Abstract:

The aim of this study was to establish an indirect ELISA to rapidly detect antibody of genotype C duck hepatitis A virus (DHAV-C).An indirect ELISA based on DHAV-C VP1 recombinant protein as coating antigen was established for rapid detection of DHAV antibody.The reaction conditions were optimized，including 5 μg•mL^－1 VP1 recombinant protein as coating antigen，1:100 dilution of testing serum and 1:5000 dilution of HRP conjugated goat anti-duck IgG with cut-off value 0.474 (OD_{450 nm}).The pH 9.6，0.05 mol•L^－1 bicarbonate buffer was used as coating antigen dilution buffer，the sealing buffer was 0.01 mol•L^－1 PBS buffer containing 10% skimmed milk，and the dilution buffer was PBS buffer containing 1% BSA.The coating condition was at 4 ℃ for a whole night，antibody reacted with coating antigen for 60 min，HRP conjugated goat anti-duck IgG incubated for 30 min and substrate reaction time was 15 min.The specific test showed that there were no cross reactions with positive sera against duck circovirus，duck plaque virus，duck hepatitis B virus，Newcastle disease virus，avian influenza virus，duck E.coli，duck Staphylococcus aureus，duck Salmonella anatis，duck Pasteurella multocida and Riemerella anatipestifer.However，DHAV-A antibody showed cross reaction with DHAV-C VP1 recombinant protein.The inter- and intra -assay demonstrated that the co-efficient variations were 2.03%-2.95% and 3.28%-7.38%，respectively.Compared with neutralization test，the detection coincidences of positive and negative sera were 80% and 100%，respectively.This method had been successfully used to detect maternal antibody and the growth and decline of immune antibody.The results revealed that the indirect ELISA could be used for sero-epidemiological survey and antibody detection of duck hepatitis A disease caused by DHAV-A or DHAV-C.