024 Detailed implementation manners

[0078] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0079] The percentage of the additive being liquid is volume percentage, and the percentage of the additive being solid is mass concentration.

[0080] 1. HCV core antigen sequence analysis and polypeptide synthesis

[0081] Bioinformatics tools are used to align and analyze the core antigen sequences of HCV with different genotypes in NCBI. The core antigen sequences of HCV with different genotypes include: the core antigen sequence of HCV genotype 1, especially the amino acid sequence positions 1 - 191 described in NCBI Reference Sequence: NP_671491.1; the core antigen sequence of HCV genotype 2, especially the amino acid sequence positions 1 - 191 described in NCBI Reference Sequence: YP_001469630.1; the core antigen sequence of HCV genotype 3, especially the amino acid sequence positions 1 - 191 described in NCBI Reference Sequence: YP_001469631.1; the core antigen sequence of HCV genotype 4, especially the amino acid sequence positions 1 - 191 described in NCBI Reference Sequence: YP_001469632.1; the core antigen sequence of HCV genotype 5, especially the amino acid sequence positions 1 - 191 described in NCBI Reference Sequence: YP_001469633.1; the core antigen sequence of HCV genotype 6, especially the amino acid sequence positions 1 - 191 described in NCBI Reference Sequence: YP_001469634.1; the core antigen sequence of HCV genotype 7, especially the amino acid sequence positions 1 - 191 described in GenBank: ABN05226.1. See specifically Figure 1 .

[0082] After comparison, the 22nd to 35th and 53rd to 63rd positions of different genotypes are common conserved sequences, and their amino acid sequences are VKFPGGGQIVGGVY (SEQ ID NO.1) and SERSQPRGRRQ (SEQ ID NO.2), respectively. The applicant named them C22-35 and C53-63, respectively. A third party agency (Jiangsu GenScript Biotechnology Co., Ltd.) was commissioned to synthesize peptides C22-35 and C53-63, and the third party agency coupled part of the synthesized peptides to KLH and part to BSA.

[0083] 2. Preparation of monoclonal antibodies against HCV core antigen conserved sequence peptides

[0084] 2.1 Animal immunization

[0085] 2.1.1 First immunization: Take the above KLH-coupled polypeptide (C22-35 or C53-63), adjust its concentration to 2 mg / ml and mix it with Freund's complete adjuvant (purchased from Sigma) in equal amounts. After being fully mixed and emulsified by an emulsifier, inject it subcutaneously on the back of 6-8 week old Balb / C female mice (Hunan Slake Jingda Experimental Animal Co., Ltd.) at several points. The immunization dose is 50 μg per mouse.

[0086] 2.1.2 Second immunization: 12 days later, KLH-coupled polypeptide emulsified with Freund's incomplete adjuvant (purchased from Sigma) was injected subcutaneously at the back, with an immunization dose of 50 μg per mouse.

[0087] 2.1.3 The third immunization: 12 days later, KLH-coupled polypeptide without adjuvant was injected subcutaneously at the back, with an immunization dose of 50 μg per mouse;

[0088] 2.1.4 Fourth immunization: 2 days after the third immunization, blood was collected from the tail vein of mice, centrifuged at 4000g for 5 min at 4°C, and the supernatant was taken for detection of serum titer by indirect ELISA. Two days before fusion, another booster immunization was performed by intraperitoneal injection of KLH-coupled polypeptide without adjuvant, with an immunization dose of 100 μg per mouse.

[0089] 2.2 Cell fusion, positive hybridoma cell screening and subcloning

[0090] Select SP2 / 0 myeloma cells in good growth condition (Yangzhou University) and spleen cells of immunized mice, mix them at a ratio of 1:5 to 1:10, centrifuge at 1500 g for 5 min, and discard the supernatant. Gently shake the centrifuge tube to evenly disperse the cells, slowly add 1 mL of pre-warmed PEG1500 (purchased from Sigma) at 37 °C and mix gently. After fusing for 90 s, add 20 mL of 1640 medium (purchased from Sigma) to terminate. After centrifuging at 800 g for 5 min, discard the supernatant, and add 20% serum-containing HAT-1640 medium (purchased from Gibco) to the cell pellet and mix well.

[0091] Mix the cells evenly and spread them on a 96-well plate. Observe the fusion effect after culturing in a 37 °C CO2 incubator for 5 to 7 days and change the medium. On the 4th day after changing the medium, detect the supernatant of hybridoma cells by the indirect ELISA method. Select the wells with high positive values and few cell colonies, and perform subcloning by the limiting dilution method. The theoretical number of cells per well is 1 to 2. After 3 to 4 subclonings and detection by indirect ELISA, screen for hybridoma cell lines that can stably secrete the corresponding monoclonal antibody.

[0092] 2.3 Indirect ELISA detection

[0093] Dilute BSA-conjugated polypeptide (C22-35 or C53-63) with carbonate coating buffer, coat the ELISA plate, with a concentration of 0.1 μg / ml, coat 100 μL per well, and incubate overnight at 4 °C; wash the plate 2 times with PBS (0.01 M, pH 7.2, the same below), 300 μL per well; add 150 μl per well of blocking solution (PBS containing 1% BSA) and block at 4 °C for 6 hours; discard the blocking solution in the wells, dry at a temperature of 18 °C to 26 °C and a humidity not higher than 30% for 4 hours, and store in an aluminum foil bag under vacuum at 2 °C to 8 °C for later use.

