Purification of epizootic haematopoietic necrosis virus and its detection using ELISA

Epizootic haematopoietic necrosis virus (EHNV) was grown in Bluegill fry (BF-2) cells and purified using differential and gradient centrifugation. The lower band (B2) from 15-60% sucrose gradients contained infective EHNV but few contaminating cell components when assessed by electron microscopy, SDS-PAGE, and Western blotting using anti-BF-2 serum and anti-B2 serum. Both rabbit and sheep anti-B2 sera precipitated B2 in agarose gel immunodiffusion and detected EHNV in cell culture supernatant when used in an indirect antigen-capture ELISA. Rabbit anti-B2 serum was used as capture antibody while sheep anti-B2 serum was used to detect viral antigen. Pre-adsorption of diluted sheep anti-B2 serum using BF-2 cell lysate greatly improved the specificity and sensitivity of the technique.