Humanized hepatitis c virus (HCV) monoclonal antibody and application thereof

Abstract

The invention discloses a humanized anti-hepatitis C virus (HCV) monoclonal antibody, the antibody is named as C00012 and is specifically combined with HCV Core protein, the amino acid sequence of a heavy chain variable region of the antibody is shown as SEQ ID NO.1 in sequence, and the amino acid sequence of a light chain variable region of the antibody is shown as SEQ ID NO.2 in sequence. The invention also discloses an application of the humanized anti-HCV monoclonal antibody in preparation of an anti-HCV indoor quality control product. Experiments prove that the humanized anti-HCV monoclonal antibody disclosed by the invention can specifically recognize Core protein of HCV, can be combined with an anti-human IgG antibody, and is an anti-HCV immunodetection kit compatible with methodology principles of a sandwich method, an indirect method and the like. The anti-HCV indoor quality control product prepared from the compound has the advantages of good uniformity, good stability, good intercommunity and the like, and has important application value in anti-HCV test quality control.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology and genetic engineering, and in particular relates to a humanized monoclonal antibody capable of specifically recognizing a hepatitis C virus (HCV) Core protein and an application thereof. Background Art

[0002] Hepatitis C virus (HCV), after infecting the human body, will cause acute (short-term) and chronic (long-term) viral hepatitis in the body. It is mainly transmitted through blood contact. In addition, it can also be transmitted through sexual contact, mother-to-child vertical transmission, etc. Acute HCV infection usually has no symptoms, but about 70% of infected people will develop chronic HCV infection. Chronic HCV infection may develop into serious liver diseases such as liver fibrosis, cirrhosis and even liver cancer. At present, there is no vaccine for HCV, but antiviral drugs can be used to treat HCV infection. Therefore, timely detection, diagnosis and treatment of HCV infection are the key to reducing HCV blood transmission and preventing and treating hepatitis C.

[0003] HCV is a single-stranded positive-strand RNA virus with a genome size of about 9500~10000bp. It contains a large coding open reading frame and encodes a polyprotein precursor of about 3000 amino acids. Under the action of proteases encoded by cells and viruses themselves, this protein precursor forms mature structural proteins and non-structural proteins, namely structural proteins such as core protein (Core) and envelope glycoprotein (E1, E2), and non-structural proteins such as P7, NS2 protease, NS3-4A complex, NS4B, NS5A and NS5B. At present, the most commonly used clinical specific diagnostic methods for HCV infection at home and abroad are anti-HCV and HCV RNA detection. Anti-HCV detection is more suitable for large-scale screening tests because of its low cost and simple operation; HCV RNA detection is of great significance for the diagnosis of HCV and guiding HCV antiviral treatment.

[0004] Anti-HCV detection is a detection method established by using genetic engineering technology to express some structural proteins and non-structural proteins in the HCV gene as coating antigens. At present, four generations of anti-HCV detection reagents have been developed. The first generation of anti-HCV detection uses the recombinant antigen C100-3 encoded in the NS4 region; the second generation uses the recombinant antigen C22-3 in the Core region, the recombinant antigen C33c in the NS3 region, and the C100-3 in the NS4 region; the third generation adds the recombinant antigen in the NS5 region, and reconfigures the recombinant antigens in the Core region and NS3 region to improve the sensitivity of the reagent; the fourth generation adds the biotin-avidin system (Biotin-Avidin System, BAS) to further improve the sensitivity of the reagent.