[0094] Usage method: Take the prepared ELISA plate above, add 100 μL of dilution solution (PBS containing 1% BSA and 0.5% Triton X-100) to each well, then add 10 μL of cell culture supernatant (or mouse serum) respectively, incubate at 37 °C for 30 min, and discard the liquid. Wash the plate with 1×PBST (pH 7.2), 300 μL per well, and repeat the washing 5 times. Invert the washed ELISA plate on absorbent paper and pat it to remove excess residual liquid. Add 100 μL per well of HRP-labeled anti-mouse IgG antibody (Luoyang Bai Aotong Experimental Materials Center), incubate at 37 °C for 30 min. Wash the plate with 1×PBST, 300 μL per well, and repeat the washing 5 times. Add 100 μL per well of TMB substrate chromogenic solution, incubate at 37 °C in the dark for 10 min. Add 50 μL per well of stop solution (2 M H2SO4), mix well to terminate the reaction. Detect the OD value at a wavelength of 450 nm with an ELISA reader and read the value within 10 minutes after termination.

[0095] A total of 11 hybridoma cell lines capable of stably secreting monoclonal antibodies against HCV core antigen (HCV-cAg mAbs) were obtained by indirect ELISA. Among them, the mAbs secreted by 5 cell lines with clone numbers 1D12, 1G11, 3C2, 4D9, and 4H7 (the numbers of the mAbs are the same as the clone numbers of the corresponding cell lines, the same below) could specifically react with C22-35, and the mAbs secreted by 6 cell lines with clone numbers 5E1, 7B11, 8A7, 8B2, 9G1, and 10B1 could specifically react with C53-63.

[0096] 3. Antibody Pair Screening

[0097] 3.1 Double Antibody Sandwich ELISA Detection

[0098] 3.1.1 Horseradish Peroxidase Labeling of HCV-cAg mAb: Referring to the periodate oxidation method in "Antibody Engineering (Second Edition)" (published by Peking University Medical Press in 2002), horseradish peroxidase (HRP) was labeled on the antibody.

[0099] 3.1.2 Coating the ELISA Plate with HCV-cAg mAb: Dilute the antibody with phosphate buffer and coat the ELISA plate at a concentration of 0.1 μg / ml, 200 μL per well, overnight at 4°C; wash the plate twice with PBS, 300 μL per well; add 220 μl / well of blocking solution (PBS containing 1% BSA) and block at 4°C for 6 hours; discard the blocking solution in the wells and dry at a temperature of 18°C - 26°C and a humidity not higher than 30% for 4 hours, then vacuum pack in an aluminum foil bag and store at 2 - 8°C for later use.

[0100] 3.1.3 Double Antibody Sandwich ELISA Detection Procedure: After adding 100 μL of dilution solution (PBS containing 1% BSA and 0.5% TritonX-100) to each well, add 100 μL of the sample to be tested respectively, incubate at 37°C for 90 min, and discard the liquid. Wash the ELISA plate with 1×PBST, 300 μL per well, and repeat the washing 5 times. Invert the washed ELISA plate on absorbent paper and pat to remove excess residual liquid. Add 200 μL / well of HRP-labeled HCV-cAg mAb and incubate at 37°C for 30 min. Wash the plate with 1×PBST, 300 μL per well, and repeat the washing 5 times. Add 200 μL / well of TMB substrate chromogenic solution and incubate at 37°C in the dark for 10 min. Add 50 μL / well of termination solution (2M H2SO4) and mix well to terminate the reaction. Measure the OD value at a wavelength of 450 nm with an ELISA reader and read the value within 10 minutes after termination.

[0101] 3.2 Antibody Pair Screening Using Recombinant Antigen

[0102] Five monoclonal antibodies against C22-35 and six monoclonal antibodies against C53-63 were paired and combined respectively (antibodies against the same immunogen were not paired for testing). Using the double-antibody sandwich ELISA detection method (i.e., one monoclonal antibody was coated on the microplate and the other was labeled with horseradish peroxidase), a solution with a certain concentration was prepared using the full-length recombinant antigen of the HCV core region (provided by Hunan Kangrun Bioengineering Co., Ltd.) as the test sample to screen for suitable antibody pairs. See Tables 1 and 2 for details.

[0103] Table 1 Detection data for antibody pair screening

[0104]

[0105] Table 2 Detection data for antibody pair screening

[0106]

[0107] From the results, the paired detection value of monoclonal antibody 3C2 and 5E1 was the highest. Especially when 3C2 was used for coating and 5E1 was used for labeling, the effect was better. The applicant deposited the hybridoma cells secreting these two monoclonal antibodies at the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on October 15, 2024. The cells secreting monoclonal antibody 3C2 were named hybridoma cell line 3C2, and the deposit number was CCTCC NO:C2024355; the cells secreting monoclonal antibody 5E1 were named hybridoma cell line 5E1, and the deposit number was CCTCC NO:C2024356.

[0108] 4. Application of antibodies in chemiluminescence immunoassay with magnetic microparticles

[0109] 4.1 Preliminary establishment of a method for chemiluminescence immunoassay of HCV core antigen with magnetic microparticles

[0110] 4.1.1 Coating magnetic microparticles with HCV-cAg monoclonal antibody: Wash the magnetic microparticles once with a 50 mmol / L 2-morpholinoethanesulfonic acid solution at pH 6.0. Mix the magnetic microparticles with a solution of 10 mg / mL N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg / mL N-hydroxysulfosuccinimide to a final concentration of 20 μL / mg, and place it on a magnetic stirrer at room temperature for rotary reaction for 30 min. Then mix it with HCV-cAg monoclonal antibody 3C2 to a final concentration of 20 μg / mg, and place it on a magnetic stirrer at room temperature for rotary reaction for 3 h. Then add a 0.01 M phosphate buffer solution (pH 7.2) containing 1% BSA, and place it on a magnetic stirrer at room temperature for rotary reaction for 3 h again. Finally, store the solution at 2°C - 8°C for standby.

