Hybridoma cell line secreting monoclonal antibody against rabies virus G protein, monoclonal antibody and application thereof

Abstract

The present invention relates to the field of virus detection, and in particular to hybridoma cell lines secreting monoclonal antibodies against rabies virus G protein, monoclonal antibodies and their applications. The present invention uses antibodies secreted by two hybridoma cell lines as coating and detection antibodies to prepare a double antibody sandwich ELISA kit, which can be used for the efficacy test of veterinary rabies vaccines, and has the advantages of broad spectrum and accurate results. It has been verified that the kit can quickly determine the efficacy of veterinary rabies vaccines within 2.5 hours, and both specificity and repeatability are good, and it can be stored for at least 6 months at 4°C. It can be seen that the present invention has developed a veterinary rabies vaccine efficacy detection kit, which is easy to operate, short in time, and has stable results. It can supplement or even replace the NIH method, which is beneficial to quality control in the production process of veterinary vaccines and shortens the efficacy test cycle of finished vaccines.

Description

Technical Field

[0001] The present invention relates to the field of virus detection, in particular to a hybridoma cell line secreting a monoclonal antibody against rabies virus G protein, a monoclonal antibody and applications thereof. Background Art

[0002] Rabies is a zoonosis caused by the rabies virus (RABV). Once clinical symptoms appear, the mortality rate is almost 100%. At present, vaccine immunization is still the most effective means to prevent rabies. The quality of the vaccine directly determines the preventive and therapeutic effect of rabies. The most important thing in the quality evaluation of rabies vaccines is the efficacy evaluation. The gold standard for evaluating the efficacy of vaccines is the NIH method. Although this method replaces a large number of clinical trials by using mouse challenge protection tests, and the efficacy tests are standardized and standardized, so that the efficacy of vaccines from different manufacturers and batches can be compared. However, this experiment is very time-consuming, labor-intensive, and the results are unstable, and it can only be used to evaluate finished vaccines, and it is impossible to measure intermediates in the vaccine production process. In addition, NIH needs to consume a large number of experimental animals and the experimental animals must be tortured by rabies before death, which seriously violates experimental ethics.

[0003] Compared with veterinary rabies vaccines, human rabies vaccines have simpler ingredients and do not contain adjuvants. Therefore, there are ELISA kits on the market for determining the efficacy of human rabies vaccines. However, such kits on the market have problems such as not being able to cover veterinary rabies vaccine strains, low compliance rates between test results and NIH tests, and no quantitative standards. Therefore, it is urgent to develop a broad-spectrum, accurate method for veterinary rabies vaccine efficacy to supplement or replace the NIH test. Summary of the invention

[0004] The purpose of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies against rabies virus G protein, monoclonal antibodies and their applications to solve the problems of the above-mentioned prior art. The present invention uses antibodies secreted by hybridoma cell lines as coating and detection antibodies, which can be used for the efficacy detection of veterinary rabies vaccines, and has the advantages of broad spectrum and accurate results.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a hybridoma cell line combination secreting monoclonal antibodies against rabies virus G protein, wherein the hybridoma cell line combination comprises a hybridoma cell line 8A5 with a deposit number of CCTCC NO: C2023382 and a hybridoma cell line 6G10 with a deposit number of CCTCC NO: C2023383.

[0007] The present invention provides a monoclonal antibody combination against rabies virus G protein, wherein the monoclonal antibody combination comprises the monoclonal antibody secreted by the hybridoma cell line 8A5 and the monoclonal antibody secreted by the hybridoma cell line 6G10.

[0008] The present invention provides the use of the above-mentioned monoclonal antibody combination in the preparation of veterinary rabies vaccines and / or veterinary rabies vaccine efficacy detection products.

[0009] Preferably, the veterinary rabies vaccine includes a veterinary rabies vaccine prepared from PV strain, r3G strain, SAD strain, Flury strain, CVS-11 strain or CTN-1 strain.

[0010] Preferably, the veterinary rabies vaccine intermediate includes a virus supernatant, a concentrated virus solution or a purified virus solution prepared from a PV strain, an r3G strain, a SAD strain, a Flury strain, a CVS-11 strain or a CTN-1 strain.

[0011] Preferably, the veterinary rabies vaccine efficacy detection product includes a veterinary rabies vaccine efficacy detection kit.

[0012] Preferably, the veterinary rabies vaccine potency detection kit is a sandwich ELISA detection kit for veterinary rabies vaccine potency detection.

[0013] The present invention provides a sandwich ELISA detection kit for veterinary rabies vaccine efficacy detection. The coating antigen of the sandwich ELISA detection kit is the monoclonal antibody secreted by the above-mentioned 8A5, and the detection antigen is the monoclonal antibody secreted by the above-mentioned 6G10.

[0014] Preferably, the concentration of the coating source is 2.69 μg / mL; the dilution multiple of the detection antibody is 1:300; and the detection antibody is labeled with horseradish peroxidase.

[0015] The present invention provides a double-antibody sandwich ELISA detection method for detecting the efficacy of rabies vaccines for animals, and the detection is performed using the above-mentioned sandwich ELISA detection kit.

[0016] The present invention discloses the following technical effects:

[0017] The present invention has established a double antibody sandwich ELISA kit. After antibody pairing test, the monoclonal antibody 8A5 with neutralizing activity was screened as the capture antibody, HRP-6G10 was used as the detection antibody, and the veterinary rabies vaccine quantitative reference (SAD strain, 19.7 IU / mL) was prepared and calibrated. The R value of the standard curve of the kit for the national standard for rabies vaccine potency test (11.4 IU / mL), the national standard for rabies vaccine (5.5 IU / mL) and the self-made quantitative reference was 0.147 ... 2 All of them are greater than 0.99, and the minimum detection amounts are 0.0006958IU / mL, 0.0006714IU / mL and 0.003078IU / mL, respectively. The kit can detect a variety of veterinary rabies vaccine strains, including PV strain, r3G strain, SAD strain, Flury strain, CTN-1 strain and CVS-11 strain. In addition, the kit can detect finished vaccines and intermediates in the vaccine production process, including viral supernatant, concentrated viral solution and purified viral solution. At the same time, in a specific embodiment of the present invention, the potency of 18 batches of veterinary rabies vaccines (SAD strain) was determined using the kit and the NIH method, respectively, with a correlation coefficient r of 0.9314, and 10 batches of vaccines were extracted and measured using commercially available similar kits. The correlation coefficient r between the commercially available kit and the NIH method was 0.9345, and the correlation coefficient r between the homemade kit and the NIH test was 0.9577, indicating that the homemade kit of this study has good consistency with the NIH method and is superior to the commercially available kit. It has been verified that this kit can quickly determine the potency of veterinary rabies vaccines within 2.5 hours, and both specificity and repeatability are good, and it can be stored for at least 6 months at 4°C. It can be seen that the present invention has developed a veterinary rabies vaccine potency detection kit, which is easy to operate, short in time, and has stable results. It can supplement or even replace the NIH method, which is beneficial to quality control in the production process of veterinary vaccines and shortens the potency inspection cycle of finished vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The purity of 8 monoclonal antibodies;

[0020] Figure 2 A bar graph showing the best antibody combinations for different strains;

[0021] Figure 3are hybridoma cell morphologies, where A is the morphology of 8A5 hybridoma cells (100×), and B is the morphology of 6G10 hybridoma cells (100×);

[0022] Figure 4 is the titer of 8A5 monoclonal antibody cell supernatant;

[0023] Figure 5 is the ascites titer of 8A5 mAb;

[0024] Figure 6 is the titer of 6G10 monoclonal antibody cell supernatant;

[0025] Figure 7 is the ascites titer of 6G10 mAb;

[0026] Figure 8 for the purity of monoclonal antibodies 8A5 and 6G10;

[0027] Fig. 9 To determine the best blocking solution;

[0028] Fig.10 To determine the sample incubation time (samples were diluted 1:10);

[0029] Fig.11 To determine the sample incubation time (samples were diluted 1:100);

[0030] Fig.12 To determine the incubation time for detection antibodies (samples were diluted 1:10);

[0031] Fig.13 To determine the incubation time for detection antibodies (samples were diluted 1:100);

[0032] Fig.14 To determine the optimal color development conditions (sample diluted 1:10);

[0033] Fig.15 To determine the optimal color development conditions (sample diluted 1:100);

[0034] Fig.16 The standard curve of the homemade rabies vaccine quantitative reference before and after freeze-drying is 875 μL reconstituted;

[0035] Fig.17 The standard curve of 675 μL reconstitution before and after freeze-drying of the homemade rabies vaccine quantitative reference product;

[0036] Fig.18 This is the standard curve of the national standard for rabies vaccine potency testing (China Food and Drug Inspection Institute, 11.4IU / mL), where China Food and Drug Inspection Institute is the China Food and Drug Inspection Institute;

[0037] Fig.19 This is the standard curve of the national standard product of rabies vaccine (China Veterinary Drug Administration, 5.5IU / mL), China Veterinary Drug Administration is the China Veterinary Drug Administration;

