Method for quantitatively detecting Japanese encephalitis virus antigen content, monoclonal antibody prepared for establishing the method, and application thereof

Abstract

The present application relates to the field of biological detection technology, and in particular to a method for quantitatively detecting the content of Japanese encephalitis virus antigen, a monoclonal antibody prepared for establishing the method, and its application. The method of the present application comprises the following steps: coating Japanese encephalitis monoclonal antibody on an enzyme-labeled plate, adding a gradient-diluted Japanese encephalitis positive reference substance, a negative control, and a diluted sample to be tested to each well after coating, cleaning the enzyme-labeled plate after incubation, adding enzyme-labeled Japanese encephalitis monoclonal antibody, cleaning the enzyme-labeled plate after incubation again, adding a substrate color developing solution for color development, stopping the enzyme reader to read the OD value, and using the OD value of the Japanese encephalitis positive reference substance to draw a regression equation, and quantitatively deriving the content of Japanese encephalitis virus antigen in the vaccine by regression calculation. Thus, the method of the present application can effectively and accurately predict the antigen content of the finished vaccine, provide a reliable quantitative numerical basis for the production of Japanese encephalitis vaccine, and can be well applied to the quality control of Japanese encephalitis vaccine production.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and specifically to a method for quantitatively detecting the content of Japanese encephalitis virus antigen, a monoclonal antibody prepared for establishing the method, and its application. Background Art

[0002] Japanese encephalitis (JE) is an acute infectious disease caused by the JE virus and transmitted by mosquitoes. With high mortality and disability rates, JE is a major infectious disease threatening the health of the general population, especially children. A nationwide epidemic occurred in the 1960s and early 1970s. However, with the widespread implementation of JE vaccination since the 1970s, the incidence of JE has declined significantly and has remained at a low level in recent years. This demonstrates the importance of JE vaccination in preventing JE epidemics.

[0003] JE virus antigen, the primary component of JE vaccines, plays a crucial role in vaccine quality. While commercially available JE virus detection kits exist, these primarily detect JE infection in body fluids. Because the level of JE virus antigen in infected body fluids is typically low, existing kits are only suitable for qualitative detection of low-concentration antigens in body fluids and struggle to quantitatively detect the high concentrations of viral antigens found in vaccine production. Summary of the Invention

[0004] The purpose of this application is to provide a method for quantitatively detecting the antigen content of Japanese encephalitis virus, the monoclonal antibodies prepared for establishing this method, and their applications. This method uses monoclonal antibodies to effectively and accurately detect the antigen content of finished vaccines, providing a reliable quantitative numerical basis for the production of Japanese encephalitis vaccines, and ensuring the stability and improvement of vaccine quality.

[0005] In the first aspect, the present application provides a method for quantitatively detecting the content of Japanese encephalitis virus antigen, comprising the following steps: coating Japanese encephalitis monoclonal antibody on an enzyme-labeled plate, adding gradiently diluted Japanese encephalitis positive reference substances, negative controls and diluted samples to be tested to each well after coating, incubating and cleaning the enzyme-labeled plate, adding enzyme-labeled Japanese encephalitis monoclonal antibody, incubating again and cleaning the enzyme-labeled plate, adding substrate color development solution for color development, stopping the enzyme reader to read the OD value, drawing a regression equation using the OD value of the Japanese encephalitis positive reference substance, and quantitatively obtaining the content of Japanese encephalitis virus antigen in the vaccine through regression calculation.

[0006] By adopting the above technical solution, the present application uses an ELISA plate as a carrier to fix the Japanese encephalitis monoclonal antibody, and utilizes the high specificity and stability of the monoclonal antibody and the antigen to capture the antigen in the Japanese encephalitis vaccine, thereby promoting the antigen to be stably adsorbed on the ELISA plate.

[0007] The ELISA plate is cleaned to remove other substances in the sample to reduce interference, and then enzyme-labeled Japanese encephalitis monoclonal antibody is added, forming an ELISA double antibody sandwich structure of "monoclonal antibody-antigen-enzyme-labeled monoclonal antibody." The present application utilizes enzyme-labeled monoclonal antibodies, as opposed to enzyme-labeled polyclonal antibodies. This not only promotes stable fixation of the enzyme-labeled monoclonal antibody on the ELISA plate, but also allows for revalidation of the adsorbed antigen, eliminating interfering factors with similar structures to the antigen. This allows for accurate and effective capture of the antigen in the Japanese encephalitis vaccine.

[0008] On this basis, the present invention adds a substrate color developing solution for color development, draws a regression equation through the OD value of the Japanese encephalitis positive reference substance, and then uses the regression equation to accurately and quantitatively calculate the antigen content, providing a reliable quantitative numerical basis for the production of Japanese encephalitis vaccine and ensuring the stability and improvement of vaccine quality.

[0009] Preferably, the coated Japanese encephalitis monoclonal antibody and the enzyme-labeled Japanese encephalitis monoclonal antibody form a monoclonal antibody pair, and the preparation method of the monoclonal antibody pair comprises the following steps:

[0010] The purified Japanese encephalitis virus solution is mixed with an adjuvant to prepare an immunogen;

[0011] Immunizing all mice with the immunogen, taking spleen cells from one group of mice for a first cell fusion, and continuously boosting the remaining mice with the immunogen, and taking spleen cells from the remaining mice for a second cell fusion;

[0012] The monoclonal cells obtained from the first cell fusion and the second cell fusion are cultured, and after repeated screening and specificity testing, positive clone cells that can secrete monoclonal antibodies that recognize Japanese encephalitis virus and have neutralizing activity are selected;

[0013] The selected positive clone cells are injected into the peritoneal cavity of mice used for antibody purification. After the monoclonal antibody is expressed and secreted, the ascites of the mice is extracted and the supernatant of the ascites is collected after centrifugation for purification to obtain the Japanese encephalitis monoclonal antibody.

