Development of an Indirect ELISA for the Detection of ARV Epidemic Strain xj-1.1

This study aimed to establish an indirect ELISA for detecting the avian reovirus (ARV) epidemic strain xj-1.1 by using the purified recombinant protein pET-σC as the coating antigen. To optimize assay performance, key parameters were systematically evaluated, including antigen-coating concentration, serum dilution, blocking reagent and duration, serum incubation time, and the dilution and reaction time of the HRP-conjugated secondary antibody. The optimized conditions identified were a coating antigen dilution of 1:100, serum dilution of 1:1600, coating at 37 °C for 1 h followed by overnight incubation at 4 °C, and blocking with 5% skim milk for 2 h. The optimal serum incubation time was 1.5 h, with the secondary antibody diluted 1:1000 and incubated for 2 h, followed by a 20-min color development step. The cut-off value for distinguishing positive and negative samples was determined to be 0.121. Validation of the assay demonstrated favorable specificity, sensitivity, and repeatability, indicating that the developed indirect ELISA provides a reliable method for detecting ARV xj-1.1 infection.