[0038] Fig. 20 The standard curve of the homemade rabies vaccine quantitative reference (SAD strain, 19.7 IU / mL);

[0039] Fig.21 These are the test results of various vaccine strains of rabies virus;

[0040] Fig. 22 The test results of samples from various stages of rabies vaccine production;

[0041] Fig.23 The specificity verification result of the kit

[0042] Fig.24 This is the specific test result of the kit batch 20220801;

[0043] Fig.25 This is the specific test result of the kit batch 20220802;

[0044] Fig.26 This is the specific test result of the 20220803 batch of test kits;

[0045] Fig. 27 The specific test results (stability) of the 20220801 batch of test kits;

[0046] Fig.28 The specific test results (stability) of the 20220802 batch of test kits;

[0047] Fig.29 The specific test results (stability) of the 20220803 batch of test kits;

[0048] Fig.30 for the correlation between the homemade kit and the NIH test (18 samples);

[0049] Fig.31 This is the consistency between the homemade test kit and the NIH test results (18 samples); from top to bottom, the first purple line is the upper limit of the 0.95 consistency limit, the second red line is the horizontal axis, the third blue dotted line is the offset, and the fourth light purple line is the lower limit of the 0.95 consistency limit; the part between the first purple line and the fourth light purple line represents the 0.95 consistency interval;

[0050] Fig.32 is the correlation between the commercially available kit and the NIH test (10 samples);

[0051] Fig.33 for the correlation between the homemade kit and the NIH test (10 samples);

[0052] Fig.34 is the correlation between the homemade kit and the commercially available kit (10 samples);

[0053] The Cut-off in the figure is the boundary line.

[0054] Biological deposit information

[0055] Hybridoma cell line 8A5 was deposited in China Center for Type Culture Collection on January 10, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: C2023382;

[0056] The hybridoma cell line 6G10 was deposited with the China Center for Type Culture Collection on January 10, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: C2023383. DETAILED DESCRIPTION

[0057] Prepare:

[0058] 1. Main instruments and equipment

[0059] Ordinary normal temperature centrifuge and low temperature refrigerated centrifuge were purchased from Eppendorf (Germany); constant voltage and constant flow electrophoresis instrument was purchased from Beijing Liuyi Instrument Factory, China; SDS-PAGE gel preparation related instruments were purchased from Wuhan Bosde Bioengineering Co., Ltd.; Syngene protein gel imager (USA) and biological safety cabinet (CLASSII A2) was purchased from LABCONCO, USA; CO2 cell culture incubator was purchased from Sanyo, Japan; fluorescent inverted microscope was purchased from OLYMPUS, Japan; ultra-clean water purifier was purchased from Milipore, USA; water bath was purchased from Shanghai Jinghong Company; 90-2 timed temperature control magnetic stirrer was purchased from Jintan Dadi Automation Instrument Factory; 98-2 magnetic stirrer was purchased from Jintan Yichen Instrument Manufacturing Co., Ltd.; electric constant temperature blast drying oven was purchased from Shanghai Yuejin Medical Instrument Co., Ltd.; MX-S adjustable mixer was purchased from Shanghai Youyi Instrument Co., Ltd.; ultra-high speed refrigerated centrifuge was purchased from Beckman Coulter, USA; fast oscillator was purchased from Haimen Qilin Bell Instrument Manufacturing Co., Ltd.; multi-function microplate reader was purchased from BMG LABTECH; protein purification instrument AKTA Pure25 was purchased from GE.

[0060] 2. Conventional biochemical reagents

[0061] Freund's incomplete adjuvant, HAT (50×), HT (50×), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA; Beyo ECL plusA, B solution, penicillin-streptomycin double antibody (100×), and BCA protein quantification kit were purchased from Beijing Biotech Biotechnology Co., Ltd.; anhydrous ethanol (C2H5OH), acetone (CH3COCH3), methanol (CH3OH), isopropanol (C3H8O), sodium chloride (NaCl), potassium chloride (KCl), disodium hydrogen phosphate (Na2HPO4), potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium periodate (NaIO4), sodium borohydride (NaBH4), ethylene glycol, disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) and sodium dihydrogen phosphate dodecahydrate (NaH2PO4·12H2O), mannitol, sucrose, trehalose, and lysine were purchased from Sinopharm Chemical Reagent Co., Ltd.; DMEM medium was purchased from GIBCO; fetal bovine serum (Fetal Bovine Serum, FBS) was purchased from Zhejiang Tianhang Biotechnology Co., Ltd.; HRP-sheep anti-mouse IgG antibody was purchased from Wuhan Boster Company; Alexa Fluor 488-labeled anti-mouse IgG fluorescent secondary antibody was purchased from Invitrogen; skim milk powder was purchased from Sigma; aluminum adjuvant, detection antibody diluent, blocking solution, substrate solution A, substrate solution B, stop solution and detachable polystyrene ELISA plate were purchased from Wuhan Keqian Biological Company; filter paper and TMB color development solution were purchased from Feiyoute Biological Company; other reagents were domestic analytical grade.

[0062] 3. Preparation of cell culture medium

[0063] 1× phosphate buffered saline (PBS): weigh 8.0 g of NaCl, 0.2 g of KCl, 1.42 g of Na2HPO4, and 0.27 g of KH2PO4, respectively, fully dissolve them in 800 mL of ultrapure water, adjust the pH to 7.4 with 5 mol / L NaOH and concentrated HCl, make up to 1 L, and store at room temperature after sterilization.

[0064] Hybridoma cell culture medium: RPMI-1640 containing 20% ​​FBS and 1% penicillin-streptomycin.

[0065] HAT cell culture medium: RPMI-1640 containing 2% HAT, 10% FBS and 1% penicillin-streptomycin.

[0066] HT cell culture medium: RPMI-1640 containing 2% HT, 10% FBS and 1% penicillin-streptomycin.

[0067] BSR and BHK-21 cell culture medium: DMEM containing 10% FBS and 1% penicillin-streptomycin.

[0068] 0.25% pancreatic enzyme digestion solution: weigh 2.5 g of pancreatic enzyme and dissolve it in 1 L of 1×PBS (pH=7.4), filter and sterilize it with a sterile 0.22 μm filter membrane, and store it at -20°C after aliquoting for later use.

[0069] Cell freezing solution: a mixture containing 80% basal culture medium (RPMI-1640 or DMEM), 10% DMSO and 10% FBS, ready for use.

[0070] 4. Preparation of Ascites Purification Reagents

[0071] 2× balance solution: Weigh 4.846 g of Tris-base and 292.5 g of NaCl and dissolve them in 800 mL of distilled water. After sufficient stirring, adjust the pH to 8.0 with NaOH and concentrated HCl, make up to 1 L with distilled water, filter and sterilize with a sterile 0.22 μm filter membrane, and store at room temperature.

[0072] 1× balance solution: weigh 2.429 g of Tris-base and 146.25 g of NaCl and dissolve them in 800 mL of distilled water. After sufficient stirring, adjust the pH to 8.0 with NaOH and concentrated HCl, make up to 1 L with distilled water, filter and sterilize with a sterile 0.22 μm filter membrane, and store at room temperature.

[0073] Washing solution: weigh 146.25 g of NaCl and dissolve it in 800 mL of 2×PBS. After thorough stirring, adjust the pH to 8.0 with NaOH and concentrated HCl, make up to 1 L with 2×PBS, filter and sterilize with a sterile 0.22 μm filter membrane, and store at room temperature.

[0074] Eluent (0.1 mol / L Glycine-HCl): Weigh 7.507 g of glycine and dissolve it in 800 mL of distilled water. After sufficient stirring to dissolve, adjust the pH to 2.7 with NaOH and concentrated HCl. Make the volume to 1 L with distilled water, filter and sterilize with a sterile 0.22 μm filter membrane, and store at room temperature.

[0075] 5. Preparation of Western-blot and SDS-PAGE reagents

[0076] 10% Ammonium Persulfate (AP): Weigh 0.1 g of AP powder and add it to 1 mL of ddH2O to dissolve. Prepare and use immediately.

[0077] 10% SDS polyacrylamide (separation gel): H2O 3.8 mL, 30% acrylamide solution 3.4 mL, 1.5 mol / L Tris (pH=8.8) 2.6 mL, 10% SDS 0.05 mL, 10% ammonium persulfate 0.05 mL, TEMED 0.005 mL.

[0078] 5% SDS polyacrylamide (concentrating gel): H2O 2.8 mL, 30% acrylamide solution 0.67 mL, 1.0 mol / L Tris (pH=6.8) 0.53 mL, 10% SDS 0.04 mL, 10% ammonium persulfate 0.04 mL, TEMED 0.004 mL.

[0079] 5×SDS gel loading buffer: add 0.25 mL 1 M Tris-HCl (pH=6.8), 0.5 g SDS, 2.5 mL glycerol and 25 mg bromophenol blue dissolved in 5 mL ddH2O.