[0014] The purified antibodies with high antibody yield and high sensitivity in binding to Japanese encephalitis virus in the ascites supernatant screened by detection were selected for enzyme labeling to obtain enzyme-labeled Japanese encephalitis monoclonal antibody;

[0015] The Japanese encephalitis monoclonal antibody is selected and coated on an ELISA plate. After blocking, a gradient dilution of Japanese encephalitis antigen is added. After incubation, the enzyme-labeled Japanese encephalitis monoclonal antibody is added. After further incubation, a display substrate is added and the OD value is measured. According to the OD value results, the coated Japanese encephalitis monoclonal antibody and the enzyme-labeled Japanese encephalitis monoclonal antibody that are paired with each other are screened, which is the monoclonal antibody pair.

[0016] By adopting the above technical solution, the dual-antibody sandwich structure has different binding effects between different antibodies and the ELISA plate, as well as between antibodies and antigens, during actual operation. In addition, the protein structures of different antibodies have certain steric hindrances. Therefore, the present application further prefers to use the coated Japanese encephalitis monoclonal antibody and the enzyme-labeled Japanese encephalitis monoclonal antibody as a pair of monoclonal antibodies that can be paired with each other, thereby ensuring that the present application can stably form a "monoclonal antibody-antigen-enzyme-labeled monoclonal antibody" dual-antibody sandwich structure.

[0017] Because the antigen in the Japanese encephalitis vaccine is the key component that stimulates the body to produce antibodies, some antigens lose their immune-stimulating effect during the vaccine production process. This inactivated antigen interferes with the determination of the effective antigen content in the vaccine. Therefore, this application utilizes neutralizing antibodies, which only bind to effective antigens, to accurately detect the effective antigen content in the Japanese encephalitis vaccine. However, monoclonal antibodies are known to be difficult and costly to obtain, and effectively obtaining neutralizing antibodies against Japanese encephalitis virus is even more challenging.

[0018] To this end, the present application uses the aforementioned preparation method to immunize mice with an immunogen containing Japanese encephalitis virus. Based on the tendency of neutralizing antibody concentrations to initially increase and then decrease in infected organisms, the present application specifically selects cells from mice that have undergone both primary and booster immunizations for cell fusion. Repeated screening and specificity testing then gradually identify monoclonal cell lines capable of secreting neutralizing antibodies. The isolated monoclonal cell lines exhibit high antibody yields and high sensitivity for binding to Japanese encephalitis virus. Finally, after obtaining purified antibodies, the present application also conducts antibody pairing tests to effectively screen for compatible antibodies and enzyme-labeled antibodies, thereby ensuring that the present application's quantitative detection method for antigen content is optimal.

[0019] Preferably, the adjuvant mixed with the Japanese encephalitis virus purified solution is adjuvant CFA and A mixture of classic adjuvants.

[0020] By adopting the above technical solution, the adjuvant CFA is a complete Freund's adjuvant, which is a mixture of heat-killed Mycobacterium tuberculosis, paraffin oil and lanolin, and mainly causes Th1 cell immune response. Its activity is a kind of sustained release of deposited antigens and stimulation of local natural immunity, thereby enhancing adaptive immunity; however, the adjuvant CFA will cause a severe inflammatory reaction and interaction with the antigen due to the influx of a large number of white blood cells into the site of antigen deposition. Therefore, the adjuvant CFA may cause local inflammation and granulomatous reaction at the injection site, changes in lymph node structure, skin ulcers and other side effects. For this reason, this application uses The combination with classic adjuvants can not only reduce the amount of adjuvant CFA added to reduce the occurrence of side effects, but also make up for the low immune response of oil-in-water adjuvant CFA in the early stage.

[0021] Preferably, the preparation steps of the immunogen are as follows:

[0022] The purified Japanese encephalitis virus solution was mixed with the adjuvant CFA in equal volumes, and the mixture was ultrasonically treated to form an emulsion to obtain immunogen A. Mix equal volumes of classic adjuvants to obtain immunogen B; mix equal volumes of A and B to obtain immunogen B.

[0023] By adopting the above technical solution, the present invention prepares the immunogen by mixing the purified solution of Japanese encephalitis virus with the adjuvant CFA and Classic adjuvant volume mixing, which is based on the difficulty of mixing the oil-in-water adjuvant CFA with the purified Japanese encephalitis virus solution, uses ultrasonic treatment to promote the adjuvant CFA to form a gel state after mixing with the viral protein, so as to facilitate the subsequent addition of the immunogen B. Classic adjuvants can quickly and evenly disperse in immunogen A, while ensuring the excellent immune response effect of the immunogen of this application.

[0024] Preferably, the mice used for antibody purification are injected with adjuvant in the abdomen before the monoclonal cells are injected.

[0025] Preferably, the adjuvant pre-injected into the antibody purification mice is the adjuvant IFA.

[0026] By adopting the above technical solution, the adjuvant IFA is an incomplete Freund's adjuvant. Compared with the adjuvant CFA, it lacks Mycobacterium tuberculosis and mainly induces a Th2 cell immune response, which is mainly used for subsequent immunization. In this application, injecting the adjuvant IFA before injecting the monoclonal cells into the mouse helps the mouse's Th2 cells to produce an immune response in advance, prompting the subsequent injection of monoclonal cells to quickly produce neutralizing antibodies, effectively improving the secretion efficiency of neutralizing antibodies and obtaining a higher antibody secretion amount.

