Establishment of a Competitive ELISA Method for Detection of African Swine Fever Virus Antibodies
To establish a competitive ELISA method for rapid and accurate detection of AFSV antibody by using recombinant proteins of African swine fever virus p30,p72 and MGF360-12L,and their enzyme-labeled monoclonal antibodies.BALB/c mice were immunized with purified recombinant proteins of ASFV p30,p72 and MGF360-12L,respectively,and positive hybridoma cells were obtained by conventional fusion method.The corresponding monoclonal antibodies were purified by caprylic acid-ammonium sulfate method,and HRP labeled monoclonal antibodies were prepared.A competitive ELISA method was established and the reaction conditions were optimized.The critical value,sensitivity,specificity and coincidence rate of this method were evaluated.The purified recombinant proteins p30,p72 and MGF360-12L were of good purity,and the fused cell lines were identified by Western blot with good reactivity and specificity.All the hybridoma cell lines screened were IgG antibodies,and the purified monoclonal antibodies met the molecular weight of antibody IgG.The monoclonal antibodies were highly active after HRP labeling.The optimized results of competitive ELISA showed that the dilution of serum and enzyme-labeled monoclonal antibody were 1∶8 and 1∶3200,respectively.The critical value of PI≤14.71％was negative,PI≥18.97％was positive,and it was suspicious when 14.71％≤PI≤18.97％.The sensitivity of the kit was 1∶128,and the coincidence rate was very high.The rapid detection method of ASFV antibody has been successfully established,which provides an effective monitoring tool for the prevention of ASFV and the detection of large-scale serum samples,and provides an effective technical means for the evaluation of the immune effect of ASFV vaccine.
万方数据
维普
