Kit based on anti-HCV core antigen monoclonal antibody and preparation method thereof

Abstract

The present invention discloses a kit based on anti-HCV core antigen monoclonal antibody and a preparation method thereof. Specifically, the selected monoclonal antibody targets the common conserved sequence of the core protein of different genotypes of HCV, can detect different genotypes of HCV, and has good tolerance. A sample diluent that is conducive to the dissociation of the antigen-antibody complex and a magnetic bead diluent that is conducive to the formation of the antigen-antibody complex are developed. By adding a specific polypeptide, the two are used in combination to effectively reduce the influence of the HCV antibody contained in the sample itself, thereby greatly improving the HCV antigen positive detection rate.

Description

Technical Field

[0001] The present invention relates to a kit based on anti-HCV core antigen monoclonal antibody and a preparation method thereof, belonging to the field of molecular biology and infection immunity technology. Background Art

[0002] Hepatitis C virus (HCV) is the causative agent of hepatitis C, which is mainly transmitted through blood transfusion. Chronic HCV infection often leads to cirrhosis, and some patients may develop hepatocellular carcinoma. There are three main methods for HCV detection:

[0003] (1) HCV antibody test, to detect HCV antibodies in the patient's serum;

[0004] (2) HCV antigen test, detecting HCV core antigen in patient serum;

[0005] (3) HCV-RNA detection: determine the presence of the virus by qualitative or quantitative detection of HCV RNA.

[0006] HCV-RNA testing is mainly used for the selection and efficacy monitoring of antiviral treatment, but it requires strict environmental control and relatively expensive equipment. Testing personnel must undergo professional training and obtain relevant qualifications, and high quality control requirements for samples are also required.

[0007] HCV antibody testing is currently the most commonly used testing method, but its fatal drawback is the existence of a "window period", that is, there is a 40-70 day period between HCV infection and the production of HCV antibodies. The human body has been infected and is contagious, 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 transfusion infection. In addition, HCV patients who have been cured or self-healed may still have high concentrations of antibodies in their bodies and can be detected, so antibody testing cannot distinguish between current infection and past infection.

[0008] HCV antigen detection can advance the window period by an average of about 50 days, shortening 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 body of the infected person, and it has a certain correlation with the level of HCV nucleic acid, and can be used as a marker for HCV detection. However, there are many genotypes of HCV, and there are large differences in the amino acid sequence of the core antigen between different genotypes, and most of these differences are located in the strong epitope region. Monoclonal antibodies are usually obtained by immunization with the core antigen of the main genotype, and a combination of multiple monoclonal antibodies with different epitopes is used for paired detection to reduce missed detections due to different genotypes, but this will increase costs and reduce production efficiency.

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

[0010] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and to provide a kit based on anti-HCV core antigen monoclonal antibodies and a preparation method thereof.

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

[0012] 1. A kit based on anti-HCV core antigen monoclonal antibody, comprising:

[0013] (A) Anti-HCV core antigen monoclonal antibody monoclonal antibody 3C2, the three complementary determining region sequences of its heavy chain variable region are:

[0014] Heavy chain CDR1: GFSLTSYG, as shown in SEQ ID NO.18;

[0015] Heavy chain CDR2: IWAGGTT, as shown in SEQ ID NO.19;

[0016] Heavy chain CDR3: ARERNGMDY, as shown in SEQ ID NO. 20;

[0017] The three complementarity determining region sequences of the light chain variable region are:

[0018] Light chain CDR1: SSITY, as shown in SEQ ID NO.21;

[0019] Light chain CDR2: DTS;

[0020] Light chain CDR3: QQWRSDPPT, as shown in SEQ ID NO.22;

[0021] (B) Anti-HCV core antigen monoclonal antibody monoclonal antibody 5E1, the three complementary determining region sequences of its heavy chain variable region are:

[0022] Heavy chain CDR1: GFSITSSYSC, as shown in SEQ ID NO. 30;

[0023] Heavy chain CDR2: ICFEGSI, as shown in SEQ ID NO.31;

[0024] Heavy chain CDR3: SRERHWGSFAMDY, as shown in SEQ ID NO.32;

[0025] The three complementarity determining region sequences of the light chain variable region are:

[0026] Light chain CDR1: KSLLHSNGITY, as shown in SEQ ID NO.33;

[0027] Light chain CDR2: QMS;

[0028] Light chain CDR3: AQNLELP, as shown in SEQ ID NO.34.

[0029] As one of the preferred technical solutions, the gene encoding the heavy chain variable region of monoclonal antibody 3C2 has the following nucleotide sequence:

[0030] Heavy chain CDR1: GGATTTAGCCTGACCTCCTACGGC, as shown in SEQ ID NO.23;

[0031] Heavy chain CDR2: ATCTGGGCTGGCGGAACCACC, as shown in SEQ ID NO.24;

[0032] Heavy chain CDR3: GCCAGGGAGAGGAATGGCATGGATTAT, as shown in SEQ ID NO.25;

[0033] Light chain CDR1: TCCTCCATCACCTAT, as shown in SEQ ID NO.26;

[0034] Light chain CDR2: GATACCTCC;

[0035] Light chain CDR3: CAGCAGTGGCGGAGCGACCCTCCTACC, as shown in SEQ ID NO.27.

[0036] As one of the preferred technical solutions, the amino acid sequence of the heavy chain variable region of monoclonal antibody 3C2 is: QVHLKESGPGLVASSQSLSITCTVSGFSLTSYGLHWVRQPPGKGLEWLGVIWAGGTTNY NSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYCARERNGMDYWGQGTSVTVSS, as shown in SEQ ID NO.28;

[0037] The amino acid sequence of the light chain variable region of mAb 3C2 is:

[0038] QIVLTQSPAIMSASPGEKVTMTCSAS SSITY MHWYQQKSGTSPKRWIY DTSKLASG VPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSDPPTFGGGTKLEIK, as shown in SEQ ID NO. 29.

[0039] As one of the preferred technical solutions, the gene encoding the heavy chain variable region of monoclonal antibody 5E1 has the following nucleotide sequence:

[0040] Heavy chain CDR1: GGATTCTCCATCACCAGCAGCTATAGCTGC, as shown in SEQ ID NO.35;

[0041] Heavy chain CDR2: ATCTGTTTCGAGGGCAGCATC, as shown in SEQ ID NO.36;

[0042] Heavy chain CDR3: TCCAGGGAGAGGCACTGGGGCAGCTTCGCTATGGATTAT, as shown in SEQ ID NO.37;

[0043] Light chain CDR1: AAGTCCCTGCTGCACTCCAACGGCATCACCTAT, as shown in SEQ ID NO.38;

[0044] Light chain CDR2: CAGATGAGC;

[0045] Light chain CDR3: GCTCAGAATCTGGAGCTGCCT, as shown in SEQ ID NO.39.

