Epizootic haematopoietic necrosis virus (EHNV): improved ELISA for detection in fish tissues and cell cultures and an efficient method for release of antigen from tissues.
An improved antigen capture ELISA for the detection of epizootic haematopoietic necrosis virus (EHNV) is described. Affinity purified rabbit anti-EHNV immunoglobulin was used as capture antibody, enabling the detection of EHNV at a concentration of 10(3.5) TCID50/ml cell culture supernatant. The sensitivity and specificity of the ELISA, determined by examination of clarified tissue homogenates from 55 infected fish and 348 uninfected fish in relation to virus isolation, were 81.2% and 98.9%, respectively. Specificity for redfin perch (Perca fluviatilis) tissue samples was 100%, however, high background optical density (OD) in samples from uninfected rainbow trout (Oncorhynchus [corrected] mykiss) resulted in lower specificity (98.5%). Specificity for rainbow trout tissues was increased to 100% by testing clarified tissue homogenates that had been diluted 1:10. Sensitivity and specificity estimates for cell culture supernatants were 96.2% and 99.9% based on the results obtained for 158 infected and 355 uninfected samples. Incubation steps in the ELISA can be shortened to enable its completion in less than 4 h if a rapid qualitative diagnosis is required. Of eight methods of releasing EHNV from infected tissue that were evaluated, manual grinding of tissue with a fitted pestle in a 1.5-ml plastic disposable tube, followed by vortexing in the same tube with 3 mm diameter glass beads and clarification in a microcentrifuge was the most efficient. Samples from 150-200 individual fish could be prepared for ELISA and cell culture by a single operator in a day. Antigen capture ELISA can replace virus isolation as the method of choice for diagnosis of EHNV.
Whittington
R J
RJ
NSW Agriculture, Microbiology and Immunology Laboratory, Elizabeth Macarthur Agricultural Institute, Camden, Australia.
Steiner
K A
KA
D000818:Animals; D000956:Antigens, Viral / Q000302:isolation & purification*; D002478:Cells, Cultured; D003531:Cypriniformes / Q000382:microbiology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005393:Fish Diseases / Q000175:diagnosis*; D010471:Perches / Q000382:microbiology; D012680:Sensitivity and Specificity; D014337:Trout / Q000382:microbiology; D014780:Viruses / Q000302:isolation & purification*
D000956:Antigens, Viral