Development of a stringent ELISA protocol to evaluate anti-viral hemorrhagic septicemia virus-specific antibodies in olive flounder Paralichthys olivaceus with improved specificity

Olive flounder were vaccinated with polyinosinic:polycytidylic acid [Poly (I:C)] to prevent viral hemorrhagic septicemia (VHS). Vaccine efficacy was verified by detection of anti- VHS virus (VHSV) antibodies using enzyme-linked immunosorbent assay (ELISA). In the study, ELISA absorbance values of the negative control group [Poly (I:C)-MEM10] were saturated when an ELISA protocol, that includes pretreatment of the fish sera with 5% skim milk, was used. However, the saturated OD values in the negative control did not correlate with a specific immune response against VHSV, because the group showed low survival rate (only 10%) following the VHSV challenge. Also, OD values of Poly (I:C)- VHSV group were high, and the group showed high survival rate (97.5%) against VHSV challenge test. It was suggested that the high OD values were possibly due to the presence of anti-fetal bovine serum (FBS) cross-reactivity. To compensate this, we subtracted the absorbance of infectious hematopoietic necrosis (IHNV)-Ag plates from those of the VHSV-Ag plates. However, the average value for the Poly (I:C)-VHSV group (0.167) was lower than expected even though high survival rate. We used an advanced ELISA system to pre-treat fish sera with 5% skim milk and two novirhabdoviruses as capture antigens as well as 50% FBS. The corrected absorbance values for pre-treated fish sera from the negative control Poly (I:C)-MEM10 and experimental Poly (I:C)-VHSV groups averaged 0.033 and 0.579, respectively. The specific VHSV antibody response of the vaccinated group was assessed using fish sera pretreated with skim milk and FBS and by calculating the corrected absorbance values from ELISA with two novirhabdovirus capture antigens.