[0005] The development and large-scale application of anti-HCV detection technology are of great significance to HCV epidemiological surveys, blood donor screening, high-risk population monitoring and clinical diagnosis of HCV patients. It also provides a solid and reliable basis for the early detection and early treatment of HCV infection. Therefore, in order to monitor and evaluate the level and quality of anti-HCV detection in each laboratory, indoor quality control products and inter-laboratory quality assessment samples have been widely used. However, the existing anti-HCV indoor quality control products and inter-laboratory quality assessment samples are mostly prepared from clinical strong positive serum / plasma samples, which involve biosafety and ethical issues and are difficult to obtain. In addition, the sample composition is complex and the differences between batches of raw materials are difficult to control, resulting in large differences in the quality of quality control products between batches. Summary of the invention

[0006] In view of the problem of raw material sources for anti-HCV quality control products, the present invention aims to provide a humanized anti-HCV monoclonal antibody that can specifically recognize HCVCore protein and its application as an anti-HCV indoor quality control product.

[0007] The humanized anti-HCV monoclonal antibody provided by the present invention is characterized in that the antibody can specifically recognize HCVCore protein, including light chain and heavy chain, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0008] Furthermore, the present invention also provides a method for preparing a humanized anti-HCV monoclonal antibody, comprising the following steps:

[0009] (1) Select HCV Core protein and immunize BALB / c mice to obtain immunized mice;

[0010] (2) Based on the immunized mice described in step (1), monoclonal antibodies are prepared by hybridoma technology, and high-titer specific monoclonal antibodies are screened by limiting dilution method and ELISA method;

[0011] (3) sequencing the hybridoma variable region gene of the monoclonal antibody described in step (2) to obtain a gene sequence, and combining the gene sequence with the human IgG1 gene structure to construct a eukaryotic expression plasmid;

[0012] (4) Transfecting the eukaryotic expression plasmid described in step (3) into CHO-S cells to construct a stable cell line, and using the stable cell line to express and prepare a humanized monoclonal antibody.

[0013] Furthermore, the present invention provides an anti-HCV indoor quality control product, which uses humanized anti-HCV monoclonal antibodies as raw materials to prepare indoor quality control products with good uniformity, good stability and good interoperability, thereby realizing the preliminary application of the humanized monoclonal antibodies.

[0014] Furthermore, the anti-HCV indoor quality control product provided by the present invention comprises components such as humanized anti-HCV monoclonal antibody, human serum matrix, sodium chloride, protein protective agent, preservative, etc. The humanized anti-HCV monoclonal antibody is a humanized monoclonal antibody that specifically recognizes HCVCore protein, and the preservative is one or both of ProClin300 and ProClin950.

[0015] Furthermore, the present invention provides a method for preparing an anti-HCV indoor quality control product, comprising the following steps:

[0016] (1) Place the human serum matrix in a (2-8)°C environment until it is completely dissolved. Then treat it at 60°C for 60 minutes, sterilize and filter, and obtain the treated human serum matrix.

[0017] (2) While stirring, add humanized anti-HCV monoclonal antibody, sodium chloride, protein protective agent and preservative to the treated human serum matrix in step (1) in sequence, then use the treated human serum matrix to make up to the target preparation volume, mix thoroughly and evenly, and obtain the semi-finished anti-HCV indoor quality control product.

[0018] (3) Use the ELISA kit to measure the anti-HCV indoor quality control semi-finished product in step (2) to determine whether the concentration of the quality control product meets the quality inspection standards. If it meets the standards, it is a qualified anti-HCV indoor quality control semi-finished product.

[0019] (4) In step (3), the qualified anti-HCV indoor quality control product semi-finished product is packaged according to the required packaging quantity and labeled to obtain the finished anti-HCV indoor quality control product. The quality control product is stored at -20℃.

[0020] Compared with the existing technology, the beneficial effects of the present invention are as follows:

[0021] 1. The present invention provides a humanized anti-HCV monoclonal antibody that specifically recognizes HCV Core protein, and discloses the amino acid sequence of the humanized anti-HCV monoclonal antibody. The humanized anti-HCV monoclonal antibody provided by the present invention specifically recognizes HCV structural proteins, which are all commonly used coating and / or labeling raw materials for anti-HCV detection kits, and after humanization, they can also specifically bind to anti-human IgG antibodies, and are compatible with commercial anti-HCV immunoassay kits based on various methodological principles such as sandwich methods and indirect methods. In addition, humanized anti-HCV monoclonal antibodies are easy to scale up, have small batch differences, and are low in cost. They have broad application prospects as materials for anti-HCV indoor quality control products and inter-laboratory quality assessment samples.