[0027] In a second aspect, the present application provides a monoclonal antibody pair suitable for establishing the above-mentioned method for quantitatively detecting the content of Japanese encephalitis virus antigen, comprising Japanese encephalitis monoclonal antibody 12B9 and enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP, wherein the Japanese encephalitis monoclonal antibody 12B9 comprises a heavy chain variable region with a base sequence as shown in SEQ ID NO: 1 and a light chain variable region with a base sequence as shown in SEQ ID NO: 2, and the enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP comprises a heavy chain variable region with a base sequence as shown in SEQ ID NO: 3 and a light chain variable region with a base sequence as shown in SEQ ID NO: 4.

[0028] Preferably, the amino acid sequence of the heavy chain variable region of the Japanese encephalitis monoclonal antibody 12B9 is shown in SEQ ID NO: 5; the amino acid sequence of the light chain variable region of the Japanese encephalitis monoclonal antibody 12B9 is shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP is shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region of the enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP is shown in SEQ ID NO: 8.

[0029] By adopting the above technical solution, the present application has found through experiments that the monoclonal antibody pair obtained by applying the above base sequence and its corresponding amino acid sequence to the detection method of the present application has a better capture effect, so the present application regards it as a further preferred solution.

[0030] Thirdly, the present application provides a method for quantitatively detecting the content of Japanese encephalitis virus antigens for use in the preparation of Japanese encephalitis vaccines. This method provides a reliable quantitative numerical basis for the production of Japanese encephalitis vaccines, facilitating the production quality control of vaccines.

[0031] In a fourth aspect, the present application provides a kit for quantitatively detecting the content of Japanese encephalitis virus antigen, characterized in that the content of Japanese encephalitis virus antigen in Japanese encephalitis vaccine is quantitatively determined by using the above-mentioned method for quantitatively detecting the content of Japanese encephalitis virus antigen.

[0032] In summary, this application has the following beneficial effects:

[0033] 1. The method of the present application utilizes the characteristics of monoclonal antibodies, such as high specificity, strong targeting, and ability to stably bind to antigens, to form an ELISA double-antibody sandwich structure of "monoclonal antibody-antigen-enzyme-labeled monoclonal antibody", thereby accurately capturing the antigens in the Japanese encephalitis vaccine. The regression relationship can then be used to quickly and quantitatively detect the Japanese encephalitis antigen content, providing favorable data support for the production of Japanese encephalitis vaccine.

[0034] 2. In this application, mice are preferably used for immunization. Cells from primary and boosted immunized mice are specifically selected for cell fusion. Monoclonal cell lines capable of secreting neutralizing antibodies are then gradually selected through repeated screening and specificity testing. This allows for the effective and accurate preparation of monoclonal antibodies against Japanese encephalitis that are suitable for the quantitative detection of Japanese encephalitis antigen content.

[0035] 3. The application of specific monoclonal antibody pairs to the method of the present application can effectively improve the capture effect of effective antigens in Japanese encephalitis vaccines and further improve the accuracy of antigen quantitative detection.

[0036] 4. The method of the present application is applied in the production process of Japanese encephalitis vaccine, which can timely know the content of effective antigen in the vaccine and provide a reliable quantitative numerical basis for the production quality control of the vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a standard curve diagram of Example 1 of the present application. DETAILED DESCRIPTION

[0038] Example 1

[0039] 1. Preparation of mouse anti-Japanese encephalitis virus monoclonal antibodies

[0040] 1. Cell fusion and screening of hybridoma cell lines

[0041] 1-1. Prepare the immunogen. The preparation steps are as follows:

[0042] ①. Mix 1 mL of purified Japanese encephalitis virus solution with 1 mL of adjuvant CFA;

[0043] ②Use ultrasonic cell disruptor to sonicate the mixture for 3 seconds, pause for 3 seconds, and perform sonication 10-15 times until a milky mixture is obtained to obtain immunogen A;

[0044] ③, 1mL of Japanese encephalitis virus purified solution and 1mL of Classical adjuvant (purchased from Sigma) was mixed;

[0045] ④. Shake the mixture on a shaker set at 80-100 rpm at room temperature for 2 hours to obtain immunogen B;

[0046] ⑤. Mix equal volumes of A and B to harvest the comprehensive immunogen.

[0047] Among them, the purified Japanese encephalitis virus solution is a concentrated solution of viral protein in the production process of Japanese encephalitis vaccine, with a concentration of 800μg / mL.

[0048] 1-2. Three groups of mice for cell fusion were immunized with the comprehensive immunogen, which were marked as 1#, 2# and 3#, respectively. Three mice were set in each group for a balanced experiment.

[0049] 1-3. On the 14th day after immunization (2 weeks after the first immunization), the tail blood of mice was drawn, and the antibody titer in the mouse serum was evaluated using the indirect ELISA method. The titer of the tail blood antibodies recognizing Japanese encephalitis virus in the three groups of mice was 1:50,000. In the next step, 1# was selected for cell fusion. At the same time, the remaining two groups of mice continued to be boosted with immunization, and the tail blood was evaluated again on the 21st day of immunization (3 weeks after the first immunization). After the boosted immunization, the antigen recognition titer of 2# and 3# mice still reached 1:50,000. In the next step, 2# mice were selected for cell fusion again, and 3# was used to monitor the immune effect (if the immune effect of 1# and 2# mice is not ideal, 3# mice have the longest immunization time and can be used as a substitute for cell fusion).