[0046] As one of the preferred technical solutions, the amino acid sequence of the heavy chain variable region of monoclonal antibody 5E1 is: QIQLKESGPAVIKPSQSLSLTCKVSGFSITSSYSCWHWIRQPPGKGLEWMGRICFEGSIFY SPSIKSRSTISRDTSLNKLFMQLSSVTREDTAMYYC SRERHWGSFAMDY WGQGTSVTVSS S, as shown in SEQ ID NO.40;

[0047] The amino acid sequence of the light chain variable region of mAb 5E1 is:

[0048] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMS NLASGVPDRFSSSGSGADFTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIK, as shown in SEQ ID NO. 41.

[0049] As one of the preferred technical solutions, the heavy chain nucleotide sequence of monoclonal antibody 3C2 is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO.4; the light chain nucleotide sequence is shown as SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO.6.

[0050] As one of the preferred technical solutions, the heavy chain nucleotide sequence of monoclonal antibody 5E1 is shown as SEQ ID NO.7, and the amino acid sequence is shown as SEQ ID NO.8; the light chain nucleotide sequence is shown as SEQ ID NO.9, and the amino acid sequence is shown as SEQ ID NO.10.

[0051] As one of the preferred technical solutions, monoclonal antibody 3C2 is obtained by secretion of hybridoma cell line 3C2. The classification of hybridoma cell line 3C2 is named Hybridoma cell line 3C2. It was deposited in the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: C2024355.

[0052] As one of the preferred technical solutions, monoclonal antibody 5E1 is obtained by secretion of hybridoma cell line 5E1. The classification of hybridoma cell line 5E1 is named Hybridoma cell line 5E1. It was deposited in the China Center for Type Culture Collection on October 15, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCCNO: C2024356.

[0053] As one of the preferred technical solutions, monoclonal antibody 3C2 has specificity for polypeptides with the following amino acid sequences:

[0054] C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO.1.

[0055] As one of the preferred technical solutions, monoclonal antibody 5E1 has specificity for polypeptides with the following amino acid sequences:

[0056] C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.2.

[0057] As one of the preferred technical solutions, monoclonal antibody 3C2 is coated with magnetic particles, and monoclonal antibody 5E1 is labeled with acridinium ester.

[0058] As one of the further preferred technical solutions, the kit further comprises a sample diluent for diluting the sample to be tested and a magnetic bead diluent for diluting the monoclonal antibody 3C2-coated magnetic microparticles, and a polypeptide comprising the C53-63 sequence but not the C22-35 sequence is added to one of the sample diluent or the magnetic bead diluent to make its concentration 1 to 8 ng / mL; the amino acid sequence of C53-63 is: SERSQPRGRRQ, as shown in SEQ ID NO.2; the amino acid sequence of C22-35 is VKFPGGGQIVGGVY, as shown in SEQID NO.1.

[0059] As one of the further preferred technical solutions, the C53-63 sequence is a single or multiple C53-63 sequences.

[0060] As one of the further preferred technical solutions, the sample diluent comprises: 0.1M Gly-HCl buffer (pH 2.2), 150mM NaCl, 1.2% CHAPS by mass concentration, 1% SDS by mass concentration, and 0.5% Triton X-100 by volume concentration.

[0061] As one of the further preferred technical solutions, the magnetic bead diluent contains: 20mM phosphate buffer (pH7.6), 150mM NaCl, 0.5% mass concentration of trehalose, 10% volume concentration of horse serum, and 1% volume concentration of TritonX-100.

[0062] As one of the further preferred technical solutions, the concentration of the polypeptide containing the C53-63 sequence but not the C22-35 sequence is 4 to 8 ng / mL.

[0063] 2. The preparation method of the aforementioned kit comprises the following steps:

[0064] (1) Magnetic particles coated with monoclonal antibody 3C2 and labeled with monoclonal antibody 5E1 with acridinium ester;

[0065] (2) preparing a sample diluent for diluting the sample to be tested and a magnetic bead diluent for diluting the monoclonal antibody 3C2-coated magnetic microparticles, and adding a polypeptide containing the C53-63 sequence but not the C22-35 sequence to one of the sample diluent or the magnetic bead diluent to a concentration of 1 to 8 ng / mL; the amino acid sequence of C53-63 is: SERSQPRGRRQ, as shown in SEQ ID NO.2; the amino acid sequence of C22-35 is VKFPGGGQIVGGVY, as shown in SEQ ID NO.1.

[0066] As one of the preferred technical solutions, the concentration of the polypeptide containing the C53-63 sequence but not the C22-35 sequence is 4 to 8 ng / mL.

[0067] Beneficial effects of the present invention:

[0068] The selected monoclonal antibody targets the common conserved sequence of the core protein of different HCV genotypes, and can detect different HCV genotypes with good tolerance. A sample diluent that is conducive to the dissociation of antigen-antibody complexes and a magnetic bead diluent that is conducive to the formation of antigen-antibody complexes have been developed. By adding specific polypeptides, the two are used in combination to effectively reduce the impact of HCV antibodies contained in the sample itself, thereby greatly improving the HCV antigen positivity detection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 It is HCV core antigen sequence analysis.

[0070] Collection Information

[0071] Classification and naming: Hybridoma cell line 3C2

[0072] Latin name: Hybridoma cell line 3C2

[0073] Deposit number: CCTCC NO: C2024355

[0074] Name of depository: China Center for Type Culture Collection (CCTCC)

[0075] Address of depository: Wuhan University, Wuhan, China

[0076] Deposit date: October 15, 2024

[0077] Classification and naming: Hybridoma cell line 5E1

[0078] Latin name: Hybridoma cell line 5E1

[0079] Deposit number: CCTCC NO: C2024356

[0080] Name of depository: China Center for Type Culture Collection (CCTCC)

[0081] Address of depository: Wuhan University, Wuhan, China

[0082] Deposit date: October 15, 2024 DETAILED DESCRIPTION

[0083] The present invention is 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 contents.

[0084] The percentage of liquid additives is volume percentage, while the percentage of solid additives is mass concentration.