[0022] 2. The anti-HCV indoor quality control product provided by the present invention uses humanized anti-HCV monoclonal antibodies and is compatible with a variety of commercial anti-HCV immunoassay kits based on sandwich methods, indirect methods, etc., and is a true third-party quality control product. In addition, the anti-HCV indoor quality control product uses human serum matrix as the quality control matrix, which is similar to the matrix of clinical samples, greatly improving the interoperability of indoor quality control products and better meeting the requirements of various laboratories for monitoring and evaluating the quality of anti-HCV tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The protein electrophoresis diagram of humanized anti-HCV monoclonal antibody is provided, wherein lane C00012 is the electrophoresis diagram of humanized anti-HCV monoclonal antibody C00012.

[0024] Figures 2A-2D The interoperability test results of anti-HCV indoor quality control products are provided. Figure 2A This is a comparison chart of the interoperability test between domestic ELISA reagent A and domestic ELISA reagent B; Figure 2B This is a comparison chart of the interoperability test between domestic ELISA reagent A and domestic luminescent reagent; Figure 2C This is a comparison chart of the interoperability test between domestic ELISA reagent A and imported luminescent reagent A; Figure 2D This is a comparison chart of the interoperability test between domestic ELISA reagent A and imported luminescent reagent B. DETAILED DESCRIPTION

[0025] The present invention is described in detail below through specific embodiments. It should be pointed out that the following embodiments are only for further explanation of the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by technicians in this field based on the above invention content still fall within the scope of protection of the present invention.

[0026] As used herein, the term "transfection" refers to the uptake of foreign or exogenous DNA into a cell, and this technique can be used to introduce one or more exogenous DNA moieties into a suitable host cell.

[0027] Example 1

[0028] Preparation of humanized anti-HCV monoclonal antibody

[0029] (1) Animal immunization: The purchased recombinant HCV Core protein expressed by genetic engineering was used as the immunogen. After being fully emulsified with an equal amount of Freund's complete adjuvant, BALB / C mice were immunized by intraperitoneal injection. After 14 days, the BALB / C mice were immunized again by intraperitoneal injection with the same dose of incomplete Freund's adjuvant. After 5 days, blood was collected from the mice through the vein, and the antibody titer in the mouse serum was detected by indirect ELISA.

[0030] (2) Cell fusion and monoclonal screening: Mouse spleen cells were mixed with mouse myeloma cells SP2 / 0 in the logarithmic phase at a ratio of 10:1, centrifuged at 2000rpm for 5 minutes, and the supernatant was discarded. The resulting cell mixture was placed in a 37°C water bath, and 50% PEG1500 was added while stirring. The addition was completed within 1-1.5 minutes, and the mixture was allowed to stand for 2 minutes. Then, within 5 minutes, 37°C preheated serum-free culture medium was added dropwise, centrifuged at 2000rpm for 5 minutes, the supernatant was discarded, and complete selection medium (20% FBS +HAT) was added. The fused cells were resuspended by gentle blowing. Then 200μL / well was inoculated into a 96-well plate with trophoblast cells, and cultured at 37°C with 5% CO2. After 7-10 days, an indirect ELISA screening test was performed to screen the hybridoma cells in the positive wells, and monoclonalization was performed by limiting dilution method, with a specific dilution concentration of 0.5-1 cell / well. After 10 days, subclone detection was started, strong positive clones were screened and expanded in 24-well plates, subcloning was repeated 2-3 times, and the expanded positive clone cells were stored in liquid nitrogen. The anti-HCV Core protein hybridoma cell line was named C00012.