[0050] 1-4. Based on the ELISA results, spleen cells from mouse #1 on day 17 after immunization (calculated from the first immunization) and mouse #2 on day 26 after immunization (calculated from the first immunization) were fused with SP2 / 0 myeloma cells. Ten days after fusion, 1222 monoclonal cells were selected and cultured in 96-well plates. After an additional 7 days of culture, the supernatant of the monoclonal cells was diluted 1:1 by volume with PBS buffer. The supernatant in the 96-well plate was evaluated by indirect ELISA to screen for monoclonal cell lines that secrete monoclonal antibodies that recognize Japanese encephalitis virus.

[0051] The screening method is as follows: JE virus protein is diluted to 1 μg / mL with PBS, 100 μL is added to each well, and the reaction is allowed to proceed overnight at 4°C. The plate is then washed three times with PBS and blocked with 5% milk-PBS at room temperature for 1 hour. The plate is then washed once with PBS. Cell supernatant from the 96-well plate is diluted 1:1 with 5% milk-PBS and added to the coated plate. The plate is then incubated at room temperature for 1 hour. The plate is then washed three times with PBS and patted dry. A 1:2000 dilution of HRP-labeled goat anti-mouse IgG (Fc) secondary antibody is added and the reaction is allowed to proceed at room temperature for 1 hour. The plate is then washed five times with PBS, patted dry, and equal volumes of colorimetric solutions A and B are added. The reaction is carried out at room temperature in the dark for 20 minutes. 50 μL of stop solution is then added, mixed, and the OD450 and OD630 values ​​are read on a microplate reader, with the output being OD = OD450 - OD630. The wells with an OD value greater than 1.0 were screened and used as positive wells. There were 13 well plates in total. The corresponding OD values ​​on these 13 well plates are shown in Table 1 below.

[0052] Table 1 OD values ​​of mouse fusion monoclonal cell lines on 13 well plates

[0053]

[0054]

[0055]

[0056]

[0057] 1-5. Based on Table 1 above, 54 positive clone cell lines with high signals (OD values ​​greater than 1.0) were selected and expanded from 96-well plates to 48-well plates. Culture was continued for 2-3 days. Rescreening and specificity detection experiments were performed by indirect ELISA method using JE virus, human serum albumin, Vero cell protein, and culture wastewater. The OD values ​​of the rescreened 54 positive clone cell lines are shown in Table 2 below.

[0058] Table 2 OD values ​​of 54 positive clone cell lines rescreened

[0059]

[0060]

[0061] Note: PC is the positive control; NC is the negative control 5% milk-PBS, and the positive judgment standard is 2.1 times the NC value.

[0062] 1-6. 37 positive clone cell lines with strong recognition of JE virus were retained and expanded from 48-well plates to 12-well plates. They were cultured for 2 days and rescreened and tested for specificity by indirect ELISA, which was coated with JE virus, human serum albumin, Vero cell protein, and culture wastewater. The OD values ​​of the specificity test of the 37 positive clone cell lines are shown in Table 3 below.

[0063] Table 3 OD values ​​of 37 positive clone cell line specific detection

[0064]

[0065] 1-7. Retain 25 positive clones (1C2, 1E9, 1F6, 1F11, 1G7, 1G10, 2G11, 3B4, 3D9, 3E12, 3H1, 4D8, 4E10, 4F12, 5E4, 6A5, 6E11, 7G8, 8A4, 8A7, 9G8, 11A1, 12B9, 13B12, and 13C2) that only recognize JE virus but not human serum albumin, Vero cell protein, or culture wastewater. In vitro antibody neutralization assays were performed on these 25 JE virus strains, and 8 positive clones were selected.

[0066] The neutralization assay used in this screening test demonstrates that viruses lose their pathogenicity to susceptible cells after binding to corresponding antibodies. Specific neutralizing antibodies bind specifically to corresponding viral particles, thereby preventing viral attachment to susceptible cells or inhibiting their invasion, rendering the virus incapable of infection. This experiment uses the neutralization assay to screen for suitable monoclonal antibodies, providing experimental data and theoretical basis for subsequent experiments.

[0067] The neutralization test method specifically includes the following steps:

[0068] ①. Preparation of six-well cell plates

[0069] Prepare BHK21 cell suspension; take BHK21 cells in logarithmic growth phase to make 8×10 4 -1.2×10 5 / mL suspension was inoculated into a six-well cell culture plate, 4mL per well, and cultured in a carbon dioxide incubator (37°C, 5% CO2) for 2 days until a full monolayer was grown for later use.

[0070] ②Virus and antibody neutralization

[0071] Dilute the test sample to a concentration of 10 μg / mL, take approximately 200 PFU / 0.4 mL of the Japanese encephalitis virus for testing, and mix 500 μL of the diluted test antibody and the diluted virus suspension in equal amounts; neutralize the above antibody-virus mixture in a 37°C water bath for 90 minutes, shaking every 30 minutes.

[0072] ③. Adsorption of cells and viruses

[0073] The culture medium from the confluent monolayer of BHK21 cells was aspirated, and the neutralizing antibody-virus mixture was added to one well per group; the virus control group had 12 wells, with 0.4 mL per well. Gently shake the six-well cell culture plate to evenly distribute the liquid. The six-well cell culture plate was placed in a carbon dioxide incubator (37°C, 5% CO2) for 90 minutes, shaking every 30 minutes for 30 seconds each time.