[0085] 1. Sequence Analysis and Peptide Synthesis of HCV Core Antigen

[0086] Bioinformatics tools were used to compare and analyze the core antigen sequences of different HCV genotypes in NCBI. The core antigen sequences of different genotypes of HCV include: the core antigen sequence of HCV genotype 1, in particular having the amino acid sequence 1-191 described in NCBI Reference Sequence: NP_671491.1; the core antigen sequence of HCV genotype 2, in particular having the amino acid sequence 1-191 described in NCBI Reference Sequence: YP_001469630.1; the core antigen sequence of HCV genotype 3, in particular having the amino acid sequence 1-191 described in NCBI Reference Sequence: YP_001469631.1; the core antigen sequence of HCV genotype 4, in particular having the amino acid sequence 1-191 described in NCBI Reference Sequence: YP_001469632.1; the core antigen sequence of HCV genotype 5, in particular having the amino acid sequence 1-191 described in NCBI Reference Sequence: YP_001469633.1; the core antigen sequence of HCV genotype 6, in particular having the amino acid sequence 1-191 described in NCBI Reference Sequence: YP_001469637.1; Sequence: amino acid sequence 1-191 described in YP_001469634.1; HCV genotype 7 core antigen sequence, in particular, has the amino acid sequence 1-191 described in GenBank: ABN05226.1. See for details Figure 1 .

[0087] 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.

[0088] Preparation of monoclonal antibodies against conserved peptide sequences of 2HCV core antigen

[0089] 2.1 Animal immunization

[0090] 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.

[0091] 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.

[0092] 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;

[0093] 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.

[0094] 2.2 Cell fusion, positive hybridoma cell screening and subcloning

[0095] Select SP2 / 0 myeloma cells (Yangzhou University) with good growth status and mix them with spleen cells of immunized mice at a ratio of 1:5 to 1:10, centrifuge at 1500g for 5 minutes, and discard the supernatant. Gently shake the centrifuge tube to evenly disperse the cells, slowly add 1mL 37℃ preheated PEG1500 (purchased from Sigma) and gently mix, add 20mL 1640 culture medium (purchased from Sigma) after 90s of fusion to terminate. Centrifuge at 800g for 5 minutes, discard the supernatant, add 20% serum HAT-1640 culture medium (purchased from Gibco) to the cell pellet and mix.

[0096] The mixed cells were evenly plated in a 96-well plate and incubated at 37°C CO 2 After culturing in the incubator for 5 to 7 days, observe the fusion effect and change the medium. On the 4th day after changing the medium, use the indirect ELISA method to detect the hybridoma cell supernatant, select the wells with high positive values ​​and small number of cell colonies, and perform subcloning by limiting dilution method. The theoretical number of cells per well is 1 to 2. After 3 to 4 subclones and indirect ELISA detection, the hybridoma cell lines that can stably secrete the corresponding monoclonal antibodies are screened.

[0097] 2.3 Indirect ELISA test

[0098] Dilute BSA-coupled polypeptide (C22-35 or C53-63) with carbonate coating buffer, coat the ELISA plate at a concentration of 0.1 μg / ml, coat 100 μL per well, and keep overnight at 4°C; wash the plate twice with PBS (0.01M, pH 7.2, the same below), 300 μL / well; add 150 μl / well blocking solution (PBS containing 1% BSA) and block at 4°C for 6 hours; discard the blocking solution in the wells, dry for 4 hours at a temperature of 18°C ​​to 26°C and a humidity of no more than 30%, and vacuum pack in aluminum foil bags and store at 2°C to 8°C for future use.

[0099] Instructions for use: Take the prepared ELISA plate, add 100 μL of diluent (PBS containing 1% BSA and 0.5% Triton X-100) to each well, then add 10 μL of cell culture supernatant (or mouse serum), incubate at 37°C for 30 minutes, and discard the liquid. Wash the plate with 1×PBST (pH7.2), 300 μL per well, and repeat the wash 5 times. Invert the washed ELISA plate on absorbent paper and tap to remove excess residual liquid. Add 100 μL / well HRP-labeled anti-mouse IgG antibody (Luoyang Bioton Experimental Materials Center) and incubate at 37°C for 30 minutes. Wash the plate with 1×PBST, 300 μL per well, and repeat the wash 5 times. Add 100 μL / well of TMB substrate colorimetric solution and incubate at 37°C in the dark for 10 minutes. Add stop solution (2MH 2 SO 4 )50 μL / well, mix well to terminate the reaction. Use an enzyme-labeled instrument to detect the OD value at a wavelength of 450 nm and read the value within 10 minutes after termination.

[0100] Through indirect ELISA detection, a total of 11 hybridoma cell lines that can stably secrete anti-HCV core antigen monoclonal antibodies (HCV-cAg monoclonal antibodies) were obtained. Among them, the monoclonal antibodies secreted by 5 cell lines with clone numbers 1D12, 1G11, 3C2, 4D9, and 4H7 (the numbers are the same as the corresponding cell line clone numbers, the same below) can specifically react with C22-35, and the monoclonal antibodies secreted by 6 cell lines with clone numbers 5E1, 7B11, 8A7, 8B2, 9G1, and 10B1 can specifically react with C53-63.

[0101] 3 Antibody pairing screening

[0102] 3.1 Double antibody sandwich ELISA test

[0103] 3.1.1 Labeling HCV-cAg monoclonal antibody with horseradish peroxidase: Horseradish peroxidase (HRP) was labeled on the antibody with reference to the periodate oxidation method in Antibody Engineering (Second Edition) (Beijing Medical University Press, 2002).

[0104] 3.1.2 HCV-cAg monoclonal antibody coated ELISA plate: dilute the antibody with phosphate buffer, coat the ELISA plate at a concentration of 0.1 μg / ml, coat 200 μL per well, and incubate at 4°C overnight; wash the plate twice with PBS, 300 μL / well; add 220 μl / well 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 of no more than 30% for 4 hours, vacuum-pack in aluminum foil bags and store at 2°C to 8°C for later use.

[0105] 3.1.3 Double antibody sandwich ELISA detection procedure: After adding 100 μL of diluent (PBS containing 1% BSA and 0.5% TritonX-100) to each well, add 100 μL of the sample to be tested, incubate at 37°C for 90 minutes, and discard the liquid. Wash the plate with 1×PBST, 300 μL per well, and repeat the wash 5 times. Invert the washed ELISA plate on absorbent paper and tap to remove excess residual liquid. Add 200 μL / well HRP-labeled HCV-cAg monoclonal antibody and incubate at 37°C for 30 minutes. Wash the plate with 1×PBST, 300 μL per well, and repeat the wash 5 times. Add 200 μL / well of TMB substrate colorimetric solution and incubate at 37°C in the dark for 10 minutes. Add stop solution (2MH 2 SO 4 )50 μL / well, mix well to terminate the reaction. Use an enzyme-labeled instrument to detect the OD value at a wavelength of 450 nm and read the value within 10 minutes after termination.