[0031] (3) Preparation of monoclonal antibody ascites and antibody purification: Healthy female BABL / C mice aged 12 weeks or older were selected and intraperitoneally injected with 0.5 mL of sterile paraffin wax. Seven days later, each mouse was intraperitoneally injected with 8 × 10 6 hybridoma cells, and collect ascites after the abdominal cavity of the mouse is obviously swollen. After the ascites is centrifuged at 10,000 rpm for 15 minutes, the supernatant is taken and purified by protein A affinity chromatography column to obtain purified antibodies. Finally, the concentration is measured by a micro-ultraviolet spectrophotometer, and the antibody purity is detected by SDS-PAGE, and the antibody concentration is fixed at 1 mg / mL.

[0032] (4) Identification of monoclonal antibodies: HCV Core protein was coated on a 96-well microplate at a protein amount of 0.5 μg / mL and 100 μL / well, respectively. The anti-HCV monoclonal antibody obtained in step (3) was diluted in a series of gradients using PBS solution as the primary antibody. HRP-labeled goat anti-mouse IgG antibody was diluted 20,000 times as the secondary antibody working solution. An indirect ELISA test was performed to test the titer of the monoclonal antibody in specific recognition of HCV Core protein.

[0033] Example 2

[0034] Preparation of humanized anti-HCV monoclonal antibody

[0035] (1) Gene sequencing: The variable region gene sequencing of C00012 cell lines was performed separately. The amino acid sequence of the heavy chain variable region of C00012 is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0036] (2) Construction of antibody expression vector: The heavy chain variable region gene and light chain variable region gene of hybridoma cells were spliced ​​with the corresponding constant region gene of human antibody, and then the complete heavy and light chain genes were connected to the eukaryotic expression vector pcDNA3.4 to construct the anti-human-mouse chimeric antibody gene eukaryotic expression plasmid.

[0037] (3) Construction of cell lines: The constructed recombinant plasmid was transfected into CHO-S cells at a rate of 5×10 5 The cells were placed in a 6-well plate at 1000 μg / mL and screened with G418 after 48 hours. After obtaining a stable cell pool, monoclonal cells were screened by limiting dilution, and the cell supernatant was taken for indirect ELISA detection. Finally, the monoclonal cells with the highest expression level were selected for expansion culture, and the cells were frozen and stored in liquid nitrogen.

[0038] (4) Antibody purification: The supernatant of the cell culture was purified using a Protein A column to obtain a humanized monoclonal antibody that specifically recognizes the HCVCore protein. Finally, a micro-UV spectrophotometer was used to measure the concentration, and SDS-PAGE was used to detect the antibody purity, and the antibody concentration was fixed at 1 mg / mL.

[0039] Example 3

[0040] Identification of humanized anti-HCV monoclonal antibodies

[0041] The HCV Core protein was coated in a 96-well microplate at a coating amount of 0.5 μg / mL and 100 μL / well, respectively. The humanized anti-HCV monoclonal antibody obtained in Example 2 was diluted in a series of gradients using PBS solution as the primary antibody. The HRP-labeled goat anti-human IgG antibody was diluted 20,000 times as the secondary antibody working solution, and an indirect ELISA test was performed to test the titer of the three monoclonal antibodies for specific recognition of the HCV Core protein. The results showed that the titer of the humanized antibody was normal and could be used. The test results are shown in Table 1.

[0042] Table 1 Humanized anti-HCV monoclonal antibody titer test results

[0043]

[0044] Example 4

[0045] Compatibility of humanized anti-HCV monoclonal antibodies in different commercial kits

[0046] The humanized anti-HCV monoclonal antibody obtained in Example 2 was diluted into three concentrations in a series gradient using PBS solution, and 5 anti-HCV positive clinical samples, anti-HCV secondary markers, and two commercial anti-HCV antibodies diluted into three concentrations were used as samples. The anti-HCV detection kits using two domestic ELISA methods, one domestic chemiluminescence method, and two imported chemiluminescence methods were used for testing. The test results are detailed in Table 2.