[0074] ④ Cover with methylcellulose covering liquid

[0075] After 90 minutes of adsorption, methylcellulose covering solution was slowly added to the six-well cell culture plate, 4 mL per well; the plate was then placed in a carbon dioxide incubator (37°C, 5% CO2) and cultured for 5 days. Shaking of the cell plate was prohibited during the culture period.

[0076] ⑤Calculation results

[0077] After 5 days of culture, the liquid in the wells of the six-well cell culture plate was aspirated and crystal violet staining solution was added at approximately 2 mL / well. The staining was carried out at room temperature for 30 minutes, and then the staining solution was discarded. The wells were gently rinsed with running water until the color disappeared. After drying, the number of plaques in each well was counted. The results of the neutralization experiment are shown in Table 4 below.

[0078] Table 4 Virus neutralization results of the first validation (25 strains of supernatants / antibodies)

[0079]

[0080]

[0081] 2. Ascites Preparation and Antibody Purification

[0082] 2-1. Based on the neutralization test results in Table 4 above, 8 positive clones with good neutralization activity (1C2, 3E12, 5E4, 8A4, 8A7, 11A1, 12B9, and 13C2) were selected to prepare purified antibodies. These 8 clones were cultured and approximately 1×10 7 The cells were injected into the peritoneal cavity of two groups of BALB / c mice that had been pre-injected with IFA adjuvant. About 10 days later, the ascites of the mice were extracted and centrifuged at 4°C and 12,000 rpm for 15 minutes to collect the ascites supernatant.

[0083] 2-2. Collect the ascites supernatant for Protein G purification and harvest the purified antibodies. The specific process is: take 1mL of column material (coupled with Protein G) and add it to an empty column. After washing with PBS solution, dilute 2mL of ascites supernatant with 8mL of PBS and load it onto the column, or directly load the cell supernatant onto the column, collect the eluate and re-load it onto the column once; then use pH 2.7 glycine eluate for elution, collect 1mL of eluate per tube (pre-add 100μL neutralizing solution (1M Tris, 0.75M NaCl, 0.5μM EDTA, pH 8.0-8.3), collect 5 tubes in total; then use pH 2.7 glycine eluate for elution, collect 1mL of eluate per tube (pre-add 100μL neutralizing solution (1M Tris, 0.75M NaCl, 0.5μM EDTA, pH 8.0-8.3)), collect 5 tubes in total; then use pH Elution was performed with 1.9 glycine eluent, collecting 1 mL of eluate per tube (pre-added with 300 μL of neutralizing solution), for a total of 3 tubes; each tube of eluate was then read at 280 nm using a UV spectrophotometer. Eluates with an OD280 greater than 0.7 were mixed and the OD280 of the mixture was re-measured. The antibody concentration was calculated using a coefficient of 1.4; antibody concentration = OD280 / 1.4.

[0084] 3. Purified Antibody HRP Detection and Labeling

[0085] 3-1. The antibody yields of the selected 8 monoclonal antibodies in the ascites supernatant and the sensitivity of the purified antibodies in binding to Japanese encephalitis virus were tested. The test results of the ascites antibody yields and sensitivities are shown in Table 5 below.

[0086] Table 5 Ascites antibody yield and sensitivity test results of 8 positive clones

[0087]

[0088]

[0089] Based on the ascites antibody yields in Table 5 and the sensitivity of purified antibodies binding to JE virus, 3E12, 8A7, 11A1, and 13C2 were selected for HRP labeling.

[0090] 3-2. Antibody titers before and after labeling were detected by indirect ELISA. JE virus was gradient coated (coating concentrations of 1000 ng / well, 300 ng / well, 100 ng / well, 30 ng / well, and 10 ng / well). The pre-labeled antibody was used as the primary antibody, and goat anti-mouse secondary antibody was used. HRP-labeled antibody titers were also detected by direct ELISA. The results of purified antibody labeling are shown in Table 6 below, and the results of HRP-labeled antibody testing are shown in Table 7 below.

[0091] Table 6 Purified antibody labeling results

[0092]

[0093] Table 7 HRP-labeled antibody detection results

[0094]

[0095] Note: NaN indicates an OD value greater than 4.0; NC is the negative control 5% milk-PBS, and the positive judgment standard is 2.1 times the NC value.

[0096] Combined with the HRP-labeled antibody detection results in Table 7, the sensitivity of the labeled antibodies 3E12, 8A7, and 13C2 was less than 3 times that of the unlabeled antibody, and they were judged to be qualified. The sensitivity of the labeled antibody 11A1 was more than 3 times different from that of the unlabeled antibody, indicating poor labeling effect. Therefore, the labeled antibodies 3E12, 8A7, and 13C2 were preferred as detection antibodies for further development of quantitative detection kits.

[0097] 4. Antibody Pairing

[0098] Based on the results of monoclonal antibody development, we conducted pairing tests with different antibodies. Following the experimental requirements of the double antibody sandwich assay, we used an unlabeled antibody as the coating antibody and paired it with another HRP-labeled antibody. The test results are shown in Tables 8-11.

[0099] Table 8 Pairing Test 1

[0100]

[0101] Table 9 Pairing Test 2

[0102]

[0103] Table 10 Pairing Test 3

[0104]

[0105]

[0106] Table 11 Pairing Test 4

[0107]

[0108] Combined with the pairing test results in Tables 8 to 11 above, the above 28 pairs of pairing curves all have gradients, and therefore the 28 pairs of monoclonal antibodies are successfully paired.