[0106] 3.2 Antibody pairing screening using recombinant antigens

[0107] Five monoclonal antibodies against C22-35 and six monoclonal antibodies against C53-63 were paired and combined (no pairing test was performed between antibodies of the same immunogen), and 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) was used. The HCV core region full-length recombinant antigen (provided by Hunan Kangrun Bioengineering Co., Ltd.) was used to prepare a solution of a certain concentration as the test sample to screen for suitable antibody pairing. See Table 1 and Table 2 for details.

[0108] Table 1 Antibody pairing screening test data

[0109]

[0110]

[0111] Table 2 Antibody pairing screening test data

[0112]

[0113] Judging from the results, the detection value of monoclonal antibody 3C2 and 5E1 pairing is the highest, especially when 3C2 is used for coating and 5E1 is used for labeling, the effect is better. The applicant deposited the hybridoma cells secreting these two monoclonal antibodies in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on October 15, 2024. The cells secreting monoclonal antibody 3C2 are named hybridoma cell line 3C2, with a preservation number of CCTCC NO: C2024355; the cells secreting monoclonal antibody 5E1 are named hybridoma cell line 5E1, with a preservation number of CCTCC NO: C2024356.

[0114] 4 Establishment of a kit for detecting HCV core antigen by magnetic microparticle chemiluminescence

[0115] 4.1 Preliminary establishment of a method for detecting HCV core antigen using magnetic microparticle chemiluminescence

[0116] 4.1.1 HCV-cAg monoclonal antibody coated magnetic particles: The magnetic particles were washed once with 50 mmol / L 2-morpholineethanesulfonic acid solution at pH 6.0, and mixed with 10 mg / mL N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mg / mL N-hydroxysulfosuccinimide solution to a final concentration of 20 μL / mg. The solution was placed on a magnetic stirrer for rotation reaction for 30 min at room temperature, and then mixed with HCV-cAg monoclonal antibody 3C2 to a final concentration of 20 μg / mg. The solution was placed on a magnetic stirrer for rotation reaction for 3 h at room temperature, and then 0.01 M phosphate buffer solution (pH 7.2) containing 1% BSA was added. The solution was placed on a magnetic stirrer for rotation reaction for 3 h at room temperature. Finally, the solution was stored at 2°C to 8°C for future use.

[0117] 4.1.2 Acridinium ester labeled HCV-cAg monoclonal antibody: HCV-cAg monoclonal antibody 5E1 was mixed with 5mmol / L acridinium ester to a solution with a final antibody concentration of 0.5mg / mL, and the solution was placed on a magnetic stirrer for rotation reaction for 30min at room temperature, and PBS containing 1% glycine was added, and the solution was placed on a magnetic stirrer for reaction for 30min at room temperature in the dark. The acridinium ester-antibody conjugate was then transferred to a dialysis bag, dialyzed with PBS overnight, and the product was collected and stored at -20°C in the dark for future use.

[0118] 4.1.3 HCV-cAg monoclonal antibody coated magnetic microparticle working solution: dilute the HCV-cAg monoclonal antibody coated magnetic microparticles prepared in 4.1.1 to a certain appropriate ratio to obtain HCV-cAg monoclonal antibody coated magnetic microparticle working solution. The preliminary formula of the diluent used is: 20mM phosphate buffer (pH7.2), 150mM NaCl, 0.5% trehalose, 10% horse serum, 1% Triton X-100.

[0119] 4.1.4 Acridinium ester labeled HCV-cAg monoclonal antibody working solution: dilute the acridinium ester labeled HCV-cAg monoclonal antibody prepared in 4.1.2 to a certain appropriate ratio to obtain the acridinium ester labeled HCV-cAg monoclonal antibody working solution. The formula of the diluent is: 20mM phosphate buffer (pH7.2), 150mM NaCl, 0.5% trehalose, 1% BSA horse serum, 0.5% Triton X-100.

[0120] 4.1.5 Preliminary establishment of the test procedure: 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), 50 μL The HCV-cAg monoclonal antibody-coated magnetic microparticle working solution was mixed, incubated at 37°C for 20 minutes, and then washed 4 times with cleaning solution (produced by the applicant, medical device registration number: Xiangyue Xiebei 20210009), and then 100 μL of acridinium ester-labeled HCV-cAg monoclonal antibody working solution was added and incubated at 37°C for 8 minutes, washed 4 times with cleaning solution, and then 50 μL each of pre-excitation solution (produced by the applicant, medical device registration number: Xiangyue Xiebei 20210008) and excitation solution (produced by the applicant, medical device registration number: Xiangyue Xiebei 20210010) were added, and the luminescence value (RLU value) was detected by chemiluminescence instrument.

[0121] 4.1.6 Detection: Using the above procedure, the HCV core region full-length recombinant antigen solutions with concentrations of 1 ng / mL and 0.5 ng / mL and two negative sera N1 and N2 were tested by the chessboard titration method. The results are shown in Table 3.

[0122] Table 3 Chessboard titration test results

[0123]

[0124] 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.

[0125] 4.2 Optimization of magnetic microparticle chemiluminescence detection of HCV core antigen

[0126] 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.

[0127] Table 4 Detection data after neutralization of antigen by antibodies at different concentrations

[0128]

[0129] 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.

[0130] 4.2.2 Study on the formula of sample diluent and magnetic bead diluent for chemiluminescence detection

[0131] 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.

[0132] Since the reaction of antigen-antibody specific binding to form antigen-antibody complex is reversible, changing the pH value, ionic strength and other conditions of the reaction system can dissociate the antibody and antigen. 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. To this end, the applicant set up different formula combinations of sample diluent and magnetic bead diluent (for the preparation of HCV-cAg monoclonal antibody coated magnetic microparticle working solution) (see Table 5 for details) for research.

[0133] Table 5 Different formula combinations of sample diluent and magnetic bead diluent

[0134]

[0135]

[0136] The HCV-cAg monoclonal antibody-coated magnetic microparticles prepared in 4.1.1 were diluted at a uniform ratio using the magnetic bead diluents of each combination to form HCV-cAg monoclonal antibody-coated magnetic microparticle working solution. The PBS solution and the unneutralized samples and completely neutralized samples prepared in 4.2.1 above were used to perform the test according to the adjusted test procedure. The results are shown in Table 6.