[0047] From the results in Table 2, it can be seen that the humanized anti-HCV monoclonal antibody obtained in Example 2 can be detected by all five kits, but there are differences in the test results of kits of different brands. Some kits tested two commercial anti-HCV antibodies as negative, and the measured values ​​of high-concentration samples were low. It can be seen that the humanized anti-HCV monoclonal antibody obtained in Example 2 has better compatibility than commercial antibodies and higher titer, and is more suitable as a raw material for anti-HCV indoor quality control products and inter-laboratory quality assessment samples.

[0048] Table 2 Compatibility test results of humanized anti-HCV monoclonal antibodies

[0049]

[0050] Example 5

[0051] Preparation of anti-HCV indoor quality control products

[0052] The components of the anti-HCV in-house quality control product are: humanized anti-HCV monoclonal antibody, human serum matrix, 0.9% sodium chloride, 1% protein protectant, 0.05% ProClin300 and 0.05% ProClin950.

[0053] The specific preparation steps are as follows:

[0054] (1) Place the frozen human serum matrix in a (2-8)°C environment until it is completely dissolved. Then place it in a 60°C water bath, heat it to 60°C, let it stand for 60 minutes, and then use a 0.22μm ultrafiltration membrane to sterilize and filter. The treated human serum matrix is ​​ready for use.

[0055] (2) According to the preparation plan, take an appropriate amount of treated human serum matrix, place it on a magnetic stirrer for stirring, and add humanized anti-HCV monoclonal antibody, sodium chloride, protein protectant, ProClin300 and ProClin950 in sequence, and then add the treated human serum matrix, make up to the target preparation volume, and mix thoroughly.

[0056] (3) Use the ELISA kit to measure the semi-finished anti-HCV indoor quality control product in step (2). The anti-HCV indoor quality control product that meets the test concentration requirements is divided into cryopreservation tubes according to the filling volume and stored at -20°C.

[0057] Example 6

[0058] In the anti-HCV indoor quality control product, the dosage ratio of humanized anti-HCV monoclonal antibody is as follows:

[0059] Table 3 Dosage ratio of humanized anti-HCV monoclonal antibody

[0060]

[0061] Example 7

[0062] Interoperability evaluation of anti-HCV indoor quality control products

[0063] The interoperability of the anti-HCV indoor quality control products prepared in Examples 5 and 6 was evaluated. Two domestic ELISA methods, one domestic chemiluminescence method, and two imported chemiluminescence anti-HCV detection kits were used for testing. The test samples included 20 fresh anti-HCV clinical serum samples (the concentrations covered the three concentration levels of anti-HCV indoor quality control products), hepatitis C virus antibody series serum (liquid) standard materials GBW(E)090154, GBW(E)090073, GBW(E)090074, GBW(E)090075, GBW(E)090076, GBW(E)090077, GBW(E)090078, GBW(E)090079, GBW(E)090070, GBW(E)090071, GBW(E)090073, GBW(E)090074 ...5, GBW(E)090076, GBW(E)090077, GBW(E)090077, GBW(E)090078, GBW(E)090079, GBW(E)090070, GBW(E)090071, GBW(E)090073, GBW(E)090073, GBW(E)090073, GBW(E)090073, GBW(E)090073, GBW(E) )090076, hepatitis C virus antibody (anti-HCV) serum (liquid) standard materials GBW(E)090854, GBW(E)090855, GBW(E)090856, GBW(E)090857 of Guangzhou Bondesun Biotechnology Co., Ltd., hepatitis C virus antibody (anti-HCV) liquid standard materials GBW(E)090210, GBW(E)090211, GBW(E)090212, GBW(E)090213 of Beijing Jinhao Pharmaceutical Co., Ltd. Each sample was tested three times, and finally the anti-HCV indoor quality control products prepared in Examples 5 to 15, certified standard materials of other companies and 20 fresh clinical patient samples were calibrated with the secondary anti-HCV standard material of Beijing Kangchestan Biotechnology Co., Ltd.

