To establish an indirect enzyme-linked immunosorbent assay(ELISA)for detecting antibodies against Senecavirus A(SVA),this study utilized an insect baculovirus expression system to express the major structural protein and protective antigen VP2 of SVA.The immunoreactivity of VP2 was confirmed by indirect immunofluorescence assay(IFA),and the recombinant VP2 protein was purified using nickel-nitrilotriacetate(Ni-NTA)resin.Purified VP2 protein was used as the coating antigen,and the optimal antigen coating concentration and serum dilution were determined through chessboard titration.By optimizing the reaction conditions and determining the cut-off value,an indirect ELISA method for detecting SVA antibodies was established,and its specificity,sensitivity,repeatability,and coincidence rate with IFA were evaluated.The results demonstrated that the recombinant VP2 protein was solubly expressed in baculovirus-infected Sf9 cells and recognized by the SVA VP2-specific monoclonal antibody 2F5.After Ni-NTA purification,the recombinant VP2 protein of approximately 31 kDa was obtained.The optimized ELISA conditions included an antigen coating concentration of 2 μg/mL,serum dilution of 1∶100,and secondary antibody dilution of 1∶10 000.The cut-off value for positive/negative discrimination was 0.287.No cross-reactivity was observed with antisera against foot-and-mouth disease virus,porcine reproductive and respiratory syndrome virus,porcine epidemic diarrhea virus,porcine deltacoronavirus,pseudorabies virus,or porcine circovirus type 2.Intra-and inter-assay coefficients of variation were both below 10%.When applied to 52 porcine serum samples stored in the laboratory,the developed ELISA showed a 96.15%coincidence rate with IFA results(50/52).In conclusion,this study successfully established an indirect ELISA,providing a robust tool for SVA antibody detection and epidemiological investigations.
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