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.
Steiner
K A
KA
Elizabeth Macarthur Agricultural Institute, New South Wales Agriculture and Fisheries, Camden, Australia.
Whittington
R J
RJ
Petersen
R K
RK
Hornitzky
C
C
Garnett
H
H
D000818:Animals; D000914:Antibodies, Viral; D000956:Antigens, Viral / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005393:Fish Diseases / Q000175:diagnosis; D005399:Fishes; D007497:Iridoviridae / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D008854:Microscopy, Electron; D014773:Virology / Q000379:methods*; D014777:Virus Diseases / Q000175:diagnosis / Q000662:veterinary
D000914:Antibodies, Viral; D000956:Antigens, Viral