[0080] 5×Tris-Glycine electrophoresis buffer: weigh 7.55 g Tris-base, 47 g Glycine, and 25 mL 10% SDS, dissolve them in ddH2O and make up to 500 mL.

[0081] Transfer buffer: Weigh 2.9 g Glycine, 5.8 g Tris-base, and 0.37 g SDS, add to 800 mL ddH2O to dissolve, then add 200 mL methanol to mix thoroughly before use.

[0082] TBST buffer: Weigh 8.8g NaCl and 20mL 1M Tris-HCl (pH=8.0) into a beaker, then add 800mL ddH2O to the beaker and stir thoroughly to dissolve. Then add 0.5mL Tween-20 and mix thoroughly, finally dilute to 1000mL with ddH2O and store at 4℃.

[0083] Blocking solution: Weigh 5 g of skim milk powder, dissolve it in TBST and make up to 100 mL. Prepare and use immediately.

[0084] Coomassie Brilliant Blue staining solution: Measure 500 mL of methanol, 100 mL of glacial acetic acid, weigh 2.5 g of Coomassie Brilliant Blue R-250, add ddH2O to make up to 1000 mL, filter with filter paper, and store at room temperature.

[0085] Decolorizing solution: Measure 300 mL of methanol and 100 mL of glacial acetic acid, add ddH2O to make up to 1000 mL.

[0086] 6. HRP labeling related reagents

[0087] 0.06mol / L NaIO4 solution: weigh 0.1283g NaIO4 and dissolve it in 10mL water for injection. Prepare and use immediately.

[0088] 0.053 mol / L ethylene glycol solution: Measure 13.41 μL of ethylene glycol and add it to 1.5 mL of water for injection. Prepare and use immediately.

[0089] 5g / L NaBH4 solution: weigh 5 mg NaBH4 and dissolve it in 1 mL water for injection. Prepare and use immediately.

[0090] Saturated ammonium sulfate solution: Weigh 90 g (NH4)2SO4, dissolve it in 100 mL water for injection in a 80°C water bath, filter while hot, cool at room temperature overnight to allow crystals to precipitate, and adjust the pH to 7.4 with 35% aqueous ammonia.

[0091] PB solution: weigh 7.16 g of Na2HPO4·12H2O and 3.12 g of NaH2PO4·12H2O, add ddH2O to dissolve and make up to 2 L, and store at room temperature.

[0092] 7. ELISA related reagents

[0093] Coating solution (pH = 9.6): weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them in water for injection and make up to 1000 mL, adjust the pH to 9.6 with NaOH and concentrated HCl, and store at room temperature for later use.

[0094] Sample diluent: Weigh 8.0 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, and 0.27 g KH2PO4, respectively, fully dissolve them with 800 mL ultrapure water, adjust the pH to 7.4 with 5 mol / L NaOH and concentrated HCl, make up to 1 L, and store at room temperature after sterilization.

[0095] 20-fold concentrated washing solution: weigh 170.00 g of NaCl and 10.00 mL of Tween-20, dissolve in water for injection and make up to 1000 mL, store at room temperature for later use.

[0096] 8. Statistical methods

[0097] GraphPad Prism version 6.0 software (GraphPad Software, Inc., USA) was used to analyze and count the data.

[0098] Example 1

[0099] Preparation, purification and concentration detection of monoclonal antibodies

[0100] Eight hybridoma cell lines (clone numbers are: 1E11, 2B10, 7A3, 8A5, 6G10, 9A7, 9F7 and 9F10, screened by our laboratory through mice immunized with RABV whole virus protein, see patent CN110218704A for details) were cultured in 6-well plates, and each well was supplemented with 2.5 mL of culture medium. They were placed in a 37°C 5% CO2 incubator for expansion culture;

[0101] After the expansion culture was completed, 8-10 week old BALB / c female mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were intraperitoneally injected with Freund's incomplete adjuvant (0.5 mL / mouse) 7 days before inoculation of hybridoma cells. The well-growing hybridoma cells in Example 3 were collected and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the precipitate was resuspended in serum-free medium and the cell density was adjusted to (1-2)×10 6 Each mouse was intraperitoneally injected with (0.5-1)×10 6 cells. 7-10 days after the cells were inoculated, the abdomen of the mouse was obviously swollen, and the ascites was collected. The ascites was centrifuged at 1200rpm for 10 minutes, and the upper layer of fat was carefully aspirated. The yellowish ascites in the middle was taken out, packaged, and stored at -20℃ for later use; then the ascites was purified by Protein A+G affinity chromatography. The specific steps are as follows: 1) First, rinse the column with 10 column volumes of PBS, and then rinse with 10 column volumes of 1× equilibrium solution; 2) Aspirate 10mL of 1× equilibrium solution and 12mL of 2× equilibration solution, add the thawed ascites (8A5 ascites for 2 batches, 6G10 ascites for 1 batch) to the above equilibration solution, mix well and filter with a 0.45μm filter; 3) Slowly add the filtered liquid to the column for antibody purification, and repeat the loading 3 times; 4) After the loading is completed, rinse the column with 10 column volumes of 1× equilibration solution; 5) Rinse the column with 10 column volumes of washing solution, and then rinse the column with 10 column volumes of 1× equilibration solution; 6) Elute the protein with 60mL elution solution, and collect the antibody-containing solution in 2mL centrifuge tubes (each centrifuge tube has been pre-added with pre-cooled Tris-HCl 200μL / tube); then verify the purity of the purified and concentrated monoclonal antibody by SDS-PAGE. The results are as follows Figure 1 As shown, the heavy chains of the eight monoclonal antibodies are all located at 50 kDa, and the light chains are all located between 20-25 kDa, and there are no obvious mixed bands, indicating that these eight monoclonal antibodies are of high purity and can meet the requirements of subsequent pairing experiments.

[0102] The eluted monoclonal antibody is ultrafiltered and concentrated. The specific steps are as follows: precool the centrifuge to 4°C, set to 4500rpm, 60min, and the speed of increase and decrease are both 9. After the ultrafiltration tube is balanced, place it symmetrically in the centrifuge and start centrifugation. Observe it every once in a while to prevent all the liquid from being centrifuged. When there is only 1.5mL of liquid left in the ultrafiltration tube, stop centrifugation, use a protein concentration measuring instrument to detect whether there is monoclonal antibody in the inner tube, and then add precooled PBS (0.22μm filter) to 15mL in the ultrafiltration tube, and centrifuge again after balancing. A total of PBS replacement is required 3 times, and the last time to ensure that the liquid in the ultrafiltration tube is 1-1.5mL. Aspirate the liquid in the inner tube and use a 200μL pipette to absorb the liquid in the ultrafiltration tube, then repeatedly and gently rinse the ultrafiltration membrane (10-20 times), and then aspirate the liquid in the ultrafiltration tube into a 2mL EP tube. In addition, 200 μL of PBS can be added to repeatedly rinse the ultrafiltration membrane, and the rinse liquid can be sucked into a new EP tube, and then the IgG concentration of the above 8 monoclonal antibodies can be measured using a micro-spectrophotometer (Nanodrop), as shown in Table 1.

[0103] Table 1 Concentrations of 8 monoclonal antibodies

[0104] Monoclonal antibodies 1E11 2B10 7A3 8A5 6G10 9A7 9F7 9F10 IgG concentration (mg / mL) 2.318 3.731 3.218 2.380 1.410 1.967 0.893 1.709

[0105] 2. HRP labeled 8 monoclonal antibodies and their concentration detection

[0106] Eight strains of rabies virus G protein monoclonal antibodies (1E11, 2B10, 7A3, 8A5, 6G10, 9A7, 9F7 and 9F10) were coupled to HRP. The specific steps were as follows: 5 mg of horseradish peroxidase (HRP) was dissolved in 1 mL of water for injection, 0.5 mL of NaIO4 solution was added, and the reaction was carried out at 2-8°C for 30 minutes. The solution was grass green, and 0.5 mL of ethylene glycol was added. After mixing, the reaction was carried out at room temperature in the dark for 30 minutes. Then 5 mg of purified 6G10 monoclonal antibody was added and dialyzed in carbonate buffer at 2-8°C for 15 hours. Add 0.2 mL of freshly prepared NaBH4 solution to the solution, mix well and react at 2-8°C for 2 h, then add an equal volume of saturated ammonium sulfate solution, let stand at 2-8°C for 30 min, centrifuge at 7000 rpm for 10 min, discard the supernatant, resuspend in PB solution, dialyze in PB solution at 2-8°C for 15 h, and measure OD using a spectrophotometer. 403nm Value and OD 280nm Calculate the concentration of the enzyme marker using the following formula: IgG amount (mg / mL) = (OD 280nm -OD 403nm ×0.34)×0.62, the IgG concentrations of 8 enzyme-labeled antibodies are shown in Table 2.

