Comparison of ELISA formats for detection of antibodies specific for nervous necrosis virus (Betanodavirus) in the serum of immunized barramundi Lates calcarifer and Australian bass Macquaria novemaculeata

Addressing the need for validated tests for the study of Nervous Necrosis Virus (NNV, Betanodavirus) immunity, ELISA protocols were developed, optimized and compared for detection of serum antibodies against the virus in vaccinated fish. Competitive and indirect ELISA were performed using several combinations of immunoreagents, pursuing the highest specific reactivity. Reference sera were obtained by immunizing and longitudinally sampling Australian bass Macquaria novemaculeata (n=10) and barramundi Lates calcarifer (n=5) with native viral or recombinant coat protein antigens. Optimal performance was achieved with an indirect sandwich ELISA using sheep anti-NNV antibodies and non-purified live NNV from cell culture supernatant as the capture system, and rabbit anti-fish immunoglobulin antibodies for the detection system. This optimized format had greater analytical sensitivity than competitive ELISA. An increase in reactivity of 3.5–8.7 times was detected post-immunization (P<0.001), and titres persisted for at least 28weeks. The assay discriminated non-specific reactions and was considered practical for routine use as it does not require purified virus or recombinant NNV capsid protein (rCP) as antigen. Moreover, the format was versatile as it could be adapted to a wider range of fish species. We believe that this optimized assay is suitable for the study of NNV vaccinal responses and has the potential to be used for NNV surveillance in subclinically infected fish populations after further validation.