[0111] 4.1.2 Acridinium Ester-Labeled Anti-HCV-cAg Monoclonal Antibody: Mix anti-HCV-cAg monoclonal antibody 5E1 with 5 mmol / L acridinium ester to a solution with a final antibody concentration of 0.5 mg / mL. Place it on a magnetic stirrer and rotate for 30 min at room temperature. Add PBS containing 1% glycine, and place it on a magnetic stirrer and react for 30 min at room temperature in the dark. Then transfer the obtained acridinium ester-antibody conjugate into a dialysis bag, dialyze it overnight with PBS, and collect the product and store it in the dark at -20 °C for standby.

[0112] 4.1.3 Working Solution of Anti-HCV-cAg Monoclonal Antibody Coated Magnetic Particles: Dilute the anti-HCV-cAg monoclonal antibody coated magnetic particles prepared in 4.1.1 to a suitable proportion to obtain the working solution of anti-HCV-cAg monoclonal antibody coated magnetic particles. The preliminary formula of the diluent used is: 20 mM phosphate buffer (pH 7.2), 150 mM NaCl, 0.5% trehalose, 10% horse serum, 1% Triton X-100.

[0113] 4.1.4 Working Solution of Acridinium Ester-Labeled Anti-HCV-cAg Monoclonal Antibody: Dilute the acridinium ester-labeled anti-HCV-cAg monoclonal antibody prepared in 4.1.2 to a suitable proportion to obtain the working solution of acridinium ester-labeled anti-HCV-cAg monoclonal antibody. The formula of its diluent is: 20 mM phosphate buffer (pH 7.2), 150 mM NaCl, 0.5% trehalose, 1% BSA, 0.5% Triton X-100.

[0114] 4.1.5 Preliminary Establishment of Detection Procedure: Mix 100 μL of the sample to be tested, 50 μL of sample diluent (preliminary formula: 20 mM phosphate buffer (pH 7.2), 150 mM NaCl, 1.2% CHAPS, 1% SDS, 0.5% Triton X-100), and 50 μL of the working solution of anti-HCV-cAg monoclonal antibody coated magnetic particles. Incubate at 37 °C for 20 min, then wash 4 times with the washing solution (produced by the applicant, medical device record number: Xiangyue Yubei 20210009). Then add 100 μL of the working solution of acridinium ester-labeled anti-HCV-cAg monoclonal antibody and incubate at 37 °C for 8 min, wash 4 times with the washing solution, and then add 50 μL each of the pre-excitation solution (produced by the applicant, medical device record number: Xiangyue Yubei 20210008) and the excitation solution (produced by the applicant, medical device record number: Xiangyue Yubei 20210010), and detect the luminescence value (RLU value) with a chemiluminescence analyzer.

[0115] 4.1.6 Detection: Using the above procedure, detect the solutions with full-length recombinant antigen concentrations of 1 ng / mL and 0.5 ng / mL of the HCV core region and two negative sera N1 and N2 by the checkerboard titration method, and the results are shown in Table 3.

[0116] Table 3 Chessboard titration test results

[0117]

[0118] It can be seen from the data in Table 3 that the initially established magnetic particle chemiluminescence method for detecting hepatitis C virus core antigen can well detect HCV core antigen in samples.

[0119] 4.2 Optimization of magnetic microparticle chemiluminescence detection of HCV core antigen

[0120] 4.2.1 Preparation of HCV core antigen antibody complex: Add equal volume of 2 ng / mL HCV core region full-length recombinant antigen to PBS solutions containing HCV core region polyclonal antibody (provided by Hunan Kangrun Bioengineering Co., Ltd.) at concentrations of 200, 100, 50, 25, 10, 5, 2.5, and 0 ng / mL, respectively, and neutralize at 37°C for 60 min before using the method established in 3.1 above (3C2 coating, 5E1 enzyme labeling) to detect HCV core antigen (HCV-cAg). The results are shown in Table 4. When the concentration of the fixed HCV core region full-length recombinant antigen was 1 ng / mL, the amount of antigen that could be detected (OD value) gradually decreased with the increase in the concentration of the polyclonal antibody, and when the detected OD value was less than 0.12, it indicated that the antigen was completely neutralized.

[0121] Table 4 Detection data after neutralization of antigen by antibodies at different concentrations

[0122]

[0123] As shown in Table 4, the solutions with final antibody concentrations of 0 ng / mL and 100 ng / mL and antigen concentrations of 1 ng / mL were used as unneutralized samples and completely neutralized samples, respectively.

[0124] 4.2.2 Study on the formula of sample diluent and magnetic bead diluent for chemiluminescence detection

[0125] Since most clinically HCV-positive samples are antibody-positive samples, the HCV core antigen in the sample exists in the form of an antigen-antibody complex. The applicant envisions dissociating the antigen-antibody complex to make the HCV core antigen free. Therefore, the applicant adjusted the detection procedure: 100 μL of the sample to be tested was mixed with 50 μL of the sample diluent, incubated at 37°C for 5 minutes, and then 50 μL of HCV-cAg monoclonal antibody coated magnetic microparticle working solution was added to mix, incubated at 37°C for 15 minutes, washed 4 times with cleaning solution, and then 100 μL of acridinium ester labeled HCV-cAg monoclonal antibody working solution was added to incubate at 37°C for 8 minutes, washed 4 times with cleaning solution, and then 50 μL of pre-excitation solution and excitation solution were added, and the RLU value was obtained by chemiluminescence detection.

[0126] Since the reaction of specific binding between antigen and antibody to form an antigen-antibody complex is reversible, by changing conditions such as the pH value and ionic strength of the reaction system, the antibody and antigen can be dissociated. Therefore, the sample diluent should be conducive to the dissociation of the antigen-antibody complex, while the magnetic bead diluent should make the reaction environment conducive to the formation of the antigen-antibody complex. For this purpose, the applicant set different formulation combinations of the sample diluent and the magnetic bead diluent (for preparing the working solution of HCV-cAg monoclonal antibody-coated magnetic microparticles) for research (see Table 5 for details).