[0109] Among them, the monoclonal antibody pair consisting of the Japanese encephalitis monoclonal antibody 12B9 and the enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP in this example has a better curve gradient than other monoclonal antibody pairs and is more capable of capturing Japanese encephalitis virus antigens. Therefore, this pair is further preferred in this example. The above monoclonal antibody pair was commissioned to a third-party institution for gene sequencing, and the Japanese encephalitis monoclonal antibody 12B9 includes a heavy chain variable region with a base sequence as shown in SEQ ID NO: 1 and a light chain variable region with a base sequence as shown in SEQ ID NO: 2. The enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP includes a heavy chain variable region with a base sequence as shown in SEQ ID NO: 3 and a light chain variable region with a base sequence as shown in SEQ ID NO: 4.

[0110] The amino acid sequence of the heavy chain variable region of the Japanese encephalitis monoclonal antibody 12B9 was further obtained as shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the Japanese encephalitis monoclonal antibody 12B9 was shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP was shown in SEQ ID NO: 7, and the amino acid sequence of the light chain variable region of the enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP was shown in SEQ ID NO: 8.

[0111] It should be noted that the above-mentioned method for preparing monoclonal antibody pairs is a preferred method in this application. In addition, the target monoclonal antibody pair can be obtained by gene editing and other means based on the above-mentioned known antibody gene fragments. However, the antibodies obtained by gene editing have certain mutability and it is difficult to ensure the stability of the antibodies. Therefore, this application prefers to obtain them by cell line screening. However, the above-mentioned method for preparing monoclonal antibody pairs is not the only preferred option, and the reagent process parameters and raw materials can be appropriately replaced or adjusted as needed.

[0112] This application uses the monoclonal antibody 12B9 and the detection antibody 13C2-HRP as examples to further explain in detail the method for quantitatively detecting the content of Japanese encephalitis virus antigen.

[0113] 2. Quantitative detection of Japanese encephalitis virus antigen content (specific experimental sample testing)

[0114] A method for quantitatively detecting the content of Japanese encephalitis virus antigen comprises the following steps:

[0115] 1. Prepare a coating solution (5 μg / mL) of Japanese encephalitis monoclonal antibody (monoclonal antibody 12B9), add 50 μL of coating solution to each well, seal with sealing film, and place at 4°C overnight. Remove the plate and empty it. Add 300 μL / well of blocking solution (1% BSA bovine serum albumin in PBS solution), seal with sealing film, and place in a humidified chamber at 37°C for 1 hour. Wash four times with a plate washer, using 300 μL / well of washing solution. Empty the plate, seal with sealing film, and store at 2-8°C.

[0116] 2. Take the pre-coated ELISA plate out of the refrigerator and equilibrate it at room temperature for 30 minutes.

[0117] 3. Dilute the reference sample to 6 concentrations by 2-fold method, and dilute the sample to the appropriate concentration. Set up two blank control wells and two negative control wells for each experiment.

[0118] 4. Add 100 μL / well of sample, seal the plate with sealing film, incubate at 37°C for 60 minutes, and wash the plate three times with 300 μL / well;

[0119] 5. Add 100 μL / well of enzyme conjugate working solution (detection antibody 13C2-HRP), seal the ELISA plate with sealing film after adding the sample, and incubate it in a 37°C wet box for 60 minutes; wash the plate three times with 300 μL / well of washing solution.

[0120] 6. Add substrate solution A, 50 μL / well, and substrate solution B, 50 μL / well, and develop color at 25°C in the dark for 10 minutes.

[0121] 7. Add 50 μL / well of stop solution (1 mol / L sulfuric acid solution), select the main wavelength of 450 nm and the reference wavelength of 630 nm on the microplate reader, and measure the absorbance (OD value) of each well.

[0122] 3. Specificity test

[0123] Human serum albumin for production (negative control), combined vaccine diluent (2% human serum albumin PBS solution), rabies vaccine produced by our company (production batch number 202101) and Japanese encephalitis virus vaccine (production batch numbers B20210101X and 202008B11) were selected, and the test was carried out according to the above-mentioned "method for quantitative detection of Japanese encephalitis virus antigen content". The test results are shown in Table 12 below, which shows that only Japanese encephalitis samples can be detected, and combined vaccine diluent, human serum albumin, and rabies vaccine samples were not detected, indicating that this method is suitable for the detection of Japanese encephalitis antigens and will not produce non-specific binding with other matrices or other types of antigens.

[0124] Table 12 Specificity test results

[0125]

[0126]

[0127] 4. Result calculation

[0128] The Japanese encephalitis potency standard was selected as the reference product, and the Japanese encephalitis vaccines B20210101X and 202008B11 were selected as samples. The experiment was carried out according to the method in the above-mentioned "Quantitative detection of Japanese encephalitis virus antigen content".

[0129] The reference material is used with OD value as Y and concentration as X, and a four-parameter fitting method is used to perform linear fitting to obtain a standard curve. Substitute the sample OD value into the fitting curve calculation result, and then multiply it by the sample dilution factor to obtain the sample content relative to the reference material. Figure 1 , parameters are shown in Table 13 below, and curve formula (regression equation) is shown in Table 14 below.

[0130] Concentration assignment / ng / well 1 2 blank - 0 0 ST1 2 2.988 3.047 ST2 1 2.232 2.172 ST3 0.5 1.4 1.264 ST4 0.25 0.666 0.672 ST5 0.125 0.323 0.302 ST6 0.0625 0.198 0.183 Negative - 0.005 0.006

[0131] Curve Name Curve formula A B C D <![CDATA[R 2 ]]> Standard curve <![CDATA[Y=(A-D) / (1+(X / C) ^B )+D]]> 0.0805 1.39 0.846 3.9 0.998

[0132] This method was used to test the finished product of inactivated Japanese encephalitis vaccine, and the experimental data results were shown in Table 15 below by substituting the above standard curve formula.

