Development of an indirect ELISA method based on the VP3 protein of duck hepatitis A virus type 1 (DHAV-1) for dual detection of DHAV-1 and DHAV-3 antibodies.

An indirect enzyme-linked immunosorbent assay (I-ELISA) based on the recombinant VP3 protein of duck hepatitis A virus type 1 (DHAV-1) was developed and evaluated in this study. The optimal antigen, serum and enzyme-labeled antibody dilutions were 1:160 (0.94μg), 1:160 and 1:2000, respectively. The optimal blocking buffer was 1% gelatin. The cutoff value was determined to be 0.332, and the analytical sensitivity was 1:1280 (OD450-630=0.37). The results of the specificity evaluation showed that no cross-reactivity existed between DHAV-1 antiserum and other common duck-sensitive pathogens, except for duck hepatitis A virus type 3 (DHAV-3), suggesting that this could be a common approach for the simultaneous detection of DHAV-1 and DHAV-3 antibodies. The coefficients of variation (CVs) for all of the tested samples were lower than 10%. The concordance between the I-ELISA based on the VP3 subunit of DHAV-1 and that based on the whole DHAV-1 particle was 96%. These results indicate that the VP3-based I-ELISA method has high sensitivity, specificity, and repeatability and is as effective as the DHAV-1-based I-ELISA method for sero-surveillance. Thus, it may be a convenient and novel method for DHAV antibody detection and epidemiological surveillance of DHAV prevalence.