[0127] Table 5 Different formulation combinations of the sample diluent and the magnetic bead diluent

[0128]

[0129]

[0130] The HCV-cAg monoclonal antibody-coated magnetic microparticles prepared in 4.1.1 were diluted into the HCV-cAg monoclonal antibody-coated magnetic microparticle working solution at a unified ratio with the magnetic bead diluent of each combination. Using the PBS solution and the unneutralized sample and the fully neutralized sample prepared in the above 4.2.1, the detection was carried out according to the adjusted detection procedure. The results are shown in Table 6 for details.

[0131] Table 6 Detection results of different formulation combinations

[0132]

[0133] From the analysis of the above results, it can be seen that when the sample diluent is acidic and the magnetic bead diluent is alkaline, it is conducive to dissociating the antigen-antibody complex and detecting HCV-cAg in the fully neutralized sample. The combination of the sample diluent prepared with 0.1M Gly-HCl buffer with a pH value of 2.2 - 3.6 and the magnetic bead diluent prepared with 20mM phosphate buffer with a pH value of 7.2 - 8.0, or the combination of the sample diluent prepared with 0.1M Gly-HCl buffer with a pH value of 3.0 - 3.6 and the magnetic bead diluent prepared with 0.1M Gly-NaOH buffer with a pH value of 8.8 - 9.2, can both detect HCV-cAg (RLU≥6000) in the fully neutralized sample. In particular, the combination of the sample diluent prepared with 0.1M Gly-HCl buffer with a pH value of 2.2 - 3.0 and the magnetic bead diluent prepared with 20mM phosphate buffer with a pH value of 7.2 - 7.6 has a better effect.

[0134] From the perspective of the mean ratio of the detection results of the fully neutralized sample to the un-neutralized sample (M2 / M1), the highest is only 40%. The possible reason for the analysis is that the mixing of the sample to be tested and the sample diluent dissociates the antigen-antibody complex. When the working solution of HCV-cAg monoclonal antibody-coated magnetic microparticles is added, the reaction environment is conducive to antigen-antibody binding at this time. The HCV-cAg monoclonal antibody has a competitive advantage compared with the antibodies originally present in the sample and can bind to the corresponding epitopes on the HCV core antigen, capturing most of the antigens. However, at this time, the acridinium ester-labeled HCV-cAg monoclonal antibody has not been added, and most of the epitopes recognized by the acridinium ester-labeled HCV-cAg monoclonal antibody on the antigen are re-bound by the antibodies originally present in the sample. The applicant boldly hypothesizes that a certain amount of HCV-cAg fragment containing the epitope recognized by the acridinium ester-labeled HCV-cAg monoclonal antibody but not containing the epitope recognized by the HCV-cAg monoclonal antibody coated on the magnetic microparticles is added to the sample diluent or (and) the magnetic bead diluent to bind the antibodies originally present in the sample that can bind to the epitope recognized by the acridinium ester-labeled HCV-cAg monoclonal antibody.

[0135] Based on this, the inventor used antibody 3C2 to coat magnetic microparticles, labeled antibody 5E1 with acridinium ester, and selected polypeptide C53-63 for further optimization research based on combination C in Table 5. See Table 7 for details.

[0136] Table 7 Different formulation optimization combinations of sample diluent and magnetic bead diluent

[0137]

[0138]

[0139] Table 8 Detection results of different formulation optimization combinations

[0140]

[0141] By analyzing the detection results (Table 8), the applicant surprisingly found that adding a certain amount of HCV-cAg fragment C53-63 containing the epitope recognized by the acridinium ester-labeled HCV-cAg monoclonal antibody 5E1 but not containing the epitope recognized by the HCV-cAg monoclonal antibody 3C2 coated on the magnetic microparticles to the sample diluent or the magnetic bead diluent is indeed beneficial to the detection of HCV-cAg in the fully neutralized sample.

[0142] 4.2.3 Accelerated stability test

[0143] Select the above combinations C4 and C8 to prepare the corresponding solutions, store them at 37°C for 0 days, 3 days, and 6 days, and then use the detection procedure described in 4.2.2 to detect 5 positive sera (P1 - P5) and 5 negative sera (N1 - N5).

[0144] Table 9 Results of accelerated stability test

[0145]

[0146] It is not difficult to see from the detection results (Table 9) that combination C8 has better stability.

[0147] 4.2.4 Summary

[0148] In summary, monoclonal antibody 3C2 is used for coating magnetic microparticles, monoclonal antibody 5E1 is used for labeling acridinium ester, sample diluent and magnetic bead diluent are prepared using formulation B, and C53-63 with a concentration of 4 - 8 ng / mL is added to the sample diluent or magnetic bead diluent. During detection, the sample and the sample diluent are first mixed and reacted, and then the working solution of HCV-cAg monoclonal antibody-coated magnetic microparticles is added for reaction, which has good HCV-cAg detection effect. Accordingly, the applicant trial-produced an HCV-cAg magnetic microparticle chemiluminescence detection kit and further carried out performance evaluation.

[0149] 4.3 Detection performance evaluation

[0150] 4.3.1 Inclusivity

[0151] The detection results of 15 clinical serum samples of hepatitis C virus covering different genotypes such as 1a, 1b, 2a, 3a, 3b, genotype 4, genotype 5, and genotype 6 were all positive, indicating that the HCV-cAg magnetic microparticle chemiluminescence detection kit has good inclusivity for samples of the main hepatitis C virus subtypes. The results are shown in Table 10.