[0107] Table 2 Concentrations of 8 enzyme-labeled antibodies

[0108]

[0109] 3. Screening of the best monoclonal antibody combination

[0110] Initial screening: The purified rabies virus G protein monoclonal antibodies 1E11, 6G10, 2B10, 7A3, 8A5, 9A7, 9F7, and 9F10 were diluted to 2.5 μg / mL with coating solution, and coated on ELISA plates at 100 μL / well at 4°C overnight. The enzyme-labeled antibodies HRP-1E11, HRP-6G10, HRP-2B10, HRP-7A3, HRP-8A5, HRP-9A7, HRP-9F7, and HRP-9F10 were diluted to the same concentration, and the coating antibodies and enzyme-labeled antibodies were combined in pairs. The test samples were all 100-fold diluted inactivated rabies virus, and the OD 630nm The higher one is the best antibody combination. The test results are shown in Table 3.

[0111] Table 3 OD of antibody combinations initially screened by matrix method 630nm result

[0112]

[0113] The results are shown in Table 3. Based on the experimental results, the best capture antibody was preliminarily determined to be one of 2B10, 7A3, 8A5 and 9F7, and the best detection antibody was preliminarily determined to be one of HRP-6G10 and HRP-9A7.

[0114] Further screening: Based on the preliminary screening results of monoclonal antibodies, the purified rabies virus G protein monoclonal antibodies 2B10, 7A3, 8A5, and 9F7 were diluted to 2.5 μg / mL with coating solution, coated on ELISA plates at 100 μL / well, and coated at 4°C overnight. The enzyme-labeled antibodies HRP-6G10 and HRP-9A7 were diluted to the same concentration, and the coated antibodies and enzyme-labeled antibodies were combined in pairs. The test samples were 10-fold and 100-fold diluted inactivated rabies viruses, respectively. At the same time, PBS was used as the negative control group. The one with the highest P (positive value) / N (negative value) was the best antibody combination. The results are shown in Tables 4 and 5.

[0115] Table 4 P / N results of further screening of antibody combinations by the matrix method (testing 100-fold diluted samples)

[0116]

[0117] Table 5 P / N results of further screening of antibody combinations by the matrix method (testing 10-fold diluted samples)

[0118]

[0119]

[0120] As shown in Tables 4 and 5, the antibody pairing combinations using 7A3 as the capture antibody can be further eliminated.

[0121] Final screening: Considering that the kit must be able to detect a broad spectrum of rabies virus strains, based on the results of further monoclonal antibody screening, the purified rabies virus G protein monoclonal antibodies 2B10, 8A5, and 9F7 were diluted to 2.5μg / mL with coating solution, coated on the ELISA plate at 100μL / well, and coated at 4°C overnight. The enzyme-labeled antibodies HRP-6G10 and HRP-9A7 were diluted to the same concentration, and the coating antibodies and enzyme-labeled antibodies were combined in pairs. The test sample was a 100-fold diluted veterinary rabies vaccine (including domestic SAD strains, domestic PV strains, and imported PV strains). At the same time, PBS was used as the negative control group, and the OD 630nm The antibody combination with the largest value is the best antibody combination. Figure 2 As shown, the antibody pairing combination with 8A5 as the capture antibody and HRP-6G10 as the detection antibody when detecting different strains 630nm The values ​​were all the highest, so 8A5 was finally determined as the capture antibody and HRP-6G10 as the detection antibody.

[0122] Therefore, hybridoma cell line 8A5 and hybridoma cell line 6G10 were biologically deposited with the deposit numbers CCTCC NO: C2023382 and CCTCC NO: C2023383, respectively, and both hybridoma cell lines were stored in liquid nitrogen.

[0123] Example 2 Recovery and expansion of hybridoma cells

[0124] Take out 6G10 hybridoma cells and 8A5 hybridoma cells from the liquid nitrogen tank, quickly melt them in a 37℃ water bath, centrifuge at 1000rpm for 5min, discard the supernatant, gently resuspend the cells with 1mL hybridoma cell culture medium, and then culture 6G10 hybridoma cells and 8A5 hybridoma cells in 6-well plates, and add 2.5mL of culture medium to each well. Culture them in a 37℃ 5% CO2 incubator and observe their status the next day. The morphology of the two hybridoma cells of 6G10 and 8A5 is as follows: Figure 3 As shown, both cells appeared semi-adherent, round, and translucent.

[0125] Example 3 Preparation of large amounts of ascites by in vivo culture

[0126] 8-10 week old BALB / c female mice (purchased from Hubei Provincial Center for Disease Control and Prevention) were intraperitoneally injected with Freund's incomplete adjuvant (0.5 mL / mouse) 7 days before inoculation of hybridoma cells. The well-growing hybridoma cells in Example 3 were collected and centrifuged at 1000 rpm for 5 min. The supernatant was collected and the precipitate was resuspended in serum-free medium and the cell density was adjusted to (1-2)×10 6 Each mouse was intraperitoneally injected with (0.5-1)×10 6 7-10 days after inoculation, the abdomen of the mouse was obviously swollen, and the ascites was collected. The ascites was centrifuged at 1200rpm for 10min, and the upper layer of fat was carefully sucked off. The yellowish ascites in the middle was taken out, packaged, and stored at -20℃ for later use.

[0127] Example 4 Hybridoma Cell Supernatant and Ascites Titer Test

[0128] The hybridoma cell supernatant and pale yellow ascites obtained in Example 3 were tested for monoclonal antibody titer using indirect ELISA. The SFV-RVG (1.9 mg / mL, a virus-like vesicle containing only rabies virus G protein, see patent CN113234692A) harvested by ultracentrifugation was diluted 1:500 and coated on an ELISA plate. Two wells of PBS negative control were set up, and the blocking solution was added and placed at 37°C for 2h. The hybridoma cell supernatant was diluted twice continuously, and the concentration was 2 1 , 2 2 , 2 3 , 2 4 , 2 5 , 2 6 , 2 7 , 2 8 , 2 9 , 2 10 The collected ascites was diluted tenfold in succession, with a concentration of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 The cell supernatant and ascites of each dilution were added to the ELISA plate, placed at 37°C for 1 hour, washed 5 times, and allowed to stand for 30 seconds each time. HRP-sheep anti-mouse IgG diluted 1:5000 was added, placed at 37°C for 1 hour, washed 5 times, and allowed to stand for 30 seconds each time. 50 μL of substrate A and B were added to each well, and the color was developed at room temperature in the dark for 10 minutes. After the color development was completed, 50 μL of stop solution was added to each well to stop the reaction, and the OD was measured on the ELISA reader within 10 minutes. 630nm value, calculate the P / N value.

[0129] 8A5 hybridoma cell supernatant and ascites titer Figure 4 and Figure 5 The results showed that the ELISA titer of 8A5 hybridoma cell supernatant was 2 11 , 8A5 ascites ELISA titer was 10 6 6G10 hybridoma cell supernatant and ascites titer Figure 6 and Figure 7 The results showed that the ELISA titer of 6G10 cell supernatant was 2 11 , 6G10 ascites ELISA titer is 10 6 .

[0130] Example 5 Purification of Ascites

[0131] Protein A+G affinity chromatography was used to purify ascites, and the specific steps were the same as those in Example 1. The eluted protein was then subjected to SDS-PAGE electrophoresis, and after the electrophoresis was completed, Coomassie Brilliant Blue staining was performed to identify the purity of the eluted antibody. Figure 8 As shown, the heavy chains of 8A5 and 6G10 are both located at 50 kDa, and the light chains are both located between 20-25 kDa, and there are no obvious mixed bands, indicating that the purity of the two monoclonal antibodies is very good and meets the standards for use as capture antibodies and preparation of detection antibodies.

[0132] Example 6 Ultrafiltration Concentration of Monoclonal Antibodies

[0133] The two batches of 8A5 monoclonal antibodies eluted in Example 5 were combined and then ultrafiltered and concentrated. At the same time, the 6G10 monoclonal antibody eluted in Example 5 was ultrafiltered and concentrated. The specific steps were the same as those in Example 1. The IgG concentrations of the purified and concentrated 8A5 and 6G10 were then measured using a micro-spectrophotometer. The test results are shown in Table 6.

[0134] Table 6 Concentrations of 8A5 and 6G10 mAbs

[0135] Monoclonal antibodies 8A5 6G10 Protein concentration (mg / mL) 2.152 2.730

[0136] As shown in Table 6, the concentration of the 8A5 monoclonal antibody after mixing was 2.152 mg / mL, and the concentration of the 6G10 monoclonal antibody was 2.730 mg / mL.

[0137] Example 7 Horseradish peroxidase labeled monoclonal antibody

[0138] The purified 6G10 monoclonal antibody was labeled with horseradish peroxidase to obtain HRP-6G10. The specific steps were the same as those in Example 1. The OD of HRP-6G10 was measured using a spectrophotometer. 403nm =2.11, OD 280nm=2.81, calculate the enzyme marker concentration and coupling rate, the calculation formula is as follows:

[0139] HRP amount (mg / mL) = OD 403nm × 0.4; IgG amount (mg / mL) = (OD 280nm -OD 403nm ×0.34)×0.62; molar ratio (mol) = HRP amount / IgG amount ×4; coupling rate (%) = OD 403nm / OD 280nm ×100%.