[0133] Table 15. Experimental data results of Japanese encephalitis vaccine

[0134]

[0135]

[0136] The experimental data in Table 15 show that the JE samples can be quantitatively detected by this method, and the results of each concentration are stable and consistent. 2 It can reach 0.998 and above, and the precision CV is less than 15% (n=10).

[0137] Example 2

[0138] In this example, based on the method of Example 1, only the monoclonal cell line obtained from the second cell fusion was used for culture when preparing the monoclonal antibody pair, and a preferred monoclonal antibody pair was screened.

[0139] Example 3

[0140] In this example, based on the method of Example 1, immunogen A was used for primary immunization and booster immunization when preparing monoclonal antibody pairs, and a preferred monoclonal antibody pair was screened out.

[0141] Example 4

[0142] In this example, based on the method of Example 1, when preparing a monoclonal antibody pair, immunogen A is used for primary immunization, and immunogen B is used for booster immunization to screen out a preferred monoclonal antibody pair.

[0143] Example 5

[0144] In this example, based on the method of Example 1, adjuvant IFA was used to replace Classic adjuvants, screen out a pair of preferred monoclonal antibodies.

[0145] Comparative Example 1

[0146] In this comparative example, based on the method of Example 1, the detection antibody 13C2-HRP was replaced with an indirect ELISA sandwich method (Japanese encephalitis monoclonal plus goat anti-mouse IgG (H+L)). The goat anti-mouse IgG (H+L) was purchased from Jackson ImmunoResearch with the catalog number 115-035-003.

[0147] The monoclonal antibody pairs obtained in Examples 2-5 and Comparative Example 1 were applied to the "Method for Quantitative Detection of Japanese Encephalitis Virus Antigen Content" in Example 1 to form a corresponding test method. A Japanese encephalitis potency standard was used as a reference substance, and Japanese encephalitis vaccines B20210101X and 202008B11 were used as samples for testing. A four-parameter linear fit was performed using the reference substance, with the OD value as Y and the concentration as X, to obtain the standard curve formula. The corresponding linear relationship and precision results are shown in Table 16 below.

[0148] Curve Name <![CDATA[R 2 ]]> CV((n=10)) / % Example 1 0.998 15 Example 2 0.991 17 Example 3 0.985 18 Example 4 0.990 17 Example 5 0.988 17 Comparative Example 1 0.978 25

[0149] As shown in Table 16, the detection method of the present application has a higher linear relationship and feasibility than the comparative example, and can be better applied to the determination of antigen content in Japanese encephalitis vaccine. In the present application, the linear relationship and precision of Example 1 are more excellent, so it is further preferred.

[0150] In summary, the method of quantitatively detecting the content of Japanese encephalitis virus antigen in the present application utilizes the ELISA double antibody sandwich structure of "monoclonal antibody-antigen-enzyme-labeled monoclonal antibody". The concentration of the sample can be quantitatively calculated based on the actual production situation of the manufacturer and the concentration curve of the reference product. It can more effectively predict the antigen content of the finished vaccine and provide a more reliable quantitative numerical basis for the production of finished vaccine products. It has good application prospects in the production of Japanese encephalitis vaccines.

[0151] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law. Sequence Listing <110> Liaoning Chengda Biological Co., Ltd. <120> Method for quantitatively detecting Japanese encephalitis virus antigen content, monoclonal antibody prepared for establishing the method, and application thereof <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 358 <212> DNA / RNA <213> Artificial Sequence <400> 1 gaggtgcagc tggagaggagtc aggacctagc ctcgtgaaac cttctcagac tctgtccctc 60 acctgttctg tcactggcga ctccatcacc agtggttat ggatctggat ccggaaattc 120 ccaggaaata aacttgagta catggggtac ataagctaca gtggtaccac ttactacaat 180 ccatctctca aaagtcgaat ctacatcact cgagacacat ccaggaacca atattacctg 240 cagttgaatt ctgtgactac tgaggacaca gccacatatt actgtgcaag aggattacga 300 cgggattact atgctatgga ctactggggt caaggacct cagtcaccgt ctcctcag 358 <210> 2 <211> 322 <212> DNA / RNA <213> Artificial Sequence <400> 2 gacatccagc tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gaacaagtga aaatatttac aattatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg gaagttatta ctgtcaacat cattatggta ttcctccgac gttcggtgga 300 ggctccaagg tggatctcaa ac 322 <210> 3 <211> 349 <212> DNA / RNA <213> Artificial Sequence <400> 3 caggtgcagc tgaagcagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg 60 tcctgcacag cttctggctt caacattaaa gacacctata tgcactgggt gaagcagagg 120 cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggcaa tactaaatat 18o gacccgaagt tccagggcaa ggccactata acagcagaca cattttccaa tacagcctat 240 It should be noted that there may be an error in the original text where "18o" in line 29 should probably be "180". This has been left as is in the translation for the purpose of following the instruction to preserve the original text exactly.ttgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc cctacaacga 300 catgctttgg actactgggg tcaaggaacc tcagtcaccg tctcctcag 349 <210> 4 <211> 322 <212> DNA / RNA <213> Artificial Sequence <400> 4 gacattgtga tgacccagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct 60 ctttcctgca gggccagcca gagtattagc gaccacttac actggtatca acaaaaatca 120 catgagtctc caaggcttct catcaaatat gtttcccatt ccatctctgg gatcccctcc 180 aggttcagtg gcagtggatc agggtcagat ttcactctca gtatcaacag tgtgcaacct 240 gaagatgttg gcgtgtatta ttgtcaacat ggtcacagct ttccgctcac gttcggtgct 300 gggaccaagc tggagctgaa ac 322 <210> 57] <211> 119 <212> PRT <213> Artificial Sequence <400> 5 Glu Val Gln Leu Glu Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Ile Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Ile Tyr Ile Thr Arg Asp Thr Ser Arg Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Gly Leu Arg Arg Asp Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <400> 六 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Ile Pro Pro 85 90 95 Thr Phe Gly Gly Gly Ser Lys Val Asp Leu Lys 100 105 <210> 7 <211> 116 <212> PRT <213> Artificial Sequence <400> 7 Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Phe Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Leu Gln Arg His Ala Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val 100 105 110 Thr Val Ser Ser 115 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp His 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Val Ser His Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly​​​​​​​Glu Asp Val Gly Val Tyr Tyr Cys Gln His Gly His Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105