[0152] Table 10 Detection results of clinical serum samples of hepatitis C virus with different genotypes

[0153] Sample Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Genotype 1a 1a 1b 1b 1b 2a 2a 2a 3a 3b 3b Type 4 Type 5 Type 6 Type 6 Test Result + + + + + + + + + + + + + + +

[0154] 4.3.2 Sensitivity

[0155] 4.3.2.1 Detection of HCV serum conversion panel samples

[0156] The HCV-cAg detection was performed on samples of the HCV serum conversion panel (SeraCare Life Sciences, USA, catalog number PHV929). Starting from the 14th day, the samples were positive (see Table 11 for details).

[0157] Table 11 Results of detecting samples of HCV serum conversion panel PHV929

[0158]

[0159] 4.3.2.2 Detection of HCV antibody-positive samples

[0160] A total of 100 HCV antibody-positive samples were respectively tested for HCV-RNA and HCV-cAg. Among them, 72 samples were HCV-RNA positive, and 69 of them were HCV-cAg positive. Taking the HCV-RNA test result as the standard, the positive detection rate of the HCV-cAg magnetic particle chemiluminescence detection kit reached 95.83% (69 / 72). See Table 12 for details.

[0161] Table 12 Detection Results of HCV Antibody-Positive Samples

[0162]

[0163] 4.3.3 Specificity

[0164] Using the above HCV-cAg magnetic particle chemiluminescence detection kit to detect 52 serum samples positive for hepatitis B surface antigen (HBsAg), 36 serum samples positive for Treponema pallidum antibody, 46 serum samples with high triglyceride (≤30mg / ml), 25 serum samples with high bilirubin (≤0.2mg / ml), 33 serum samples with high hemoglobin (≤5mg / ml), and 18 serum samples positive for a series of pregnancy teratogenic pathogens (3 cases of Toxoplasma gondii, 7 cases of cytomegalovirus, 3 cases of herpes simplex virus, 5 cases of rubella virus), the results were all negative, proving that the HCV-cAg magnetic particle chemiluminescence detection kit has extremely high specificity.

[0165] 5. HCV Monoclonal Antibody Sequencing Analysis

[0166] 5.1 Antibody Gene Sequencing

[0167] Total mRNA of hybridoma cells 3C2 and 5E1 was extracted by the Trizol method, and the first-strand cDNA of the antibody-encoding gene was reverse-transcribed using the PrimeScript TM RT reagent Kit with gDNA Eraser kit (TaKaRa, Dalian). Using the reverse-transcribed cDNA as a template, polymerase chain reaction (PCR) amplification was performed using the Trans Start Fast Pfu Fly DNA Polymerase kit (TransGen Biotech Co., Ltd., Beijing) with the forward primer Forward-V in the primer sequence table H and the reverse primer Reverse-V H to amplify the murine heavy-chain variable region gene fragment (V H ); using the forward primer Forward-V in the primer sequence table L and the reverse primer Reverse-V LAmplify the gene fragment of the variable region of the murine heavy chain antibody (V L ).

[0168] The primer sequences are shown in Table 13.

[0169] Table 13. Primer sequences

[0170]

[0171] Recover the PCR target product, clone and ligate it to the T vector, transform Escherichia coli E. coli DH5α, select positive clones and entrust Nanjing Zhongding Biotechnology Co., Ltd. to perform sequencing. The sequencing results are as follows (the underlined part is the antibody variable region sequence):

[0172] Nucleotide sequence of the heavy chain of monoclonal antibody 3C2 (SEQ ID NO.3):

[0173] CAGGTTCACCTGAAGGAGAGCGGCCCAGGACTGGTTGCTTCCTCTCAGAGCCTGTCCATCACCTGCACC GTGTCCGGATTTAGCCTGACCTCCTACGGCCTGCACTGGGTTAGACAGCCTCCTGGAAAGGGCCTGGAGTGGTTGGG AGTGATCTGGGCTGGCGGAACCACCAATTACAATTCCGCTCTGATGAGCAGGCTGTCCATCTCCAAGGACAAGAGCA AGAGCCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGATGATACCGCCATGTATTACTGCGCCAGGGAGAGGAAT GGCATGGATTATTGGGGCCAGGGCACCTCCGTGACCGTTTCCTCCGCTAAGACCACCCCACCCAGCGTTTACCCCTTGGCTCCTGGAAGCGCTGCTCAGACAAACAGCATGGTGACCCTGGGCTGTCTGGTGAAGGGCTATTTCCCCGAGCCCGTGACCGTGACATGGAATTCCGGCTCCCTGAGCTCCGGCGTTCACACCTTTCCTGCTGTGCTGCAGTCCGACCTGTACACCCTGTCCAGCTCCGTGACCGTGCCTTCTAGCACCTGGCCATCCGAGACCGTGACCTGTAATGTGGCTCACCCTGCCTCCAGCACCAAGGTTGACAAGAAGATCGTGCCTAGGGATTGTGGCTGTAAGCCTTGCATCTGCACCGTGCCTGAGGTGTCCAGCGTGTTCATCTTCCCTCCTAAGCCTAAGGATGTGCTGACCATCACCCTGACCCCTAAGGTGACCTGCGTGGTGGTGGACATCTCCAAGGATGACCCCGAGGTGCAGTTCAGCTGGTTCGTGGACGACGTGGAGGTGCACACCGCTCAGACACAGCCTAGAGAGGAGCAGTTCAACTCCACCTTCCGGTCCGTGAGCGAGCTGCCTATCATGCACCAGGACTGGCTGAACGGCAAGGAGTTTAAGTGCCGGGTGAATTCCGCTGCTTTTCCCGCCCCAATCGAGAAGACCATCTCCAAGACCAAGGGCCGGCCCAAGGCTCCTCAGGTGTATACAATCCCTCCTCCCAAGGAGCAGATGGCTAAGGACAAGGTGTCCCTGACCTGCATGATCACCGACTTCTTTCCTGAGGACATCACCGTGGAGTGGCAGTGGAACGGCCAGCCTGCTGAAAACTACAAGAACACCCAGCCCATCATGGACACCGATGGCAGCTATTTCGTGTACTCCAAGCTGAACGTGCAGAAGAGCAACTGGGAGGCTGGCAATACCTTTACCTGTTCCGTGCTGCACGAGGGCCTGCATAATCACCACACCGAGAAGAGCCTGTCCCACTCCCCTGGAAAG