[0140] According to the formula, it can be calculated that: HRP amount (mg / mL) = 0.844 mg / mL, IgG amount (mg / mL) = 1.35 mg / mL, molar ratio (gram molecule) = 2.5, labeling rate (%) = 75.09%.

[0141] Add an equal volume of glycerol to the labeled HRP-6G10, 500 μL / tube, and store in aliquots at -20°C for use as enzyme marker stock solution.

[0142] Example 8 Determination of Dilution Factors of Capture Antibody and Detection Antibody

[0143] According to the square array titration method, the purified rabies virus G protein monoclonal antibody 8A5 was diluted 400, 500, 800, 1000, 1600, 2000, 3200, and 4000 times with the coating solution, and coated with 100 μL / well ELISA plate, and coated overnight at 4°C. Shake off the liquid in the ELISA plate and pat dry. Add blocking solution for blocking, 150 μL / well, and block overnight at 4°C. Shake off the liquid in the ELISA plate, wash the plate 5 times with washing solution, 200 μL / well, shake off the liquid in the plate and pat dry, add 100-fold diluted rabies virus, 100 μL / well, and incubate in a 37°C incubator for 1 hour. At the same time, set PBS as the negative control group. Shake off the liquid in the ELISA plate, wash the plate 5 times with washing solution, 200 μL / well. Then add horseradish peroxidase-labeled glycoprotein monoclonal antibody HRP-6G10 diluted 100, 150, 200, 250, 300, and 350 times, 100μl / well, and incubate in a 37℃ incubator for 1h. Shake off the liquid in the ELISA plate and wash the plate 5 times with washing solution, 200μL / well. Add 50μL of substrate solution A and 50μL of substrate solution B to each well for color development. After color development, add 50μL of stop solution to each well and read on the microplate reader. The best P / N value is the best, and the best dilution multiples of capture antibody and detection antibody are determined.

[0144] The experimental results are shown in Tables 7 and 8. It was determined from the experimental results that the P / N value was the largest when the capture antibody 8A5 was diluted 800 times (working concentration was 2.69 μg / mL) for coating and the detection antibody HRP-6G10 was diluted 300 times. It was finally determined that the optimal dilution of the capture antibody was 800 times, and the optimal dilution of the detection antibody was 300 times.

[0145] Table 7 Matrix titration to determine the dilution multiples of capture antibody and detection antibody (absorbance value)

[0146]

[0147] Table 8 Matrix titration to determine the dilution multiples of capture antibody and detection antibody (P / N)

[0148]

[0149]

[0150] Example 9 Determination of the Optimal Blocking Solution

[0151] 2% sucrose, 4% sucrose, 5% sucrose, 0.6% BSA, 1% BSA, 1.5% BSA, 5% skim milk powder and commercial blocking solution (Wuhan Keqian Biological Company) were added for blocking, 150 μL / well, and blocked overnight at 4°C. The sample was a 10-fold diluted inactivated rabies virus. The sample incubation conditions were 37°C for 1h, the detection antibody incubation conditions were 37°C for 1h, and the color development conditions were room temperature and dark for 10min. The best P / N value was determined to be the best blocking solution.

[0152] In order to determine the best blocking solution, 2% sucrose, 4% sucrose, 5% sucrose, 0.6% BSA, 1% BSA, 1.5% BSA, 5% skim milk powder and commercial blocking solution were used, blocked at 4°C overnight, and double antibody sandwich ELISA was performed. The results are shown in Fig. 9 As shown, when the commercial blocking solution is used for blocking, the positive value is higher, the negative value is the lowest, and the P / N value is the highest, so the commercial blocking solution is determined to be the best blocking solution.

[0153] Example 10 Determination of the optimal sample incubation time

[0154] Add 10-fold and 100-fold diluted rabies virus, 100 μL / well, and incubate in a 37°C incubator for 30 min, 45 min, 60 min, and 90 min, respectively. Set PBS as a negative control group. The blocking condition is to block overnight at 4°C with commercial blocking solution, the detection antibody incubation condition is 37°C, 1h, and the color development condition is room temperature and dark for 10 min. The optimal sample incubation time is determined by taking the maximum P / N value as the best.

[0155] In this example, the samples were placed in a 37°C incubator and reacted for 30 min, 45 min, 60 min, and 90 min, respectively, and high-concentration samples (i.e., 1:10 diluted samples) and low-concentration samples (i.e., 1:100 diluted samples) were tested to determine the optimal sample reaction time. The results are shown in Figure 2. Fig.10 and 11 As shown in the figure, when testing high-concentration samples, the P / N value is the largest when the sample incubation time is 37°C and 60 minutes. When testing low-concentration samples, although the P / N value is the largest when the sample incubation time is 37°C and 90 minutes, the P / N value does not increase significantly compared to 37°C and 60 minutes, while the reaction time increases by 30 minutes. Therefore, after comprehensive consideration, the optimal sample incubation time is determined to be 37°C and 60 minutes.

[0156] Example 11 Determination of the optimal detection antibody incubation time

[0157] Add 300-fold diluted HRP-6G10, 100 μL / well, and incubate in a 37°C incubator for 30 min, 45 min, 60 min, 90 min, and 120 min. The samples were 10-fold and 100-fold diluted inactivated rabies virus, respectively. The blocking conditions were commercial blocking solution at 4°C overnight, the sample incubation conditions were 37°C, 1 hour, and the color development conditions were room temperature and dark for 10 min. The optimal detection antibody incubation time was determined based on the maximum P / N value.

[0158] In order to determine the optimal incubation time for the detection antibody, the detection antibody was diluted to the working concentration and incubated at 37°C for 30 min, 45 min, 60 min, 90 min, and 120 min, respectively, to detect high concentration samples (i.e., 1:10 diluted samples) and low concentration samples (i.e., 1:100 diluted samples). The results are shown in Fig.12 and Fig.13 As shown, when testing low-concentration samples, the P / N value is the largest when the detection antibody incubation time is 37°C and 60 minutes. When testing high-concentration samples, although the P / N value is the largest when the sample incubation time is 37°C and 45 minutes, considering that the P / N value does not decrease significantly at 37°C and 60 minutes, the optimal detection antibody incubation time is determined to be 37°C and 60 minutes.

[0159] Example 12 Determination of the Optimal Color Development Time

[0160] Add substrate solution for color development, and react at room temperature in the dark for 5min, 10min, and 15min. Finally, read the results on an ELISA reader. The samples were 10-fold and 100-fold diluted inactivated rabies virus, respectively. The blocking conditions were commercial blocking solution at 4°C overnight, the sample incubation conditions were 37°C, 1h, and the detection antibody incubation conditions were 37°C, 1h. The optimal color development time was determined by taking the maximum P / N value as the best.

[0161] In this example, the ELISA plate was placed at room temperature and color was developed for 5 min, 10 min, and 15 min in the dark, respectively, and high concentration samples (i.e., 1:10 diluted samples) and low concentration samples (i.e., 1:100 diluted samples) were tested respectively to determine the optimal color development time. The results are shown in FIG. Fig.14 and Fig.15 It can be seen that whether it is detecting high-concentration samples or low-concentration samples, the color development condition is room temperature and dark, and the P / N value is the largest at 10 minutes. Therefore, it is determined that the best color development condition is room temperature and 10 minutes.

[0162] Example 13 Preparation and calibration of rabies vaccine quantitative reference material

[0163] Preparation of rabies vaccine potency quantitative reference material:

[0164] Take the same batch of rabies inactivated vaccine (SAD strain), mix the vaccine and gelatin sucrose protective agent (gelatin 2wt.%, sucrose 5wt.% and thiourea 1wt.%) in a volume ratio of 7:1, stir for 10min, and pack 1mL / bottle, freeze-dry 200 pieces in total, put them into a freeze dryer for freeze drying, cap them after freeze drying, and store them at -20°C.

[0165] Calibration of rabies vaccine potency quantitative reference material:

[0166] The NIH method was used to calibrate the potency of the quantitative reference product before lyophilization. The specific steps are as follows:

[0167] Dilution of reference vaccine: The inactivated rabies vaccine (SAD strain) is used as a quantitative reference for rabies vaccine and is diluted with PBS to dilutions of 1:25, 1:125, 1:625, etc.

[0168] Preparation of test solution: The test sample (specifically, inactivated rabies vaccine (SAD strain)) was diluted 5-fold in series with PBS.

[0169] Determination method: 16 mice weighing 12-14 g were immunized with different dilutions of the test sample and reference vaccine, each mouse was intraperitoneally injected with 0.5 mL, and immunized again after an interval of 1 week. 14 days after the first day of immunization, the mice were challenged intracerebrally with a pre-determined virus containing 5-100 LD50, 0.03 mL per mouse; at the same time, the challenge virus was diluted to 10 0 , 10 -1 , 10 -2 and 10 -3 Virulence titration was performed, with no less than 8 mice for each dilution. Mice were observed daily for 14 days after the attack, and the mortality was recorded. The number of mice that died after the 5th day and showed typical rabies brain symptoms was counted. The ED of the test article and the reference vaccine was calculated. 50 value.