[0064] After the experiment, the experimental data were collated and analyzed and the evaluation results were judged according to the health industry standard WS / T 356-2011 of the People's Republic of China "Guidelines for Matrix Effect and Interoperability Evaluation". The specific results are shown in Tables 4 to 8 and Appendix Figures 2A to 2D .

[0065] Table 4 Results of interoperability test of anti-HCV indoor quality control products determined by different kits (mean, NCU / mL)

[0066]

[0067] Table 5 Interoperability evaluation results - domestic ELISA reagent A and domestic ELISA reagent B (mean, NCU / mL)

[0068]

[0069] Table 6 Interoperability evaluation results - domestic ELISA reagent A and domestic luminescence reagent (mean, NCU / mL)

[0070]

[0071] Table 7 Interoperability evaluation results - domestic ELISA reagent A and imported luminescent reagent A (mean, NCU / mL)

[0072]

[0073] Table 8 Interoperability evaluation results - domestic ELISA reagent A and imported luminescent reagent B (mean, NCU / mL)

[0074]

[0075] Table 4 to Table 8 and Appendix Figures 2A to 2D The experimental results show that the measured mean values ​​of the anti-HCV indoor quality control products of Example 6 are within the 95% confidence interval of the clinical sample mean values, that is, the anti-HCV quality control products of this example and the anti-HCV positive clinical samples perform consistently in immunoassays, without obvious matrix effects, and are interoperable among two domestic ELISA methods, one domestic chemiluminescence method, and two imported chemiluminescence methods.

[0076] Example 8

[0077] Uniformity evaluation of anti-HCV indoor quality control products

[0078] The uniformity evaluation test of the anti-HCV indoor quality control product was carried out in accordance with the pharmaceutical industry standard of the People's Republic of China YY / T1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents" for experimental design, data processing and result determination. The specific experimental process is as follows.

[0079] The anti-HCV indoor quality control products of Example 6 were randomly selected, 20 tubes were selected for each concentration level, and numbered 1 to 20. Each tube of quality control product was tested three times using domestic ELISA reagent A. The three repeated tests were performed in different orders, such as 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 for the first time; 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 for the second time; and 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 for the third time.

[0080] The uniformity test results and analysis results of the anti-HCV indoor quality control products are detailed in Tables 9 and 10.

[0081] Table 9 Anti-HCV indoor quality control product uniformity test results (S / CO value)

[0082]

[0083] Table 10 Analysis of uniformity results of anti-HCV indoor quality control products

[0084]

[0085] The experimental results in Tables 9 and 10 show that the uniformity test data of the anti-HCV indoor quality control product of Example 6 was analyzed by variance analysis using the F test, and the F values ​​of the analysis results of each example were all less than F crit The value indicates that the anti-HCV indoor quality control product of Example 6 has good uniformity.

[0086] Example 9

[0087] Long-term stability evaluation of anti-HCV indoor quality control products

[0088] The anti-HCV indoor quality control product of Example 6 was stored in liquid nitrogen. Two bottles were taken out from each concentration level at the 0th month, the 2nd month, the 4th month, the 6th month, the 8th month, the 10th month, the 11th month, and the 12th month of storage, respectively, and stored in an environment of (2-8)°C as samples for the (2-8)°C stability study. The synchronous stability study method was used until the 12th month, and each stability study sample was tested using the domestic ELISA reagent A under repeatability conditions.

[0089] The anti-HCV indoor quality control product of Example 6 stored in liquid nitrogen was taken out of 2 bottles from each concentration level at the 0th month, 6th month, 12th month, 15th month, 18th month, 20th month, 22nd month, and 24th month of storage, and stored in an environment of (-20±5)°C as samples for -20°C stability study. Until the 24th month, the synchronous stability study method was adopted, and the domestic ELISA reagent A was used to test each stability study sample under repeatability conditions.