[0174] Amino acid sequence of the heavy chain of monoclonal antibody 3C2 (SEQ ID NO.4):

[0175] QVHLKESGPGLVASSQSLSITCTVSGFSLTSYGLHWVRQPPGKGLEWLGVIWAGGTTNYNSALMSRLSI SKDKSKSQVFLKMNSLQTDDTAMYYCARERNGMDYWGQGTSVTVSS AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK

[0176] Nucleotide sequence of the light chain of monoclonal antibody 3C2 (SEQ ID NO.5):

[0177] CAGATCGTGCTGACCCAGTCCCCAGCTATCATGTCCGCCAGCCCAGGAGAGAAGGTGACCATGACCTGC TCCGCTTCCTCCTCCATCACCTATATGCACTGGTACCAGCAGAAGAGCGGCACCAGCCCAAAGAGATGGATCTACGA TACCTCCAAGCTGGCCTCCGGCGTGCCTGCTAGGTTTTCTGGAAGCGGCTCCGGCACCAGCTACTCTTTGACCATCT CCAGCATGGAGGCCGAGGACGCTGCTACATACTATTGCCAGCAGTGGCGGAGCGACCCTCCTACCTTTGGAGGAGGA ACCAAGCTGGAGATCAAG GCTGATGCTGCTCCCACCGTGAGCATCTTCCCTCCTTCCTCCGAGCAGCTGACCTCCGGAGGAGCTTCTGTGGTGTGCTTTCTGAATAATTTCTACCCTAAGGACATCAACGTGAAGTGGAAGATCGATGGCTCCGAGCGGCAGAATGGCGTGTTGAACTCCTGGACCGATCAGGATAGCAAGGATAGCACCTACAGCATGTCCTCCACCCTGACCCTGACCAAGGACGAGTATGAGCGGCACAACTCCTACACCTGCGAGGCTACCCACAAGACCAGCACCAGCCCTATCGTGAAGAGCTTTAACCGGAACGAGTGC

[0178] Amino acid sequence of the light chain of monoclonal antibody 3C2 (SEQ ID NO.6):

[0179] QIVLTQSPAIMSASPGEKVTMTCSASSSITYMHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTS YSLTISSMEAEDAATYYCQQWRSDPPTFGGGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0180] Monoclonal antibody 5E1 heavy chain nucleotide sequence (SEQ ID NO.7):

[0181] CAGATCCAGCTGAAGGAGTCCGGCCCAGCTGTGATCAAGCCTAGCCAGTCCCTGAGCCTGACCTGCAAG GTGAGCGGATTCTCCATCACCAGCAGCTATAGCTGCTGGCACTGGATCCGGCAGCCTCCTGGAAAGGGACTGGAGTG GATGGGCAGGATCTGTTTCGAGGGCAGCATCTTTTACTCCCCTAGCATCAAGAGCCGGAGCACCATCTCCCGGGACA CATCTCTGAATAAGCTGTTCATGCAGCTGAGCTCCGTGACCAGGGAGGACACAGCTATGTATTACTGTTCCAGGGAG AGGCACTGGGGCAGCTTCGCTATGGATTATTGGGGCCAGGGCACCTCCGTGACCGTTTCCTCCGCTAAGACCACCGCCCCAAGCGTTTACCCCTTGGCTCCAGTGTGTGGCGACACCACAGGCTCTAGCGTGACCTTGGGCTGCCTGGTTAAGGGCTACTTCCCTGAGCCTGTGACCCTGACCTGGAATAGCGGCAGCCTGTCCTCCGGAGTGCACACCTTTCCCGCTGTGCTGCAGTCCGATCTGTACACCCTGAGCAGCAGCGTGACCGTGACCTCTTCCACCTGGCCTTCCCAGTCCATCACCTGTAACGTGGCCCACCCCGCTAGCTCTACCAAGGTTGACAAGAAGATCGAGCCTCGGGGCCCTACCATCAAGCCATGTCCACCTTGCAAGTGTCCCGCTCCCAACCTGCTGGGAGGACCTTCCGTTTTCATCTTTCCTCCTAAGATCAAGGATGTGCTGATGATCTCCCTGTCCCCAATCGTGACCTGTGTGGTGGTGGATGTGAGCGAGGACGACCCAGACGTGCAGATCAGCTGGTTTGTGAACAATGTGGAGGTGCACACCGCTCAGACCCAGACACACAGGGAGGACTATAATTCCACCCTGAGGGTGGTGAGCGCCCTGCCTATTCAGCACCAGGACTGGATGAGCGGCAAGGAGTTCAAGTGTAAGGTGAACAACAAGGACCTGCCCGCCCCTATCGAGAGAACCATCAGCAAGCCCAAGGGCTCCGTGAGGGCTCCTCAGGTGTACGTTCTGCCTCCTCCTGAGGAGGAGATGACCAAGAAGCAGGTGACCCTGACATGTATGGTGACCGATTTCATGCCTGAGGACATCTATGTGGAGTGGACCAACAACGGCAAGACCGAGCTGAACTATAAGAATACCGAGCCCGTGCTGGACAGCGACGGATCCTATTTTATGTACAGCAAGCTGAGGGTGGAGAAGAAGAACTGGGTGGAGCGGAATAGCTACAGCTGCAGCGTGGTGCACGAGGGACTGCATAACCACCACACCACCAAGTCCTTCAGCCGGACCCCAGGAAAG