[0137] Table 6 Test results of different formula combinations

[0138]

[0139] From 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 completely neutralized samples. The combination of sample diluent prepared with 0.1M Gly-HCl buffer at pH 2.2-3.6 and magnetic bead diluent prepared with 20mM phosphate buffer at pH 7.2-8.0, or the combination of sample diluent prepared with 0.1M Gly-HCl buffer at pH 3.0-3.6 and magnetic bead diluent prepared with 0.1M Gly-NaOH buffer at pH 8.8-9.2, can detect HCV-cAg (RLU ≥ 6000) in completely neutralized samples. In particular, the combination of sample diluent prepared with 0.1M Gly-HCl buffer at pH 2.2-3.0 and magnetic bead diluent prepared with 20mM phosphate buffer at pH 7.2-7.6 has a better effect.

[0140] The mean ratio of the test results of the completely neutralized samples to the unneutralized samples (M 2 / M 1), the highest is only 40%. The possible reason for the analysis is that the sample to be tested is mixed with the sample diluent to dissociate the antigen-antibody complex, and the HCV-cAg monoclonal antibody coated magnetic microparticle working solution is added. At this time, the reaction environment is conducive to the binding of antigen and antibody. HCV-cAg monoclonal antibody has a competitive advantage over the antibodies already present in the sample, and can bind to the corresponding epitope on the HCV core antigen to capture most of the antigen. However, at this time, the acridinium ester-labeled HCV-cAg monoclonal antibody has not yet 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 already present in the sample. The applicant boldly envisions that a certain amount of HCV-cAg fragments containing acridinium ester-labeled HCV-cAg monoclonal antibody recognition epitopes but not containing HCV-cAg monoclonal antibody recognition epitopes coated on magnetic microparticles are added to the sample diluent or (and) magnetic bead diluent to bind to the antibodies already present in the sample that can bind to the acridinium ester-labeled HCV-cAg monoclonal antibody recognition epitopes.

[0141] Based on this, the inventors used combination C in Table 5 as a basis, coated magnetic particles with antibody 3C2, labeled antibody 5E1 with acridinium ester, and selected polypeptide C53-63 for further optimization research. See Table 7 for details.

[0142] Table 7 Optimized combinations of different formulas for sample diluent and magnetic bead diluent

[0143]

[0144] Table 8 Test results of different formula optimization combinations

[0145]

[0146]

[0147] After analyzing the test results (Table 8), the applicant surprisingly found that adding a certain amount of HCV-cAg fragment C53-63 containing the recognition epitope of acridinium ester-labeled HCV-cAg monoclonal antibody 5E1 but not containing the recognition epitope of HCV-cAg monoclonal antibody 3C2 coated on magnetic particles to the sample diluent or magnetic bead diluent is indeed beneficial to the detection of HCV-cAg in completely neutralized samples.

[0148] 4.2.3 Accelerated stability test

[0149] The above combinations C4 and C8 were used to prepare the corresponding solutions, which were stored at 37°C for 0, 3 and 6 days, and then the detection procedure described in 4.2.2 was used to detect 5 positive sera (P1-P5) and 5 negative sera (N1-N5).

[0150] Table 9 Accelerated stability test results

[0151]

[0152] It is not difficult to see from the test results (Table 9) that combination C8 has better stability.

[0153] 4.2.4 Summary

[0154] In summary, monoclonal antibody 3C2 is used to coat magnetic microparticles, monoclonal antibody 5E1 is used to label acridinium ester, and the sample diluent and magnetic bead diluent are prepared using the combination C formula, and C53-63 at a concentration of 4 to 8 ng / mL is added to the sample diluent or magnetic bead diluent. During the test, the sample and the sample diluent are first mixed and reacted, and then the HCV-cAg monoclonal antibody coated magnetic microparticle working solution is added to react, which has a good HCV-cAg detection effect. Based on this, the applicant trial-produced the HCV-cAg magnetic microparticle chemiluminescence detection kit and further evaluated its performance.

[0155] 4.3 Detection performance evaluation

[0156] 4.3.1 Inclusiveness

[0157] The test results of 15 clinical serum samples of hepatitis C virus covering different genotypes such as 1a, 1b, 2a, 3a, 3b, 4, 5, and 6 were all positive, indicating that the HCV-cAg magnetic microparticle chemiluminescence detection kit has good tolerance for major hepatitis C virus subtype samples. The results are shown in Table 10.

[0158] Table 10 Detection results of clinical serum samples of different genotypes of hepatitis C virus

[0159] 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 + + + + + + + + + + + + + + +

[0160] 4.3.2 Sensitivity

[0161] 4.3.2.1 HCV seroconversion plate sample testing

[0162] HCV seroconversion plate (SeraCare Life Sciences, USA, number PHV929) samples were tested for HCV-cAg, and starting from day 14, the samples were positive (see Table 11 for details).

[0163] Table 11 Results of testing HCV seroconversion plate PHV929 samples

[0164]

[0165] 4.3.2.2 HCV antibody positive sample detection

[0166] 100 HCV antibody positive samples were tested for HCV-RNA and HCV-cAg respectively. There were 72 HCV-RNA positive samples, of which 69 HCV-cAg test results were positive. Based on the HCV-RNA test results, the positive detection rate of the HCV-cAg magnetic microparticle chemiluminescence detection kit reached 95.83% (69 / 72). See Table 12 for details.

[0167] Table 12 HCV antibody positive sample test results

[0168]

[0169] 4.3.3 Specificity

[0170] The above-mentioned HCV-cAg magnetic microparticle chemiluminescence detection kit was used to detect 52 serum samples positive for HBV surface antigen (HBsAg), 36 serum samples positive for Treponema pallidum antibody, 46 serum samples with high triglycerides (≤30 mg / ml), 25 serum samples with high bilirubin (≤0.2 mg / ml), 33 serum samples with high hemoglobin (≤5 mg / ml), and 18 serum samples positive for a series of pregnancy teratogenic pathogens (3 Toxoplasma gondii, 7 cytomegalovirus, 3 herpes simplex virus, and 5 rubella virus). The results were all negative, demonstrating that the HCV-cAg magnetic microparticle chemiluminescence detection kit has extremely high specificity.

