Development of an indirect ELISA diagnostic method for detecting avian metapneumovirus using recombinant attachment protein and its preliminary application

The ELISA plate was coated with the purified avian metapneumovirus(aMPV) vaccine strain(VIR-115 B) recombinant attachment(G) antigen and the optimal reaction conditions of indirect ELISA were determined by experiments.The indirect ELISA method for detecting aMPV antibody was established by determining the cut off value.The recombinant E.coli strain Rosetta containing the recombinant plasmid pET-32α-G was induced by IPTG.The recombinant G protein was expressed at high level,and then it was purified by affinity chromatography method.The recombinant G protein was identified by Western-blotting.Taking the purified recombinant G protein as diagnostic antigen,the indirect ELISA method for detecting aMPV antibody was established.The recombinant G protein was successfully expressed and purified,it was proved that the recombinant protein has good reactogenicity by Western-blotting.Taking the purified recombinant G protein as diagnostic antigen,the indirect ELISA method for detecting aMPV antibody was established.The results showed that there was no cross reaction between the recombinant G protein and the positive serum of NDV,H9N2,IBV and IBDV.Compared with the aMPV antibody detection kit of IDEXX in French,the results indicated that the specificity,sensitivity and coincidence of the developed indirect ELISA was 98.3%,92.5% and 96.0%,respectively.A total of 1 139 sera of chicken,which obtained from seven cities in Shandong province,were tested by the developed indirect ELISA method and the positive rate was 37.05%.The developed indirect ELISA has good specificity,sensitivity and reproducibility,it proved that aMPV existed in some areas in Shandong province.