[0170] Calculate relative potency:

[0171]

[0172] Where, P is the test sample potency, IU / mL; T is the test sample ED 50 The reciprocal of; S is the reference vaccine ED 50 The reciprocal of dT; dT ​​is the veterinary dose of the test article, mL; dS is the veterinary dose of the reference vaccine, mL; D is the potency of the reference vaccine, IU / mL.

[0173]

Notes

[0174] Determine the reconstitution volume of the rabies vaccine potency quantitative reference:

[0175] During freeze-drying, the vaccine and the lyophilization protective agent are mixed in a volume ratio of 7:1 and packaged at 1 mL / bottle. Therefore, under ideal conditions, when the antigen content is not reduced, reconstitution with 875 μL can theoretically keep the standard curves before and after freeze-drying consistent. Therefore, firstly, the rabies vaccine potency quantitative reference is reconstituted with 875 μL of sample diluent, and then the sample diluent is used to dilute the vaccine before freeze-drying and the rabies vaccine potency quantitative reference after freeze-drying by 2 times. With ln (dilution multiple) as the horizontal axis, ln (OD 630nm ) as the ordinate, and select the 5 points with the best linear relationship (R 2 >0.99), draw a trend line, and obtain a straight line formula. The ln(OD 630nm ) into the linear equation of the vaccine before lyophilization to obtain the corresponding dilution multiple, calculate the ratio of the dilution multiples before and after lyophilization at the same point, and then calculate the theoretical reconstitution volume. Re-verify the theoretical reconstitution volume according to the above steps, and determine the optimal reconstitution volume based on the fit of the standard curve before and after lyophilization.

[0176] Results: In order to determine the efficacy of veterinary rabies vaccine, a batch of veterinary rabies vaccine (SAD strain) was freeze-dried and calibrated as a quantitative reference for rabies vaccine. A total of 200 lyophilized tubes were tested, 1 mL / tube. The NIH titer of the vaccine was 19.7 IU / mL using the NIH test before freeze-drying. After freeze-drying, the calculated theoretical reconstitution volume of 875 μL was first used for reconstitution, and the results were compared before and after freeze-drying at the same OD 630nm The dilution factor is as follows: Fig.16As shown in the figure, it can be seen that the two standard curves are basically in parallel, indicating that the reconstitution volume is not appropriate. Therefore, the theoretical reconstitution volume is calculated to be between 640μL and 725μL by the calculation method in Table 9. Then, the intermediate value was verified first, and 675μL was used for reconstitution, and the standard curves before and after lyophilization were compared. The results are as follows Fig.17 As shown, it can be seen that the two standard curves are highly overlapped, so the final reconstitution volume of the rabies vaccine quantitative reference material was determined to be 675 μL.

[0177] Table 9 Determination of theoretical reconstitution volume of rabies vaccine quantitative reference material

[0178]

[0179] Example 14 Composition and use of veterinary rabies vaccine efficacy sandwich ELISA test kit

[0180] ELISA plate; the coating agent is 8A5 monoclonal antibody, the coating concentration is 2.69μg / mL; the detection antibody is 6G10 monoclonal antibody labeled with horseradish peroxidase, the dilution of the detection antibody is 1:300; the blocking solution is a commercial blocking solution; washing solution; substrate solution A; substrate solution B; stop solution. The entire detection process can be completed within 2.5 hours.

[0181] Directions:

[0182] 1) Antibody coating: dilute the rabies virus G protein monoclonal antibody to a certain working concentration, add the antibody dilution solution to the wells of the ELISA plate, 100 μL / well, and incubate at 4°C overnight.

[0183] 2) Blocking: After coating, discard the liquid in the ELISA plate and pat dry on absorbent material. Add blocking solution to each well, 150 μL / well, and incubate at 4°C overnight. After blocking, discard the liquid in the well.

[0184] 3) Washing: Add 1× washing solution to each well, 200 μL / well, repeat washing 5 times, and pat dry on absorbent material for the last time.

[0185] 4) Incubate sample antigen: Add pre-diluted antigen or quantitative reference to each well, 100 μL / well, gently shake to mix, cover the ELISA plate with sealing film, and incubate in a 37°C constant temperature incubator for 1 hour. After incubation, discard the liquid in the well and perform washing.

[0186] 5) Incubate the enzyme-labeled antibody: Add the enzyme-labeled working solution to each well (no solution is added to the blank well), 100 μL / well, cover the plate with a sealing film, and incubate in a 37°C constant temperature incubator for 1 hour. After the incubation, discard the liquid in the well and perform washing.

[0187] 6) Color development: First add 50 μL of substrate solution A to each well, then add 50 μL of substrate solution B, and incubate at 20-25°C in the dark for 10 min.

[0188] 7) Stop: Add 50 μL of stop solution to each well and measure the OD of each well on a microplate reader within 10 minutes. 630nm value.

[0189] 8) Vaccine potency calculation:

[0190] a) Drawing of standard curve: The potency value of the standard product is used as the vertical axis, and its corresponding OD 630nm The (standard product - blank) value is used as the horizontal axis to draw a scatter plot (usually 4-6 points are used for drawing), and the linear regression equation of the standard product is Y=ax+b, R 2 >0.99, the test is established (it is recommended to select a dilution with an OD value between 0.1-1.5 for the standard curve).

[0191] b) Titer of the sample to be tested (IU / mL): 630nm After the (sample-blank) value is substituted into the linear regression equation, the obtained value is multiplied by the dilution factor corresponding to the sample (it is recommended that the OD value of the sample be within the range of 0.7-1.5).

[0192] Example 15 Methodology Verification

[0193] 1. Linear

[0194] In order to fully verify the linearity of the kit prepared in Example 14, the national standard for rabies vaccine potency testing of the China Food and Drug Inspection Institute (CTN-1 strain, 11.4 IU / mL), the national standard for rabies vaccine of the China Veterinary Drug Administration (5.5 IU / mL), and the homemade rabies vaccine quantitative reference (SAD strain, 19.7 IU / mL) were diluted twice, and then the kit was used for detection, with ln (dilution factor) as the horizontal axis and ln (OD 630nm ) as the ordinate, select the 4-5 points with the best linear relationship to add a trend line, establish a regression equation, and obtain the correlation coefficient R of the regression equation 2 , R 2 >0.99 indicates a good linear relationship. The results are as follows Fig.18 , Fig.19 and Fig. 20 As shown in the figure, it can be seen that the kit has good linearity for the above three standards, R 2 They are 0.9989, 0.9995 and 0.9983 respectively.

[0195] 2. Broad spectrum of virus detection

[0196] In order to investigate the applicability of the kit prepared in Example 14 to rabies vaccines from different manufacturers and different strains, rabies vaccines from 6 different manufacturers were purchased in batches, including SAD strain, PV strain, Flury strain, CTN-1 strain, r3G strain and CVS-11 strain. The 6 inactivated rabies vaccines were diluted twice, and then tested using the kit. A standard curve was established according to the steps described in step 1 "Linearity" to see if there was a dose-dependent relationship. The results are shown in the figure. Fig.21 As shown, it can be seen that the kit shows a dose-dependent relationship when detecting vaccines of the above 6 rabies virus strains, so the kit can detect PV strain, r3G strain, SAD strain, Flury strain, CVS-11 strain and CTN-1 strain.

[0197] 3. Applicability

[0198] In order to test whether the kit prepared in Example 14 can be used for samples in all links of vaccine production, the virus supernatant, virus concentrate, purified virus liquid and finished vaccine (the virus supernatant, virus concentrate and purified virus liquid are samples provided by Wuhan Keqian Biological Co., Ltd.) in the vaccine production process were tested respectively, and the above samples were diluted twice to detect OD 630nm , follow the steps described in step 1 "Linearity" to establish a standard curve to see if there is a dose-dependent relationship. The results are as follows Fig. 22 As shown, the results show that the kit has a good linear relationship in detecting virus supernatant, virus concentrate, purified virus liquid and finished vaccine, so the kit can be used for samples in all links of vaccine production.

[0199] 4. Specificity

[0200] In order to test the specificity of the test kit prepared in Example 14, a comprehensive investigation was conducted to determine whether the test kit has cross-reactions with common canine viruses, other finished canine vaccines, and some raw materials required for vaccine production. Canine Parvovirus (CPV), Canine Distemper Virus (CDV), Canine Adenovirus (CAV), Canine Parainfluenza Virus (CPIV), lysine, sorbitol, trehalose, sucrose, culture medium, new bovine serum, aluminum gel adjuvant, and rabies virus inactivated vaccine positive control and PBS negative control were used as test samples. The above samples were diluted in two-fold ratios and tested using the test kit. The specificity of the test kit was verified. The results are as follows: Fig.23As shown in the figure, it can be seen that this method detects possible ingredients in veterinary vaccines, such as lysine, sorbitol, trehalose, sucrose, culture medium, new bovine serum, and aluminum gel adjuvant, and the results are all negative. There is no cross reaction to the four common viruses in dogs, namely CPV, CPIV, CAV, and CDV. It is only dose-dependent when detecting rabies vaccines. Therefore, the kit has good specificity.