[0171] 5 HCV monoclonal antibody sequencing analysis

[0172] 5.1 Antibody gene sequencing

[0173] Total mRNA of hybridoma cells 3C2 and 5E1 was extracted by Trizol method and PrimeScript TM The first-strand cDNA of the antibody encoding gene was obtained by reverse transcription using the RT reagent Kitwith gDNA Eraser Kit (Dalian TaKaRa Company). The cDNA synthesized by reverse transcription was used as a template for polymerase chain reaction (PCR) amplification using the Trans Start Fast Pfu Fly DNA Polymerase Kit (Beijing Quanshijin Biotechnology Co., Ltd.), and the forward primer Forward-V in the primer sequence list was used. H and reverse primer Reverse-V H Amplify the variable region gene fragment of mouse antibody heavy chain (V H );Forward primer Forward-V in the primer sequence table L and reverse primer Reverse-V LAmplify the variable region gene fragment of mouse antibody heavy chain (V L ).

[0174] The primer sequences are shown in Table 13.

[0175] Table 13. Primer sequences

[0176]

[0177] The PCR target product was recovered, cloned and connected to the T vector, transformed into E. coli DH5α, and the positive clones were selected and commissioned to Nanjing Zhongding Biotechnology Co., Ltd. for sequencing. The sequencing results are as follows (the underlined part is the antibody variable region sequence):

[0178] Monoclonal antibody 3C2 heavy chain nucleotide sequence (SEQ ID NO.3):

[0179] CAGGTTCACCTGAAGGAGAGCGGCCCAGGACTGGTTGCTTCCTCTCAGAGCCTGTCC

[0180] ATCACCTGCACCGTGTCCGGATTTAGCCTGACCTCCTACGGCCTGCACTGGGTTAGA

[0181] CAGCCTCCTGGAAAGGGCCTGGAGTGGTTGGGAGTGATCTGGGCTGGCGGAACCAC

[0182] CAATTACAATTCCGCTCTGATGAGCAGGCTGTCCATCTCCAAGGACAAGAGCAAGA

[0183] GCCAGGTGTTTCTGAAGATGAATAGCCTGCAGACCGATGATACCGCCATGTATTACT

[0184] GCGCCAGGGAGAGGAATGGCATGGATTATTGGGGCCAGGGCACCTCCGTGACCGTT

[0185] TCCTCCGCTAAGACCACCCCACCCAGCGTTTACCCCTTGGCTCCTGGAAGCGCTGCT

[0186] CAGACAAACAGCATGGTGACCCTGGGCTGTCTGGTGAAGGGCTATTTCCCCGAGCC

[0187] CGTGACCGTGACATGGAATTCCGGCTCCCTGAGCTCCGGCGTTCACACCTTTCCTGC

[0188] TGTGCTGCAGTCCGACCTGTACACCCTGTCCAGCTCCGTGACCGTGCCTTCTAGCAC

[0189] CTGGCCATCCGAGACCGTGACCTGTAATGTGGCTCACCCTGCCTCCAGCACCAAGGT

[0190] TGACAAGAAGATCGTGCCTAGGGATTGTGGCTGTAAGCCTTGCATCTGCACCGTGCC

[0191] TGAGGTGTCCAGCGTGTTCATCTTCCCTCCTAAGCCTAAGGATGTGCTGACCATCAC

[0192] CCTGACCCCTAAGGTGACCTGCGTGGTGGTGGACATCTCCAAGGATGACCCCGAGG

[0193] TGCAGTTCAGCTGGTTCGTGGACGACGTGGAGGTGCACACCGCTCAGACACAGCCT

[0194] AGAGAGGAGCAGTTCAACTCCACCTTCCGGTCCGTGAGCGAGCTGCCTATCATGCAC

[0195] CAGGACTGGCTGAACGGCAAGGAGTTTAAGTGCCGGGTGAATTCCGCTGCTTTTCCC

[0196] GCCCCAATCGAGAAGACCATCTCCAAGACCAAGGGCCGGCCCAAGGCTCCTCAGGT

[0197] GTATACAATCCCTCCTCCCAAGGAGCAGATGGCTAAGGACAAGGTGTCCCTGACCT

[0198] GCATGATCACCGACTTCTTTCCTGAGGACATCACCGTGGAGTGGCAGTGGAACGGCC

[0199] AGCCTGCTGAAAACTACAAGAACACCCAGCCCATCATGGACACCGATGGCAGCTAT

[0200] TTCGTGTACTCCAAGCTGAACGTGCAGAAGAGCAACTGGGAGGCTGGCAATACCTT

[0201] TACCTGTTCCGTGCTGCACGAGGGCCTGCATAATCACCACACCGAGAAGAGCCTGTC

[0202] CCACTCCCCTGGAAAG

[0203] Amino acid sequence of monoclonal antibody 3C2 heavy chain (SEQ ID NO.4):

[0204] QVHLKESGPGLVASSQSLSITCTVSGFSLTSYGLHWVRQPPGKGLEWLGVIWAGGTTNY

[0205] NSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYCARERNGMDYWGQGTSVTVSSA

[0206] KTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSD

[0207] LYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPP

[0208] KPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSE

[0209] LPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVS

[0210] LTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNT

[0211] FTCSVLHEGLHNHHTEKSLSHSPGK

[0212] Nucleotide sequence of monoclonal antibody 3C2 light chain (SEQ ID NO.5):

[0213] CAGATCGTGCTGACCCAGTCCCCAGCTATCATGTCCGCCAGCCCAGGAGAGAAGGT

[0214] GACCATGACCTGCTCCGCTTCCTCCTCCATCACCTATATGCACTGGTACCAGCAGAA

[0215] GAGCGGCACCAGCCCAAAGAGATGGATCTACGATACCTCCAAGCTGGCCTCCGGCG

[0216] TGCCTGCTAGGTTTTCTGGAAGCGGCTCCGGCACCAGCTACTCTTTGACCATCTCCA

[0217] GCATGGAGGCCGAGGACGCTGCTACATACTATTGCCAGCAGTGGCGGAGCGACCCT

[0218] CCTACCTTTGGAGGAGGAACCAAGCTGGAGATCAAGGCTGATGCTGCTCCCACCGT

[0219] GAGCATCTTCCCTCCTTCCTCCGAGCAGCTGACCTCCGGAGGAGCTTCTGTGGTGTG

[0220] CTTTCTGAATAATTTCTACCCTAAGGACATCAACGTGAAGTGGAAGATCGATGGCTC

[0221] CGAGCGGCAGAATGGCGTGTTGAACTCCTGGACCGATCAGGATAGCAAGGATAGCA

[0222] CCTACAGCATGTCCTCCACCCTGACCCTGACCAAGGACGAGTATGAGCGGCACAAC

[0223] TCCTACACCTGCGAGGCTACCCACAAGACCAGCACCAGCCCTATCGTGAAGAGCTTT

[0224] AACCGGAACGAGTGC

[0225] Amino acid sequence of monoclonal antibody 3C2 light chain (SEQ ID NO.6):

