Cell Culture Medium Inhibits Antigen Binding Used in an ELISA for Detection of Antibodies against Nervous Necrosis Virus

Abstract

We investigated the optimum dilution of nervous necrosis virus (NNV) for use as antigens to detect antibodies by an enzyme-linked immunosorbent assay (ELISA) in the Sevenband Grouper Epinephelus septemfasciatus. The ELISA values for a standardized suspension of antigens diluted with L-15 medium containing 1% fetal bovine serum decreased gradually with the dilution of the antigens, whereas those for the antigens diluted with distilled water (DW) initially increased with the dilution of the antigens, peaked at a 320-fold dilution, and then decreased thereafter. Additional studies revealed that binding of NNV antigens to ELISA wells was inhibited by fetal bovine serum and other substances in the L-15 medium. Sera obtained from Sevenband Grouper vaccinated with live NNV vaccine and survivors from natural NNV-infection were subjected to antibody detection by ELISA. All of the sera were positive by ELISA when the standardized suspension was diluted 320-fold, whereas sera from five out of the six survivors and two out of the six vaccinated fish were negative or weakly positive by ELISA using NNV antigens diluted 10-fold. We therefore concluded that cultured NNV solutions prepared in cell culture media may need to be diluted with distilled water for use in ELISA. Received January 28, 2014; accepted April 16, 2014.

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

“…In contrast, assays in which the plate was coated directly with CCSN performed poorly, most likely due to inhibition of antigen binding by the L-15 medium components (Choi et al, 2014). As previously observed (Espelid et al, 1987), there was a good level of correlation between results for indirect sandwich and competitive ELISA.…”

Altered conformational structures of nervous necrosis virus surface protrusions and free coat proteins after incubation at moderate-low temperatures

“…Each heat-treated NNV suspension was subjected to titration of NNV infectivity and ELISA to detect NNV antigens. Prior to be their use as ELISA antigens, cultured NNV suspensions after heat-treatments were diluted 320-fold with DIW because adsorption of NNV antigens to ELISA plates could be inhibited by FBS and other components present in the culture medium 42 .…”

Viral encephalopathy and retinopathy in aquaculture: a review

“…The major reasons for this are poor knowledge on the kinetics of the antibody response in fish at various water temperatures and lack of validation data. Nevertheless, several ELISA or serum neutralization tests described and improved over time proved their efficiency to detect antibodies specific to VNN (Watanabe et al 1998;Huang et al 2001;Fenner et al 2006b;Scapigliati et al 2010;Choi et al 2014;Jaramillo et al 2016b). For ELISA tests, the determination of the cut-off point is critical to make the distinction between virus free status and viral infection.…”