Development and application of a recombinant protein-based indirect ELISA for detection of anti-tilapia lake virus IgM in sera from tilapia

Abstract

Serological assays for indirect screening of tilapia lake virus (TiLV) are currently not available. We examined the potential use of an antigenic protein encoded by genome segment 8 of TiLV, tentatively called S8 protein, as a coating antigen for development of indirect enzyme-linked immune-sorbent assay (iELISA) for the detection of antibody against TiLV. After optimization of antigen and antibody concentration, determination of a cutoff value, reproducibility, sensitivity and specificity of the iELISA were evaluated. We found that the optimal concentration of the coating S8 protein antigen was 1.0 μg/mL with a secondary antibody dilution of 1:10.000, and the cut-off value was defined to be 0.258 OD units. The iELISA also showed high analytic specificity and was able to distinguish between antibodies against TiLV and other pathogens. It was more sensitive than the current IFA technique for detecting TiLV. In addition, for detection of artificial infection samples, the results showed that the diagnostic sensitivity and specificity of the iELISA compared with IFA was 100% and 92.6%, respectively; while the diagnostic sensitivity and specificity of the iELISA versus semi-nested PCR was 80.8% and 95.6%, respectively. A serological survey was performed using the iELISA with tilapia sera samples from the field compared with an unknown status regarding TiLV, 13/73 serum samples tested positive by iELISA, indicating a 17.8% TiLV antibody positives. In conclusion, our newly developed iELISA was specific and sensitive, and it may be useful for large-scale investigations of TiLV infections and monitoring trials of future vaccination protocols.