[0090] All stability study test data were tested for trend significance using the t-test. The data analysis and judgment criteria for stability studies were conducted in accordance with YY / T 1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents". Specific test results and data analysis results are detailed in Tables 11 to 14.

[0091] Table 11 Test results of storage stability of anti-HCV indoor quality control products at (2~8)℃ (S / CO value)

[0092]

[0093] Table 12 Analysis results of storage stability study of anti-HCV indoor quality control products at (2~8)℃ (S / CO value)

[0094]

[0095] Table 13 Test results of storage stability study of anti-HCV indoor quality control products at -20℃ (S / CO value)

[0096]

[0097] Table 14 Analysis results of storage stability study of anti-HCV indoor quality control products at -20℃ (S / CO value)

[0098]

[0099] The experimental results in Tables 11 and 12 show that the stability results of the anti-HCV indoor quality control product of Example 6 under the storage condition of (2~8)℃ were subjected to linear regression analysis. Referring to the calculation process of YY / T 1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents", the slope (β1) of concentration 1 was 0.0054, the slope (β1) of concentration 2 was 0.0015, and the slope (β1) of concentration 3 was 0.0004. The corresponding t 0.05, n-2 ×s(β 1 ) are 0.0086, 0.0020, and 0.0262, respectively. Therefore, it can be seen that the absolute values ​​of the slopes (|β1|) of the three concentration levels of the anti-HCV indoor quality control product in Example 12 are all smaller than the corresponding t 0.05, n-2 ×s(β1 ), indicating that the slope trend of the linear regression analysis of the three concentrations of the anti-HCV indoor quality control products in Example 12 is not significant, no instability is observed, and the shelf life when stably stored at (2~8)°C is not less than 12 months.

[0100] The experimental results in Tables 13 and 14 show that the stability results of the anti-HCV indoor quality control product of Example 6 under the storage condition of -20°C were subjected to linear regression analysis. Referring to the calculation process of YY / T 1652-2019 "General Technical Requirements for Quality Control Materials for In Vitro Diagnostic Reagents", the slope (β1) of level 1 was 0.0022, the slope (β1) of concentration 2 was -0.0002, and the slope (β1) of concentration 3 was 0.0052. The corresponding t 0.05, n-2 ×s(β 1 ) are 0.0048, 0.0062, and 0.0270, respectively. Therefore, it can be seen that the absolute values ​​of the slopes (|β1|) of the three concentration levels of the anti-HCV indoor quality control product in Example 12 are all smaller than the corresponding t 0.05, n-2 ×s(β 1 ), indicating that the slope trend of the linear regression analysis of the three concentrations of the anti-HCV indoor quality control products in Example 12 is not significant, no instability is observed, and the shelf life when stably stored at -20°C is not less than 24 months.

[0101] Example 10

[0102] Evaluation of the Repeated Freeze-Thaw Stability of Anti-HCV In-House Quality Control Products

[0103] Randomly select the anti-HCV indoor quality control products of Example 6, extract 10 tubes at each concentration level, and store them in an environment of -20°C. After all the anti-HCV indoor quality control products are frozen, take out 5 tubes at each concentration level, place them at room temperature, wait for them to completely melt into liquid, and balance to room temperature, and then place them at -20°C again, and repeat the above freeze-thaw operation 10 times.

[0104] Domestic ELISA reagent A was used to test the above research samples that had and had not been subjected to repeated freeze-thaw treatment, and the mean and standard deviation were calculated. The data were then analyzed by t-test to determine the repeated freeze-thaw stability of the anti-HCV indoor quality control product. The specific repeated freeze-thaw stability evaluation test results and analysis results are detailed in Table 15.

