Optimization of enzyme-linked immunosorbent assay for analyzing surface structures of nervous necrosis virus and...

Enzyme-linked immunosorbent assay (ELISA) is a quantitative immunoassay used to detect antigens and antibodies. In the field of fish diseases, the usefulness of ELISA has been largely overlooked due to its low reproducibility and high background optical density (OD). Nevertheless, ELISA is indispensable for evaluating specific immunity in vaccinated fishes, tracking infection history and analyzing the conformational structures and functions of the surface proteins of fish pathogens. Therefore, a highly quantitative and reproducible ELISA is important and necessary for fish disease research and diagnosis. In this technical review, we used nervous necrosis virus (NNV) as a model to compile the existing knowledge on improving background OD and increasing the reproducibility of ELISA. The issues associated with this method are primarily caused by non-specific reactions of immunoglobulins (Igs) to viral particles and changes in the aggregation states of viral particles. The countermeasures include methods for in vitro virus culture, purification of virus particles, immobilization of virus particle antigens and fish Igs on ELISA plate wells, consideration of physicochemical properties of virus particles and reaction order of the antigens and antibodies. This information will improve the detection of fish viruses other than NNV and specific antibodies against them using ELISA.