An indirect enzyme-linked immunosorbent assay (iELISA) was developed for detecting antibody to duck swollen head hemorrhagic disease virus (DSHDV) using purified whole virus by sucrose density gradient ultracentrifugation as a coating antigen. Antiserum against DSHDV strains HY-99 (hyperimmume serum) was prepared in 30-day-old ducks by vaccination with inactivated DSHDV and used as positive sera. The iELISA test was optimized with different reagents or dilutions. The validation results showed that this assay was only specific for antibodies against duck viral swollen head hemorrhagic disease. The OD450 value for positive serum diluted 1:800 was also determined to be greater than the positive threshold. The highest coefficient of variation value was 3.66% for the intra-assay and 5.79% for the interassay, which were all less than 10%. This assay has been successfully applied to the examination of the duck sera clinically. These results in this study indicate that the newly-developed iELISA offers a precise, specific, sensitive, and reproducible means of measuring DSHDV antibodies in duck sera.
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