[0201] 5. Minimum detection amount

[0202] In order to comprehensively test the minimum detection amount of the kit prepared in Example 14, the kit was used to detect the national standard for rabies vaccine potency testing of the China Food and Drug Inspection Institute (11.4 IU / mL), the national standard for rabies vaccine of the China Veterinary Drug Administration (5.5 IU / mL), and the homemade rabies vaccine potency quantitative reference (19.7 IU / mL). After reconstitution, the pre-dilution was followed by a 2-fold dilution, and the kit was used for detection. At the same time, a negative control was set. According to the Cut-off value, the average absorbance value of the negative control wells × 2.1 was used as the Cut-off value, and the absorbance value of the positive control wells should not be less than 2.1 times the average absorbance value of the negative control wells, and the OD value was calculated. 630nm ≥2.1×negative control OD 630nm The corresponding potency value is the minimum detection amount. The results are shown in Tables 10, 11 and 12. The minimum detection amount of the kit for the national standard product (11.4 IU / mL) for the potency test of rabies vaccine of the China National Institute for Food and Drug Control is 0.0006958 IU / mL, the minimum detection amount of the kit for the national standard product (5.5 IU / mL) of rabies vaccine of the Central Prison is 0.0006714 IU / mL, and the minimum detection amount of the kit for the homemade rabies vaccine quantitative reference product (19.7 IU / mL) is 0.003078 IU / mL.

[0203] Table 10 Minimum detection amount test results (National standard for rabies vaccine potency test of China Food and Drug Inspection Institute, 11.4IU / mL)

[0204] Dilution multiple <![CDATA[OD 630nm ]]> Potency (IU / mL) 256 3.860 0.0445313 512 2.877 0.0222656 1024 1.790 0.0111328 2048 0.898 0.0055664 4096 0.477 0.0027832 8192 0.271 0.0013916 16384 0.169 0.0006958 32768 0.133 0.0003479 Negative control 0.077 0

[0205] Table 11 Minimum detection amount test results (rabies vaccine national standard product of China Veterinary Drug Administration, 5.5 IU / mL)

[0206]

[0207]

[0208] Table 12 Minimum detection results (self-made standard, 19.7 IU / mL)

[0209] Dilution multiple <![CDATA[OD 630nm ]]> Potency (IU / mL) 200 2.885 0.0985 400 1.945 0.04925 800 0.956 0.02462 1600 0.401 0.01231 3200 0.207 0.006156 6400 0.134 0.003078 12800 0.099 0.001539 25600 0.075 0.0007695 Negative control 0.063 0

[0210] 6. Repeatability

[0211] (1) Intraplate repeatability: In order to preliminarily examine the repeatability of the kit prepared in Example 14, the homemade rabies vaccine titer quantitative reference material (SAD strain) was diluted to high (stock solution), medium (2 times), and low (4 times) dilutions, respectively, so that the sample concentrations were 19.7 IU / mL, 9.85 IU / mL, and 4.925 IU / mL, respectively. Then, the ELISA plate with synchronous coating and sealing was used to detect the above three samples, and the titer value of each sample was calculated and the intraplate coefficient of variation was calculated. The results are shown in Table 13, and the results show that the intraplate coefficient of variation for detecting high-concentration samples is 2.61%, the intraplate coefficient of variation for detecting medium-concentration samples is 5.36%, and the intraplate coefficient of variation for detecting low-concentration samples is 8.97%.

[0212] Table 13 Intra-plate repeatability test results

[0213]

[0214] (2) Inter-plate repeatability: In order to preliminarily examine the repeatability of the kit prepared in Example 14, the homemade rabies vaccine potency quantitative reference material (SAD strain) was diluted to high (stock solution), medium (2 times), and low (4 times) dilutions, so that the sample concentrations were 19.7 IU / mL, 9.85 IU / mL, and 4.925 IU / mL, respectively. Then, the ELISA plates that were coated and blocked at different times were used to detect the above three samples, and the potency value of each sample was calculated and the inter-plate variation coefficient was calculated. The results are shown in Table 14, which show that the inter-plate variation coefficient for detecting high-concentration samples was 5.71%, the inter-plate variation coefficient for detecting medium-concentration samples was 9.53%, and the inter-plate variation coefficient for detecting low-concentration samples was 6.89%.

[0215] In summary, the intra-plate coefficient of variation and the inter-plate coefficient of variation of the kit are both less than 10%, so the intra-plate repeatability and inter-plate repeatability of the kit are good.

[0216] Table 14 Inter-plate repeatability test results

[0217]

[0218] 7. Accuracy

[0219] The homemade rabies vaccine potency quantitative reference material (SAD strain) was diluted high (original solution), medium (2 times), and low (4 times) to make the sample concentrations 19.7 IU / mL, 9.85 IU / mL, and 4.925 IU / mL, respectively. Then the kit was used to detect these three samples separately. Each sample was tested three times in succession, and the difference between the test results and the actual concentration of the reference material was calculated, and the recovery rate was calculated.

[0220] In order to test the accuracy of the kit, three ELISA plates were used to detect high, medium and low samples respectively, and the sample titers were detected and calculated. The recovery rate was calculated according to the recovery rate formula (recovery rate = measured titer / theoretical titer × 100%). The results are shown in Table 15. The results show that the recovery rate of the kit in detecting high-concentration samples is 99.2% to 111%, the recovery rate of detecting medium-concentration samples is 71.5% to 78.9%, and the recovery rate of detecting low-concentration samples is 71.2% to 79.1%. In summary, the kit has good accuracy.

[0221] Table 15 Accuracy test results

[0222]

[0223] Example 16 Clinical Trial

[0224] 1. Trial production of 3 batches of finished test kits

[0225] The kit consists of the following components:

[0226] Antibody coated plate (the coating material is 8A5 monoclonal antibody, the coating concentration is 2.69 μg / mL, and it has been blocked with commercial blocking solution); the detection antibody is 6G10 monoclonal antibody labeled with horseradish peroxidase (enzyme marker), and the dilution of the detection antibody is 1:300; 20-fold concentrated washing solution; sample treatment solution; substrate solution A; substrate solution B; stop solution.

[0227] In accordance with the above conditions, three batches of test kits were trial-produced on the diagnostic reagent GMP production line in August 2022, with 50 boxes in each batch, with batch numbers 20220801, 20220802 and 20220803 respectively.

[0228] 2. Quality control of 3 batches of finished test kits

[0229] (1) Property inspection: Take 5 samples from each batch of finished test kits and conduct property inspection on the test kits and their components. The outer packaging of the test kit should be clean and intact, the label should comply with relevant national regulations, the inner packaging should be sealed intact, without damage or leakage; the product name, batch number, storage conditions, and expiration date should be clear. The packaging film in the antibody-coated plate should be well sealed, and the bottom of the well should be clean and free of foreign matter. Enzyme markers, substrate solution A, substrate solution B, stop solution, 20-fold concentrated washing solution, etc. should all be clear liquids. Eliminate changes in clarity caused by reagent deterioration or contamination. When conducting property inspections, any test samples with broken packaging must be discarded, and attention should be paid to whether the seal is tight and whether the label is incorrect or unclear.

[0230] The three batches of test kits were sampled and the test results of various supporting reagents in the three batches of test kits are shown in Table 16. The results showed that the properties of the components of the three batches of test kits did not change.

[0231] Table 16 Characteristic test results

[0232]

[0233] Note: “s” means that the properties of the components of the kit have not changed and meet the standards.

[0234] (2) Specificity: One kit was taken from each of the three batches of kits and its specificity was tested. Four specific reference products (including CPV, CPIV, CAV and CDV) were tested after two-fold dilution. The results were as follows: Fig.24 , Fig.25 and Fig.26 As shown, the results show that the OD values ​​of 4 specific quality control products diluted 2-fold were detected by 3 batches of kits respectively. 630nm The values ​​were all less than 0.1, and the dose-dependent relationship was only shown when the positive control was tested. In summary, the specificity of the 20220801, 20220802 and 20220803 batches of kits was good.

[0235] (3) Repeatability: including intra-batch repeatability and inter-batch repeatability. Intra-batch repeatability means diluting the homemade rabies vaccine potency quantitative reference material into high (stock solution), medium (2 times), and low (4 times) dilutions, so that the sample concentrations are 19.7 IU / mL, 9.85 IU / mL, and 4.925 IU / mL, respectively. Then, 5 test kits are drawn from each of the three batches of test kits, 20220801, 2022080, and 20220803, and the above three samples are tested respectively. The potency value of each sample is calculated and the intra-batch coefficient of variation is calculated. The batch repeatability is to dilute the homemade rabies vaccine potency quantitative reference material into high (original solution), medium (2 times), and low (4 times) dilutions to make the sample concentrations 19.7 IU / mL, 9.85 IU / mL, and 4.925 IU / mL, respectively. Then, one box of test kit is taken from each of the three batches of test kits, 20220801, 20220802, and 20220803, and the above three samples are tested respectively. The potency value of each sample is determined and the batch variation coefficient is calculated.

