The Development Of The African Swine Fever Virus Antibody Rapid Detection Kit

Objective: In this study,the recombinant proteins of African swine fever virus(ASFV)p30,p72 and MGF360-12 L with good antigenicity and highly conserved structure were expressed and purified,and the three recombinant proteins were used as immunogens to prepare p30,p72 and MGF360-12 L respectively.The combination of three monoclonal antibodies was used to develop an African swine fever virus antibody competition ELISA rapid detection kit.Methods:1.Expression and purification of ASFV p30,p72 and MGF360-12 L recombinant proteins The genetic engineering bacteria containing the recombinant plasmids p MAL-c6T-p30,p MAL-c6T-p72 and p ET-28a-MGF360-12 L were induced to express by IPTG,the recombinant protein was purified by Ni-NTA resin affinity chromatography,and its expression was identified by SDS-PAGE analysis.Finally the the concentration of recombinant proteins was determined by BCA method.2.Preparation of ASFV p30,p72 and MGF360-12 L monoclonal antibodiesUsing purified ASFV p30,p72 and MGF360 proteins as immunogens,6-7week-old female BALB/C mice were immunized by conventional methods,After the first immunization,the same dose was boosted on the 14 th and 28 th day,and the tail was docked after the last booster immunization.The antibody titer was measured by indirect ELISA,and intraperitoneal injection was performed for shock immunization3 days before fusion.Mice with high immune titer were selected,and PEG 1500 was used to conduct cell fusion experiments with SP2/0 myeloma cells and spleen cells at a ratio of 1:5.On the 10 th day after fusion,the hybridoma cells were screened by indirect ELISA,the positive hybridoma cells were cloned by limiting dilution method,and the culture supernatant was taken for cross-reaction test to test its specificity;A large number of monoclonal antibodies were prepared by the ascites induction method,the monoclonal antibodies were purified by the n-octanoic acid ammonium sulfate method,HRP-labeled monoclonal antibodies,and the titers of the labeled antibodies were detected by indirect ELISA.3.Development of ASFV Antibody Competitive ELISA Rapid Detection KitASFV p30,p72 and MGF360-12 L proteins were used as coating agents,and the reaction conditions were optimized by indirect ELISA method.The prepared ASFVp30,p72 and MGF360-12 L specific enzyme-labeled monoclonal antibodies were used in combination as competitive antibodies to establish the African swine fever virus antibody competition.ELISA rapid detection method,SIV,PRV,PCV,PPV,PRRSV-positive sera and several ASFV-negative sera were detected by optimized competitive ELISA kits,and the critical value,sensitivity,specificity and coincidence rate of the method were evaluated.Results: IPTG induced expression of p30,p72,MGF360-12 L recombinant proteins,purified by Ni-NTA resin affinity chromatography,and SDS-PAGE electrophoresis showed that the molecular weights were about 65 KDa,73.2 KDa,43.2 KDa.The concentration of purified protein was determined by BCA method,and the purified protein concentrations of p30,p72 and MGF360-12 L were 6.30 mg/m L,10.18 mg/m L and 6.18 mg/m L,respectively.The titers of the tail blood of mice after immunization were all greater than 1:16000.Indirect ELISA was used to detect the culture supernatant of hybridoma cells and subcloned cells,and obtained hybridoma cell lines that could stably secrete specific monoclonal antibodies,have no crossover,have strong specificity and belong to the Ig G subclass,respectively named p30-35,p30-80,p72-37,p72-41 and MGF360-12L-8,MGF360-12L-20,the ascites titers were all after indirect ELISA purification Greater than 1:32000,the titers of HRP-labeled antibodies are all greater than 1:3200.The reaction conditions were optimized by competitive ELISA.The optimal coating concentrations of p30,p72 and MGF360-12 L purified proteins were 0.5 μg/m L,0.5 μg/m L and 0.25 μg/m L,respectively,and the optimal coating conditions were carbonate buffer.Buffer solution(Carbonate Buffer solution,CBS),coated at 4℃ for 12 h,the optimal blocking solution conditions were 5%nonfat milk powder,blocked at 37℃ for 2 h,serum samples were diluted 1:8,and the optimal dilution ratio of enzyme-labeled antibodies was 1: 3200,the best competition time is 1 h,the best color development conditions are 37℃ dark for 20 min,and the test results of SIV,PRV,PCV,PPV,PRRSV are all negative,and the critical value determination PI≤25.68% is judged as negative,PI≥27.27% was judged positive,25.68% ≥ PI ≥ 27.27% was judged suspicious,the sensitivity was 1:128,and the coincidence rate was 97.5%.Conclusion: The competitive ELISA developed for the preparation of specific monoclonal antibodies against recombinant proteins p30,p72 and MGF360-12 L has good specificity and sensitivity,and can provide an effective monitoring tool for the prevention of ASFV and the detection of large-scale serum samples.Effective technical means are provided for effect evaluation.