Claims

1. A method for quantitatively detecting the content of Japanese encephalitis virus antigen, characterized in that: The following steps are involved: A Japanese encephalitis monoclonal antibody is coated on an ELISA plate. After coating, a gradient dilution of a Japanese encephalitis positive reference substance, a negative control, and a diluted sample to be tested are added to each well. After incubation, the ELISA plate is washed, and the enzyme-labeled Japanese encephalitis monoclonal antibody is added. After incubation again, the plate is washed, and a substrate color development solution is added for color development. The OD value of the ELISA reader is stopped and the OD value is read. A regression equation is plotted using the OD value of the Japanese encephalitis positive reference substance. The content of Japanese encephalitis virus antigen in the vaccine is quantitatively determined by regression calculation. The monoclonal antibody pair prepared for establishing a method for quantitatively detecting the content of Japanese encephalitis virus antigen includes Japanese encephalitis monoclonal antibody 12B9 and enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP. The Japanese encephalitis monoclonal antibody 12B9 includes a heavy chain variable region with a base sequence as shown in SEQ ID NO: 1 and a light chain variable region with a base sequence as shown in SEQ ID NO:

2. The enzyme-labeled Japanese encephalitis monoclonal antibody 13C2-HRP includes a heavy chain variable region with a base sequence as shown in SEQ ID NO: 3 and a light chain variable region with a base sequence as shown in SEQ ID NO:

4.

2. The method for quantitatively detecting Japanese encephalitis virus antigen content according to claim 1, wherein The coated Japanese encephalitis monoclonal antibody and the enzyme-labeled Japanese encephalitis monoclonal antibody form a monoclonal antibody pair. The preparation method of the monoclonal antibody pair comprises the following steps: The purified Japanese encephalitis virus solution is mixed with an adjuvant to prepare an immunogen; Immunizing all mice with the immunogen, taking spleen cells from one group of mice for a first cell fusion, and continuously boosting the remaining mice with the immunogen, and taking spleen cells from the remaining mice for a second cell fusion; The monoclonal cells obtained from the first cell fusion and the second cell fusion are cultured, and after repeated screening and specificity testing, positive clone cells that can secrete monoclonal antibodies that recognize Japanese encephalitis virus and have neutralizing activity are selected; The selected positive clone cells are injected into the peritoneal cavity of mice used for antibody purification. After the monoclonal antibody is expressed and secreted, the ascites of the mice is extracted and the supernatant of the ascites is collected after centrifugation for purification to obtain the Japanese encephalitis monoclonal antibody. The purified antibodies with high antibody yield and high sensitivity in binding to Japanese encephalitis virus in the ascites supernatant screened by detection were selected for enzyme labeling to obtain enzyme-labeled Japanese encephalitis monoclonal antibody; The JE monoclonal antibody is selected and coated on an ELISA plate. After blocking, a gradient dilution of JE antigen is added. After incubation, the enzyme-labeled JE monoclonal antibody is added. After further incubation, a display substrate is added and the OD value is measured. The coated JE monoclonal antibody and the enzyme-labeled JE monoclonal antibody that are paired with each other are screened according to the OD value results, which is the monoclonal antibody pairing.

3. The method for quantitatively detecting Japanese encephalitis virus antigen content according to claim 2, wherein: The adjuvant mixed with the purified Japanese encephalitis virus solution is a mixture of adjuvant CFA and TiterMax® classic adjuvant.

4. The method for quantitatively detecting Japanese encephalitis virus antigen content according to claim 2, wherein The preparation steps of the immunogen are as follows: Mix equal volumes of purified Japanese encephalitis virus liquid and adjuvant CFA, and sonicate the mixture until it becomes an emulsion to obtain immunogen A. Mix equal volumes of purified Japanese encephalitis virus liquid and TiterMax® classic adjuvant to obtain immunogen B. Mix equal volumes of A and B to obtain the immunogen.

5. The method for quantitatively detecting Japanese encephalitis virus antigen content according to claim 2, wherein: The mice used for antibody purification were injected with adjuvant in the abdomen before the monoclonal cells were injected.

6. The method for quantitatively detecting Japanese encephalitis virus antigen content according to claim 5, wherein: The adjuvant pre-injected into the mice for antibody purification was IFA.

7. Use of the method for quantitatively detecting the content of Japanese encephalitis virus antigen according to any one of claims 1 to 6 in the preparation of Japanese encephalitis vaccine.

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