[0236] The results of intra-batch repeatability are shown in Tables 17, 18 and 19. The intra-batch coefficients of variation of the three batches of kits are all less than 10%. In summary, the intra-batch repeatability of the 20220801 batch, 20220802 batch and 20220803 batch kits is good.

[0237] Table 17 Intra-batch repeatability test results of 20220801 batch

[0238]

[0239] Table 18 Intra-batch repeatability test results of 20220802 batch

[0240]

[0241] Table 19 20220803 batch repeatability test results

[0242]

[0243] The results of inter-batch repeatability are shown in Table 20. The inter-batch coefficients of variation of the three batches of kits were all less than 10%.

[0244] Table 20 Results of inter-batch repeatability test

[0245]

[0246] In summary, the batch reproducibility of the 20220801, 20220802 and 20220803 test kits was good.

[0247] 3. Stability

[0248] Three batches of test kits were placed at 37°C for 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days and 8 days, respectively. The test kits were taken out every day to test their specificity, minimum detection amount and linear range.

[0249] (1) Specific stability

[0250] In order to test the stability of the test kit, the three batches of test kits were placed in a 37°C greenhouse for accelerated destruction. They were placed in the 37°C greenhouse for 8 consecutive days. The test kits were first taken out every day to test their specificity. The results are as follows: Fig. 27 , Fig.28 and Fig.29 The results showed that after the three batches of test kits were stored at 37°C for 8 days, the results of various negative reference products were the same as when they were just produced, and all were negative. In summary, the specificity and stability of the test kits in batches 20220801, 20220802 and 20220803 were good.

[0251] (2) Stability of minimum detection amount

[0252] In addition to the stability of detection specificity, the kit was taken out every day to test its minimum detection amount, and the results are shown in Table 21. The results showed that the minimum detection amount of the three batches of kits did not change after being stored at 37°C for 8 days. In summary, the minimum detection amount of the 20220801 batch, 20220802 batch, and 20220803 batch kits had good stability.

[0253] Table 21 Stability results of minimum detection amount (IU / mL)

[0254]

[0255] (3) Linear interval stability

[0256] In addition to testing specificity and stability of the linear range, the kit was taken out every day to test its linear range, and the results are shown in Table 22. The results showed that the linear range of the three batches of kits did not change after being stored at 37°C for 8 days. In summary, the linear range stability of the 20220801 batch, 20220802 batch, and 20220803 batch kits was good.

[0257] Table 22 Stability results in linear range (IU / mL)

[0258]

[0259] 4. Shelf life

[0260] The three batches of test kits were placed at 4°C for 6 months and then taken out to test their specificity, minimum detection amount and linear range.

[0261] (1) Specificity

[0262] In order to test the shelf life of the kit, the three batches of kits were placed in a 4℃ cold storage for 6 consecutive months. After 6 months, the kits were taken out to test their specificity. The results are shown in Table 23, Table 24 and Table 25. The results show that after the three batches of kits were stored at 4℃ for 6 months, the OD of various negative references was higher than that when they were just produced. 630nm Although the values ​​were increased, they were still lower than the OD value of the negative control. 630nm In summary, it is preliminarily shown that the 20220801, 20220802 and 20220803 batches of kits have a shelf life of up to 6 months in terms of specificity.

[0263] Table 23 Specificity test results of the 20220801 batch of test kits stored at 4°C for 6 months

[0264]

[0265] Table 24 Specificity test results of the 20220802 batch of test kits stored at 4°C for 6 months

[0266]

[0267] Table 25 Specificity test results of the 20220803 batch of test kits stored at 4°C for 6 months

[0268]

[0269]

[0270] (2) Minimum detection amount

[0271] In addition to testing the specificity of the three batches of test kits, the test kits were taken out after 6 months to test their minimum detection amount, and the results are shown in Table 26. The results show that after the three batches of test kits were stored at 4°C for 6 months, the minimum detection amount increased compared to when they were first produced. However, because the potency of the veterinary rabies vaccine detected by the test kit is generally higher than 2IU / mL, the minimum detection amount is still far lower than the actual test sample, so it will not affect normal testing. In summary, it is preliminarily shown that the minimum detection amount of the test kits in batches 20220801, 20220802 and 20220803 can be stored for 6 months.

[0272] Table 26 Test results of the minimum detectable amount of 3 batches of test kits stored at 4℃ for 6 months

[0273]

[0274] (3) Linear interval

[0275] In addition to testing the specificity and minimum detection amount of the three batches of kits, the kits were taken out after 6 months to test their linear ranges, and the results are shown in Table 27. The results show that after the three batches of kits were stored at 4°C for 6 months, their linear ranges did not change compared to when they were just produced. In summary, it is preliminarily shown that the 20220801 batch, 20220802 batch and 20220803 batch kits have a shelf life of up to 6 months in terms of linear range.

[0276] Table 27 Test results of linear range of 3 batches of test kits stored at 4℃ for 6 months

[0277]

[0278] 5. Compliance rate

[0279] (1) Correlation analysis between homemade kits and NIH tests

[0280] The potency of 18 batches of rabies inactivated vaccine (SAD strain) was tested using a homemade kit and the NIH test. The potency measured by the homemade kit was used as the horizontal axis, and the potency measured by the NIH test was used as the vertical axis. A trend line was added and r, i.e., correlation, was calculated using software. The closer r is to 1, the better the correlation between the two methods. The results are shown in Figure 2. Fig.30 As shown, the results showed that the correlation coefficient r between the homemade kit and the NIH test was 0.9314 (P<0.0001). In summary, the kit and the NIH test had a good correlation.

[0281] (2) Consistency analysis between homemade kits and NIH tests

[0282] The 18 rabies vaccine sample data measured in the "Correlation analysis between the homemade kit and the NIH test" were analyzed for consistency between the two methods using Graphpad software. The consistency results are shown in the figure below: Fig.31 As shown in the figure, it can be seen from the results that compared with the results of the homemade kit and the NIH test, 17 of the 18 data fall within the 95% consistency interval. In summary, the titer measured by the homemade kit and the NIH test is well consistent.

[0283] (3) Comparison between homemade kits and commercially available kits

[0284] Ten samples were selected from the above 18 clinical samples and tested using commercially available kits of the same type. The correlation coefficient results are as follows: Fig.32 , Fig.33 and Fig.34As shown in the results, the correlation coefficient between the commercial kit and the NIH test is 0.9345, the consistency rate between the homemade kit and the NIH test is 0.9577, which is better than the commercial kit, and the consistency rate between the homemade kit and the commercial kit is 0.9615. In summary, the homemade kit has a good correlation with the NIH test and the commercial kit, and is better than the commercial kit.

[0285] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A hybridoma cell line combination that secretes monoclonal antibodies against rabies virus G protein, characterized in that: The hybridoma cell line combination includes hybridoma cell line 8A5 with a deposit number of CCTCC NO: C2023382 and hybridoma cell line 6G10 with a deposit number of CCTCC NO: C2023383.

2. A monoclonal antibody combination against rabies virus G protein, characterized in that: The monoclonal antibody combination comprises the monoclonal antibody secreted by the hybridoma cell line 8A5 described in claim 1 and the monoclonal antibody secreted by the hybridoma cell line 6G10 described in claim 1.

3. Use of the monoclonal antibody combination according to claim 2 in the preparation of a veterinary rabies vaccine and / or a veterinary rabies vaccine intermediate for efficacy detection, characterized in that: The potency detection product is a sandwich ELISA detection kit; the coating material of the sandwich ELISA detection kit is a monoclonal antibody secreted by the hybridoma cell line 8A5, and the detection antibody is a monoclonal antibody secreted by the hybridoma cell line 6G10.

4. The use according to claim 3, characterized in that: The veterinary rabies vaccine includes the veterinary rabies vaccine prepared from PV strain, r3G strain, SAD strain, Flury strain, CVS-11 strain or CTN-1 strain.

5. The use according to claim 3, characterized in that: The veterinary rabies vaccine intermediate includes virus supernatant, concentrated virus liquid or purified virus liquid prepared from PV strain, r3G strain, SAD strain, Flury strain, CVS-11 strain or CTN-1 strain.

6. A sandwich ELISA test kit for veterinary rabies vaccine efficacy detection, characterized in that: The coating material of the sandwich ELISA detection kit is the monoclonal antibody secreted by the hybridoma cell line 8A5 described in claim 1, and the detection antibody is the monoclonal antibody secreted by the hybridoma cell line 6G10 described in claim 1.

7. The sandwich ELISA detection kit according to claim 6, characterized in that The concentration of the coating source is 2.69 μg / mL; the dilution multiple of the detection antibody is 1:300; and the detection antibody is labeled with horseradish peroxidase.

8. A double antibody sandwich ELISA test method for detecting the efficacy of rabies vaccine for animals for non-disease diagnosis and treatment purposes, characterized in that: The detection is performed using the sandwich ELISA detection kit described in claim 6 or 7.

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