Establishment Of An Indirect ELISA For Detection Of Antibodies Against African Swine Fever Virus PA104R Protein

African swine fever(ASF)is a highly lethal swine infectious disease caused by African swine fever virus(ASFV),the only member of Asfivirus and Asfarviridae.The disease is listed as notifiable disease by the World Organization for Animal Health,and it is also listed as Class A disease in China.In August 2018,ASFV was introduced into China,and then spread to 31 provinces and autonomous regions,the occurrence and prevalence of the disease has caused serious cost on pig industry and on China's economy.As there is no effective vaccine and treatment for ASF at present,the means of prevention and control mainly rely on rely on timely stamping out and cleaning.The hyperacute and acute cases caused by highly virulent strains can often lead to rapid death of pigs;however,by the time cause of the occurrence and prevalence of the disease,some animals might show subacute infection and survival.The recovered animals had high antibody levels,and the virus was usually not detectable in the blood.Therefore,antibody detection is of great significance for the prevention and cleaning of ASF.In this study,five kinds of antigens commonly used in ASF serological detection were used and were prepared by prokaryotic expression system,and the reactivity of the antigens was compared.The dominant antigens were selected for the study of ASF serological antibody detection technology.The specific contents are as follows:1.Prokaryotic expression and antigen reactivity analysis of ASFV pA104R,p B602L,P30,P72 and p K205R proteins.In this study,prokaryotic expression vectors p ET-30a-A104R,B602L,CP204L,B646L and K205R were successfully constructed.The induced expression and solubility analysis of five proteins were carried out and the proteins were purified by affinity chromatography.SDS-PAGE analysis suggested that the five proteins could react with ASFV positive sera,but not with negative sera.Using indirect ELISA,the reactivity of five antigens was compared in parallel.According to the P/N value,the pA104R protein was selected as the best antigen for the development of ASF antibody detection technology.2.Establishment of the indirect antibody detection ELISA method based on the pA104R protein of ASFV.Establishment of the indirect ELISA method for the detection of ASFV antibody,by using the ASFV pA104R protein antigen to coat the enzyme plate.The optimal concentration of antigen coating,serum dilution secondary antibody working concentration of the ELISA method were screened by using ASF negative and positive sera.The antigen coating concentration,sera dilution and The ELISA reaction conditions were:the best dilutions of pA104R antigen,sera and the best dilution of enzyme-labeled secondary antibody is 2?g/m L,1:200,1:50000 respectivety.Furthermore,the reaction conditions were screened,and 2%BSA was selected as the optimal blocking solution for the ELISA method,while the optimal reaction time for serum and HRP-labeled secondary antibody was 45 min.The optimal reaction conditions were used to determine the ELISA threshold by detecting the negative and positive sera of ASF with known background.The results showed that when the Ab R value(OD_450nmof the sample to be tested-OD_{450 nm}of the standard negative sample)/(standard positive OD_{450 nm}-standard negative OD_{450 nm})reached 16%,the sensitivity and specificity of the method were 97.2%and97.4%,respectively,No cross-reaction was detected with positive sera of known porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus,classical swine fever virus and foot-and-mouth disease virus by this method.Using the established ELISA method clinical serum samples,were examined and compared with the commercial kit.The results showed that the coincidence rate was 94.7%.In summary,five antigens of ASFV were prepared by prokaryotic expression system in this study.Through parallel comparative analysis,pA104R antigen with good reactivity was screened out,and used to establish the indirect ELISA for detecting ASF antibody,which had high sensitivity and specificity.