An indirect enzyme-linked immunosorbent assay (iELISA) was established to detect the serological prevalence of bluetongue virus (BTV) infection in ruminant populations. A recombinant NS4 (rNS4) protein was used as the encapsulated antigen. Optimization of the iELISA included the encapsulated antigen, serum dilution, blocking solution, and working concentration of a horseradish peroxidase (HRP)-labeled secondary antibody (Ab) by the square-matrix titration test. The results showed that the optimal reaction conditions for the iELISA were: volume of coating buffer, 2.5 μg/mL; serum dilution, 1:200; blocking solution, 0.25% polyvinyl alcohol + 5% skim milk powder; working concentration of the HRP-labeled secondary Ab, 1:4000; and critical values, sample-to-positive ratios 0.26 and 0.38 for determining positivity and negativity, respectively. The detection sensitivity of the iELISA was up to 1:4000, the intra- and inter-batch reproducibility coefficients of variation were less than 10%, and the positive and negative concordance rates of 56 bovine serum samples were both 100%, confirming excellent sensitivity and reproducibility. The proposed iELISA should prove useful for the diagnosis of BTV infection and future epidemiological investigations.
Clinical immunology, Immunochemistry, Immunochemistry, Immunomodulation, Immunotherapy