Reducing background optical density in enzyme-linked immunosorbent assay for detecting nervous necrosis virus (NNV)-specific IgM by immobilizing fish sera

Enzyme-linked immunosorbent assay (ELISA) for detecting fish antibodies is problematic due to its low reproducibility and high background optical density (OD). In this study, we investigated problematic sources using nervous necrosis virus (NNV, a fish pathogenic virus belonging to the genus Betanodavirus) and sevenband grouper (Hyporthodus septemfasciatus) as a model for detecting specific fish IgM through ELISA. It was revealed that both fish IgM and mammalian immunoglobulins were non-specifically adsorbed to purified NNV particles. This could be the problematic source of the high background OD and low reproducibility in ELISA. ELISA values of naive fish IgM non-specifically adsorbed to NNV particles immobilized onto ELISA plate wells ranged from 0.09 to 0.15 (high background OD). However, ELISA values of NNV antigens non-specifically adsorbed to naive fish IgM immobilized onto ELISA plate wells were all< 0.03 (low background OD). Thus, we developed a sandwich ELISA by immobilizing fish sera. NNV-specific antibodies could be indirectly detected by detecting NNV antigens captured by fish IgM immobilized onto ELISA plate wells using anti-NNV serum. When anti-NNV and naive fish sera were subjected to such sandwich ELISA, ELISA values of anti-NNV fish sera ranged from 0.24 to 2.48. Its reproducibility was sufficiently secured based on results obtained from five experiments performed on different days. Conversely, ELISA values of naive fish sera were all< 0.02, suggesting that background OD was completely regulated. The present sandwich ELISA does not require antiserum against fish IgM, meaning that NNV-specific antibodies are detectable from any fish species using only one antiserum against NNV.