[0105] Table 15 Repeated freeze-thaw stability test and analysis results of anti-HCV indoor quality control products (S / CO value)

[0106]

[0107] Table 15 shows the results of independent sample T test analysis at three concentration levels of the anti-HCV indoor quality control product of Example 6. Independent sample T test was performed between different groups of samples stored at -20°C and samples subjected to 10 repeated freeze-thaw cycles. The analysis showed that the P value of level 1 was 0.881, the P value of level 2 was 0.560, and the P value of level 3 was 0.819, all of which met the P>0.05 requirement. Therefore, it is believed that there is no significant difference in the detection values ​​of level 1, level 2 and level 3 of the anti-HCV indoor quality control product of Example 12 after 10 repeated freeze-thaw cycles when stored at -20°C and subjected to 10 repeated freeze-thaw cycles. It can be considered that the anti-HCV indoor quality control product of Example 12 can withstand 10 repeated freeze-thaw operations.

Claims

1. A humanized anti-HCV monoclonal antibody, characterized in that: The antibody can specifically recognize HCV Core protein, including light chain and heavy chain, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

2. The humanized anti-HCV monoclonal antibody according to claim 1, characterized in that The preparation method thereof comprises the following steps: (1) Selecting HCV Core protein and immunizing BALB / c mice to obtain immunized mice; (2) Based on the immunized mice described in step (1), monoclonal antibodies are prepared by hybridoma technology, and high-titer specific monoclonal antibodies are screened by limiting dilution method and ELISA method; (3) sequencing the hybridoma variable region gene of the monoclonal antibody described in step (2) to obtain a gene sequence, and combining the gene sequence with the human IgG1 gene structure to construct a eukaryotic expression plasmid; (4) Transfecting the eukaryotic expression plasmid described in step (3) into CHO-S cells to construct a stable cell line, and using the stable cell line to express and prepare a humanized monoclonal antibody.

3. An anti-HCV indoor quality control product, characterized in that: The invention comprises the humanized anti-HCV monoclonal antibody according to claim 1, a human serum matrix, sodium chloride, a protein protective agent and a preservative.

4. The anti-HCV indoor quality control product according to claim 3, characterized in that: In terms of concentration, the content of humanized anti-HCV monoclonal antibody is 1.0-200 ng / mL; in terms of weight percentage, the content of sodium chloride is 0.5-1.2%, the content of protein protectant is 0.1%-5%, and the content of preservative is 0.02%-0.3%; in terms of volume, the human serum matrix is ​​fixed to the target preparation volume.

5. The anti-HCV indoor quality control product according to claim 3 or 4, characterized in that: The preservatives are ProClin300 and ProClin950.

6. The anti-HCV indoor quality control product according to claim 3 or 4, characterized in that: The quality control product is liquid.

7. The method for preparing the anti-HCV indoor quality control product according to claim 3 or 4, characterized in that: The following steps are involved: (1) placing the frozen human serum matrix in a (2-8)°C environment until it is completely dissolved; then treating it at 60°C for 60 minutes, sterilizing and filtering, and obtaining the treated human serum matrix; (2) under stirring, sequentially adding humanized anti-HCV monoclonal antibody, sodium chloride, protein protective agent and preservative to the human serum matrix treated in step (1), then using the treated human serum matrix to make up to the target preparation volume, and fully mixing to obtain an anti-HCV indoor quality control semi-finished product; (3) Using an ELISA kit, measure the anti-HCV indoor quality control semi-finished product in step (2) to determine whether the concentration of the quality control product meets the quality inspection standard; if it meets the standard, it is a qualified anti-HCV indoor quality control semi-finished product; (4) In step (3), the qualified anti-HCV indoor quality control product semi-finished product is packaged according to the required packaging quantity and labeled to obtain the finished anti-HCV indoor quality control product; the quality control product is stored at -20°C.

8. The anti-HCV indoor quality control product according to claim 3 or 4, characterized in that: It can be stably stored at -20℃ for no less than 2 years; it can be stably stored at (2~8)℃ for no less than 1 year.

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