Development of double antibody sandwich ELISA assay for detection of African swine fever virus
Two monoclonal antibodies targeting different antigenic epitopes of African swine fever virus(ASFV)p72 protein were used to develop the sandwich ELISA for ASFV antigen.One McAb was coated on the 96-well plate as the capture antibody,and the other was labeled with HRP as the detection antibody.The square matrix method was used to optimize the ELISA reac-tion conditions.The results showed that the optimized concentration for coating antibody was 1.25 mg/L,and the optimal dilution of the enzyme-conjugate monoclonal antibody was 1∶4 000.The critical value of this method was determined to be 0.299 by test.When the D450 value of the sample was greater than or equal to 0.3 and P/N was greater than 2,it could be considered to be positive.The double antibody sandwich ELISA method can specifically detect ASFV antigen,and the re-sults of other antigens were negative.The intra-and inter-assay coefficients of variation of D450 val-ues were less than 8％,respectively.The method established in this study and fluorescent PCR method were used to detect 225 clinical samples at the same time,and the total coincidence rate was 96.86％.The ELISA assay established in this study can be used for the detection of ASFV antigen with a large scale of samples,providing technical support for the prevention and control of ASF.
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