[0182] Amino acid sequence of the heavy chain of monoclonal antibody 5E1 (SEQ ID NO.8):

[0183] QIQLKESGPAVIKPSQSLSLTCKVSGFSITSSYSCWHWIRQPPGKGLEWMGRICFEGSIFYSPSIKSRS TISRDTSLNKLFMQLSSVTREDTAMYYCSRERHWGSFAMDYWGQGTSVTVSS AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0184] Nucleotide sequence of the light chain of monoclonal antibody 5E1 (SEQ ID NO.9):

[0185] GACATCGTGATGACCCAGGCCGCTTTTAGCAATCCCGTGACCCTGGGCACCTCCGCTTCTATTAGCTGC AGGAGCTCCAAGTCCCTGCTGCACTCCAACGGCATCACCTATCTGTACTGGTACCTGCAGAAGCCTGGCCAGAGCCC ACAGTTGCTGATCTACCAGATGAGCAATCTGGCTTCCGGCGTGCCCGATAGATTCAGCAGCAGCGGATCCGGCGCTG ACTTTACCCTGAGGATCTCCAGGGTGGAGGCTGAGGATGTGGGCGTTTACTACTGTGCTCAGAATCTGGAGCTGCCT CCTACCTTTGGCGGCGGAACAAAGCTGGAGATCAAG GCCGACGCCGCTCCTACAGTGTCCATCTTTCCTCCTAGCTCCGAGCAGCTGACCAGCGGAGGAGCTTCTGTGGTGTGCTTTCTGAATAACTTCTACCCTAAGGATATCAACGTGAAGTGGAAGATCGATGGCTCCGAGCGGCAGAATGGCGTGTTGAACAGCTGGACCGACCAGGATTCCAAGGATTCCACCTATTCCATGTCCAGCACCCTGACCCTGACCAAGGATGAGTACGAGCGGCACAACTCCTACACCTGTGAGGCCACCCACAAGACCAGCACCTCCCCTATCGTGAAGAGCTTTAATCGGAACGAGTGC

[0186] Amino acid sequence of the light chain of monoclonal antibody 5E1 (SEQ ID NO.10):

[0187] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSS SGSGADFTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIKADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0188] 5.2 Antibody Variable Region Sequence Analysis

[0189] 5.2.1 Monoclonal Antibody 3C2 Variable Region Sequence Analysis

[0190] Further analyze the heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody 3C2. The heavy chain variable region belongs to the IGHV2 subgroup, and the light chain variable region belongs to the IGKV4 subgroup.

[0191] The amino acids of the heavy chain variable region of monoclonal antibody 3C2 are as follows (the heavy chain CDR regions are underlined): QVHLKESGPGLVASSQSLSITCTVS GFSLTSYG LHWVRQPPGKGLEWLGV IWAGGTT NYNSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYC ARERNGMDY WGQGTSVTVSS

[0192] The amino acids of the light chain variable region of monoclonal antibody 3C2 are as follows (the light chain CDR regions are underlined): QIVLTQSPAIMSASPGEKVTMTCSAS SSITY MHWYQQKSGTSPKRWIY DTS KLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWRS DPPT FGGGTKLEIK

[0193] The amino acid sequences and nucleotide sequences of each CDR region of monoclonal antibody 3C2 are summarized in Table 14.

[0194] Table 14

[0195] Amino acid sequence Nucleotide sequence Heavy chain CDR1 GFSLTSYG GGATTTAGCCTGACCTCCTACGGC Heavy chain CDR2 IWAGGTT ATCTGGGCTGGCGGAACCACC Heavy chain CDR3 ARERNGMDY GCCAGGGAGAGGAATGGCATGGATTAT Light chain CDR1 SSITY TCCTCCATCACCTAT Light chain CDR2 DTS GATACCTCC Light chain CDR3 QQWRSDPPT CAGCAGTGGCGGAGCGACCCTCCTACC

[0196] 5.2.2 Monoclonal Antibody 5E1 Variable Region Sequence Analysis

[0197] Further analyze the heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody 5E1. The heavy chain variable region belongs to the IGHV12 subgroup, and the light chain variable region belongs to the IGKV2 subgroup.

[0198] The amino acids of the heavy chain variable region of monoclonal antibody 5E1 are as follows (the heavy chain CDR regions are underlined): QIQLKESGPAVIKPSQSLSLTCKVS GFSITSSYSCWHWIRQPPGKGLEWMGR ICFEGSI FYSPSIKSRSTISRDTSLNKLFMQLSSVTREDTAMYYC SRERHWGSFAMDY WGQGTSVTVSS

[0199] The amino acids of the variable region of the light chain of monoclonal antibody 5E1 are as follows (the CDR regions of the light chain are underlined): DIVMTQAAFSNPVTLGTSASISCRSS KSLLHSNGITY LYWYLQKPGQSPQLLIY QMS NLASGVPDRFSSSGSGADFTLRISRVEAEDVGVYYC AQNLELP PTFGGGTKLEIK

[0200] The amino acid sequences and nucleotide sequences of each CDR region of monoclonal antibody 5E1 are summarized in Table 15.

