Establishment of indirect ELISA for detecting avian encephalomyelitis virus with VP1 as coating protein

Prokaryotic expression of VP1 gene of AEV Shaanxi strain was done.SDS-PAGE and Western blotting were employed for analyzing the recombinant protein.The purified recombinant protein was used for establishment of an indirect ELISA for detecting of anti-AE antibody.The results showed that target fusion protein was successfully expressed in E.coli,it was about 50 000 in molecular weight and has a good reactogenicity;the optimal reaction condition were determined:coating antigen at 4℃ overnight after 37℃ for 2 h at a concentration of 3.8 mg/L,1%BSA as blocking agent at 37℃ for 2 h,serum sample(1∶50) at 37℃ for 30 min,HRP labeled goat anti-chicken(1∶4 000) at 37℃ for 30 min,the substrate TMB for ELISA being incubated at 37℃ for 5 min;the threshold of positive and negative sera was 0.360 by statistical analysis.The ELISA assay was confirmed to have a good repeatability,sensitivity and specificity.The coincidence rate of the two assays was up to 91.9% compared with the bird AE-Ab ELISA kit of IDEXX by detecting 180 clinical sera.The method can be used for the detection of large quantities of clinical samples.