Preparation of Polyclonal Antibody and Development of an Indirect ELISA Method for Senecavirus A VP2 Protein
[Objective]This study was aimed to prepare polyclonal antibodies(pAbs)against the VP2 protein of Senecavirus A(SVA),and establish an indirect ELISA method to specifically detect SVA antibodiy,so as to provide a research basis for the pathogenesis and diagnosis of SVA.[Method]VP2 gene was cloned into the prokaryotic expression vector pET-28a by homologous recombination,the recombinant expression plasmid pET-28a-VP2 were designated and transformed into Escherichia coli BL21(DE3)competent cell for IPTG induced expression.The New Zealand White rabbits were subcutaneously immunized with the purified recombinant protein to prepare its pAbs.The specificity,reactivity and neutralizing activity of pAbs were analyzed via indirect immunofluorescence assay(IFA),Western blotting and neutralization test.An indirect ELISA based on VP2 coating antigen was established by matrix optimization,determination of cut-off value,specificity identification,sensitivity and repeatability analysis.A total of 50 serum samples were collected,which were tested by indirect ELISA and IFA to analyze the coincidence rate.[Result]The recombinant VP2 protein was expressed in the form of inclusion body with a size of 40 ku.The pAbs could specifically react with recombinant VP2 protein and SVA,and had no cross-reactivity with other viruses.Moreover,the pAbs had a higher neutralizing activity.After optimizing the conditions of indirect ELISA,the optimal VP2 coating concentration was measured at 4 μg/mL,and optimal serum sample dilution was 1∶250.Furthermore,the best blocking solution was selected as 5％skimmed milk+5％BSA.The optimal reaction time for serum,secondary antibodies and TMB solution were 90,90 and 10 min,respectively.Finally,the value of cut-off was calculated to 0.182.No cross-reactivity was observed with other virus antibody-positive pig sera,and indirect ELISA showed a 94.0％coincidence rate compared with IFA.[Conclusion]VP2 protein expressed in the prokaryotic expression system had good immunogenicity,and the prepared pAbs could react specifically with SVA and recombinant VP2 protein.The established indirect ELISA had high specificity and consistency with IFA,which could be suitable for clinical SVA antibody detection and vaccine efficacy evaluation.
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