[0226] QIVLTQSPAIMSASPGEKVTMTCSASSSITYMHWYQQKSGTSPKRWIYDTSKLASGVPA

[0227] RFSGSGSGTSYSLTISSMEAEDAATYYCQQWRSDPPTFGGGTKLEIK ADAAPTVSIFPPSS

[0228] EQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLT

[0229] LTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0230] Nucleotide sequence of the heavy chain of monoclonal antibody 5E1 (SEQ ID NO.7):

[0231] CAGATCCAGCTGAAGGAGTCCGGCCCAGCTGTGATCAAGCCTAGCCAGTCCCTGAG

[0232] CCTGACCTGCAAGGTGAGCGGATTCTCCATCACCAGCAGCTATAGCTGCTGGCACTG

[0233] GATCCGGCAGCCTCCTGGAAAGGGACTGGAGTGGATGGGCAGGATCTGTTTCGAGG

[0234] GCAGCATCTTTTACTCCCCTAGCATCAAGAGCCGGAGCACCATCTCCCGGGACACAT

[0235] CTCTGAATAAGCTGTTCATGCAGCTGAGCTCCGTGACCAGGGAGGACACAGCTATGT

[0236] ATTACTGTTCCAGGGAGAGGCACTGGGGCAGCTTCGCTATGGATTATTGGGGCCAG

[0237] GGCACCTCCGTGACCGTTTCCTCCGCTAAGACCACCGCCCCAAGCGTTTACCCCTTG

[0238] GCTCCAGTGTGTGGCGACACCACAGGCTCTAGCGTGACCTTGGGCTGCCTGGTTAAG

[0239] GGCTACTTCCCTGAGCCTGTGACCCTGACCTGGAATAGCGGCAGCCTGTCCTCCGGA

[0240] GTGCACACCTTTCCCGCTGTGCTGCAGTCCGATCTGTACACCCTGAGCAGCAGCGTG

[0241] ACCGTGACCTCTTCCACCTGGCCTTCCCAGTCCATCACCTGTAACGTGGCCCACCCC

[0242] GCTAGCTCTACCAAGGTTGACAAGAAGATCGAGCCTCGGGGCCCTACCATCAAGCC

[0243] ATGTCCACCTTGCAAGTGTCCCGCTCCCAACCTGCTGGGAGGACCTTCCGTTTTCATC

[0244] TTTCCTCCTAAGATCAAGGATGTGCTGATGATCTCCCTGTCCCCAATCGTGACCTGTG

[0245] TGGTGGTGGATGTGAGCGAGGACGACCCAGACGTGCAGATCAGCTGGTTTGTGAAC

[0246] AATGTGGAGGTGCACACCGCTCAGACCCAGACACACAGGGAGGACTATAATTCCAC

[0247] CCTGAGGGTGGTGAGCGCCCTGCCTATTCAGCACCAGGACTGGATGAGCGGCAAGG

[0248] AGTTCAAGTGTAAGGTGAACAACAAGGACCTGCCCGCCCCTATCGAGAGAACCATC

[0249] AGCAAGCCCAAGGGCTCCGTGAGGGCTCCTCAGGTGTACGTTCTGCCTCCTCCTGAG

[0250] GAGGAGATGACCAAGAAGCAGGTGACCCTGACATGTATGGTGACCGATTTCATGCC

[0251] TGAGGACATCTATGTGGAGTGGACCAACAACGGCAAGACCGAGCTGAACTATAAGA

[0252] ATACCGAGCCCGTGCTGGACAGCGACGGATCCTATTTTATGTACAGCAAGCTGAGG

[0253] GTGGAGAAGAAGAACTGGGTGGAGCGGAATAGCTACAGCTGCAGCGTGGTGCACG

[0254] AGGGACTGCATAACCACCACACCACCAAGTCCTTCAGCCGGACCCCAGGAAAG

[0255] Amino acid sequence of the heavy chain of monoclonal antibody 5E1 (SEQ ID NO.8):

[0256] QIQLKESGPAVIKPSQSLSLTCKVSGFSITSSYSCWHWIRQPPGKGLEWMGRICFEGSIFY

[0257] SPSIKSRSTISRDTSLNKLFMQLSSVTREDTAMYYCSRERHWGSFAMDYWGQGTSVTVS

[0258] S AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQS

[0259] DLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGG

[0260] PSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN

[0261] STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEE

[0262] MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEK

[0263] KNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0264] Nucleotide sequence of the light chain of monoclonal antibody 5E1 (SEQ ID NO.9):

[0265] GACATCGTGATGACCCAGGCCGCTTTTAGCAATCCCGTGACCCTGGGCACCTCCGCT

[0266] TCTATTAGCTGCAGGAGCTCCAAGTCCCTGCTGCACTCCAACGGCATCACCTATCTG

[0267] TACTGGTACCTGCAGAAGCCTGGCCAGAGCCCACAGTTGCTGATCTACCAGATGAG

[0268] CAATCTGGCTTCCGGCGTGCCCGATAGATTCAGCAGCAGCGGATCCGGCGCTGACTT

[0269] TACCCTGAGGATCTCCAGGGTGGAGGCTGAGGATGTGGGCGTTTACTACTGTGCTCA

[0270] GAATCTGGAGCTGCCTCCTACCTTTGGCGGCGGAACAAAGCTGGAGATCAAG GCCG

[0271] ACGCCGCTCCTACAGTGTCCATCTTTCCTCCTAGCTCCGAGCAGCTGACCAGCGGAG

[0272] GAGCTTCTGTGGTGTGCTTTCTGAATAACTTCTACCCTAAGGATATCAACGTGAAGT

[0273] GGAAGATCGATGGCTCCGAGCGGCAGAATGGCGTGTTGAACAGCTGGACCGACCAG

[0274] GATTCCAAGGATTCCACCTATTCCATGTCCAGCACCCTGACCCTGACCAAGGATGAG

[0275] TACGAGCGGCACAACTCCTACACCTGTGAGGCCACCCACAAGACCAGCACCTCCCC

[0276] TATCGTGAAGAGCTTTAATCGGAACGAGTGC

[0277] Monoclonal antibody 5E1 light chain amino acid sequence (SEQ ID NO.10):

[0278] DIVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLA

[0279] SGVPDRFSSSGSGADFTLRISRVEAEDVGVYYCAQNLELPPTFGGGTKLEIK ADAAPTVS

[0280] IFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSM

[0281] SSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0282] 5.2 Antibody variable region sequence analysis

[0283] 5.2.1 Sequence analysis of the variable region of mAb 3C2

[0284] Further analysis of the heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody 3C2 showed that the heavy chain variable region belonged to the IGHV2 subgroup, and the light chain variable region belonged to the IGKV4 subgroup.