[0201] Table 15

[0202] Amino acid sequence Nucleotide sequence Heavy chain CDR1 GFSITSSYSC GGATTCTCCATCACCAGCAGCTATAGCTGC Heavy chain CDR2 ICFEGSI ATCTGTTTCGAGGGCAGCATC Heavy chain CDR3 SRERHWGSFAMDY TCCAGGGAGAGGCACTGGGGCAGCTTCGCTATGGATTAT Light chain CDR1 KSLLHSNGITY AAGTCCCTGCTGCACTCCAACGGCATCACCTAT Light chain CDR2 QMS CAGATGAGC Light chain CDR3 AQNLELP GCTCAGAATCTGGAGCTGCCT

[0203] Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, it is not a limitation to the protection scope of the present invention. Based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. A hybridoma cell line 3C2 secreting monoclonal antibody against HCV core antigen, characterized in that, Its classification name is Hybridoma cell line 3C2, which was deposited at the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024355; HCV core antigen, whose amino acid sequence is: (A)C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.

1.

2. A hybridoma cell line 5E1 secreting monoclonal antibody against HCV core antigen, whose classification name is Hybridoma cell line 5E1, which was deposited at the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2024356; HCV core antigen, whose amino acid sequence is: (B)C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.

2.

3. Monoclonal antibody against HCV core antigen, characterized in that, It is secreted by the hybridoma cell line described in claim 1 or 2, and is specific for polypeptides selected from the following amino acid sequences: (A)C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1; or (B)C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.

2.

4. The monoclonal antibody against HCV core antigen according to claim 3, characterized in that, The monoclonal antibody against HCV core antigen is monoclonal antibody 3C2. The sequences of the three complementary determining regions of its heavy chain variable region are respectively: Heavy chain CDR1: GFSLTSYG, as shown in SEQ ID NO.18; Heavy chain CDR2: IWAGGTT, as shown in SEQ ID NO.19; Heavy chain CDR3: ARERNGMDY, as shown in SEQ ID NO.20; The sequences of the three complementary determining regions of the light chain variable region are respectively: Light chain CDR1: SSITY, as shown in SEQ ID NO.21; Light chain CDR2: DTS; Light chain CDR3: QQWRSDPPT, as shown in SEQ ID NO.

22.

5. The monoclonal anti-HCV core antigen antibody according to claim 4, characterized in that, The gene encoding the heavy chain variable region described in claim 4 has the following nucleotide sequence: Heavy chain CDR1: GGATTTAGCCTGACCTCCTACGGC, as shown in SEQ ID NO.23; Heavy chain CDR2: ATCTGGGCTGGCGGAACCACC, as shown in SEQ ID NO.24; Heavy chain CDR3: GCCAGGGAGAGGAATGGCATGGATTAT, as shown in SEQ ID NO.25; The sequences of the three complementary determining regions of the light chain variable region are respectively: Light chain CDR1: TCCTCCATCACCTAT, as shown in SEQ ID NO.26; Light chain CDR2: GATACCTCC; Light chain CDR3: CAGCAGTGGCGGAGCGACCCTCCTACC, as shown in SEQ ID NO.

27.

6. The anti-HCV core antigen monoclonal antibody according to claim 3, characterized in that, The anti-HCV core antigen monoclonal antibody described above is monoclonal antibody 5E1, and the sequences of the three complementarity-determining regions of its heavy chain variable region are respectively: Heavy chain CDR1: GFSITSSYSC, as shown in SEQ ID NO.30; Heavy chain CDR2: ICFEGSI, as shown in SEQ ID NO.31; Heavy chain CDR3: SRERHWGSFAMDY, as shown in SEQ ID NO.32; Light chain CDR1: KSLLHSNGITY, as shown in SEQ ID NO.33; Light chain CDR2: QMS; Light chain CDR3: AQNLELP, as shown in SEQ ID NO.

34.

7. The monoclonal antibody against HCV core antigen according to claim 6, characterized in that, The gene encoding the heavy chain variable region as claimed in claim 6 has the following nucleotide sequence: Heavy chain CDR1: GGATTCTCCATCACCAGCAGCTATAGCTGC, as shown in SEQ ID NO.35; Heavy chain CDR2: ATCTGTTTCGAGGGCAGCATC, as shown in SEQ ID NO.36; Heavy chain CDR3: TCCAGGGAGAGGCACTGGGGCAGCTTCGCTATGGATTAT, as shown in SEQ ID NO.37; Light chain CDR1: AAGTCCCTGCTGCACTCCAACGGCATCACCTAT, as shown in SEQ ID NO.38; Light chain CDR2: CAGATGAGC; Light chain CDR3: GCTCAGAATCTGGAGCTGCCT, as shown in SEQ ID NO.

39.

8. The anti-HCV core antigen monoclonal antibody according to claim 3, characterized in that, The anti-HCV core antigen monoclonal antibody described above is monoclonal antibody 3C2, whose heavy chain nucleotide sequence is as shown in SEQ ID NO.3 and amino acid sequence is as shown in SEQ ID NO.4; the light chain nucleotide sequence is as shown in SEQ ID NO.5 and amino acid sequence is as shown in SEQ ID NO.6; monoclonal antibody 3C2 is specific for a polypeptide with the following amino acid sequence: C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1; Or the anti-HCV core antigen monoclonal antibody described above is monoclonal antibody 5E1, whose heavy chain nucleotide sequence is as shown in SEQ ID NO.7 and amino acid sequence is as shown in SEQ ID NO.8; the light chain nucleotide sequence is as shown in SEQ ID NO.9 and amino acid sequence is as shown in SEQ ID NO.10; monoclonal antibody 5E1 is specific for a polypeptide with the following amino acid sequence: C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.

2.

9. Use of monoclonal antibody combination in preparing HCV detection kit, characterized in that, The monoclonal antibody combination is secreted by the hybridoma cell lines as claimed in claims 1 and 2, and is specific for a polypeptide with the following amino acid sequence: (A) C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1; or (B) C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.2.

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