[0285] The amino acids of the heavy chain variable region of monoclonal antibody 3C2 are as follows (the underlined part is the heavy chain CDR region):

[0286] QVHLKESGPGLVASSQSLSITCTVS GFSLTSYG LHWVRQPPGKGLEWLGV IWAGGTT NY NSALMSRLSISKDKSKSQVFLKMNSLQTDDTAMYYC ARERNGMDY WGQGTSVTVSS

[0287] The amino acids of the light chain variable region of monoclonal antibody 3C2 are as follows (the underlined part is the light chain CDR region):

[0288] QIVLTQSPAIMSASPGEKVTMTCSAS SSITY MHWYQQKSGTSPKRWIY DTS KLASGVPA RFSGSGSGTSYSLTISSMEAEDAATYYC QQWRSDPPT FGGGTKLEIK

[0289] The amino acid sequence and nucleotide sequence of each CDR region of mAb 3C2 are summarized in Table 14.

[0290] Table 14

[0291] 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

[0292] 5.2.2 Sequence analysis of the variable region of mAb 5E1

[0293] Further analysis of the heavy chain variable region sequence and light chain variable region sequence of monoclonal antibody 5E1 showed that the heavy chain variable region belonged to the IGHV12 subgroup, and the light chain variable region belonged to the IGKV2 subgroup.

[0294] The amino acids of the heavy chain variable region of monoclonal antibody 5E1 are as follows (the underlined part is the heavy chain CDR region):

[0295] QIQLKESGPAVIKPSQSLSLTCKVS GFSITSSYSC WHWIRQPPGKGLEWMGR ICFEGSI FYSPSIKSRSTISRDTSLNKLFMQLSSVTREDTAMYYC SRERHWGSFAMDY WGQGTSVTVSS

[0296] The amino acids in the light chain variable region of monoclonal antibody 5E1 are as follows (the underlined region is the light chain CDR region): DIVMTQAAFSNPVTLGTSASISCRSS KSLLHSNGITY LYWYLQKPGQSPQLLIY QMS NLASGVPDRFSSSGSGADFTLRISRVEAEDVGVYYC AQNLELP PTFGGGTKLEIK

[0297] The amino acid sequence and nucleotide sequence of each CDR region of mAb 5E1 are summarized in Table 15.

[0298] Table 15

[0299] 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

[0300] Although the above describes the specific implementation mode of the present invention in conjunction with the accompanying drawings, it is not intended to limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A kit based on anti-HCV core antigen monoclonal antibodies, characterized in that: include: (A) Anti-HCV core antigen monoclonal antibody monoclonal antibody 3C2, the three complementary determining region sequences of its heavy chain variable region are: 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 three complementarity determining region sequences of the light chain variable region are: 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; (B) Anti-HCV core antigen monoclonal antibody monoclonal antibody 5E1, the three complementary determining region sequences of its heavy chain variable region are: 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; The three complementarity determining region sequences of the light chain variable region are: 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.

2. The kit according to claim 1, characterized in that Monoclonal antibody 3C2 is obtained by secretion of hybridoma cell line 3C2. The classification of hybridoma cell line 3C2 is named Hybridoma cell line 3C2. It was deposited in 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.

3. The kit according to claim 1, characterized in that Monoclonal antibody 5E1 is obtained by secretion of hybridoma cell line 5E1. The classification of hybridoma cell line 5E1 is named Hybridoma cell line 5E1. It was deposited in 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.

4. The kit according to claim 1, characterized in that Monoclonal antibody 3C2 is specific for peptides with the following amino acid sequences: C22-35: VKFPGGGQIVGGVY, as shown in SEQ ID NO. 1; Monoclonal antibody 5E1 is specific for peptides with the following amino acid sequences: C53-63: SERSQPRGRRQ, as shown in SEQ ID NO.

2.

5. The kit according to claim 1, characterized in that The monoclonal antibody 3C2 was coated on magnetic microparticles, and the monoclonal antibody 5E1 was labeled with acridinium ester.

6. The kit according to claim 5, characterized in that The kit also comprises a sample diluent for diluting the sample to be tested and a magnetic bead diluent for diluting the monoclonal antibody 3C2-coated magnetic microparticles, and a polypeptide comprising the C53-63 sequence but not the C22-35 sequence is added to one of the sample diluent or the magnetic bead diluent to make its concentration 1 to 8 ng / mL; the amino acid sequence of C53-63 is: SERSQPRGRRQ, as shown in SEQ ID NO.2; the amino acid sequence of C22-35 is VKFPGGGQIVGGVY, as shown in SEQ ID NO.

1.

7. The kit according to claim 6, characterized in that The sample diluent comprises: 0.1M Gly-HCl buffer, 150mM NaCl, 1.2% CHAPS by mass concentration, 1% SDS by mass concentration, and 0.5% Triton X-100 by volume concentration.

8. The kit according to claim 6, characterized in that The magnetic bead diluent contains: 20 mM phosphate buffer, 150 mM NaCl, 0.5% mass concentration of trehalose, 10% volume concentration of horse serum, and 1% volume concentration of Triton X-100.

9. The kit according to claim 6, characterized in that The concentration of the polypeptide containing the C53-63 sequence but not the C22-35 sequence was 4 to 8 ng / mL.

10. The method for preparing the kit according to any one of claims 1 to 9, characterized in that: The following steps are involved: (1) Magnetic microparticles coated with monoclonal antibody 3C2 and labeled with acridinium ester monoclonal antibody 5E1; (2) preparing a sample diluent for diluting the sample to be tested and a magnetic bead diluent for diluting the monoclonal antibody 3C2-coated magnetic microparticles, and adding a polypeptide containing the C53-63 sequence but not the C22-35 sequence to one of the sample diluent or the magnetic bead diluent to a concentration of 1 to 8 ng / mL; the amino acid sequence of C53-63 is: SERSQPRGRRQ, as shown in SEQ ID NO.2; the amino acid sequence of C22-35 is VKFPGGGQIVGGVY, as shown in SEQ ID NO.1.

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