Development of a Highly Sensitive ELISA for Detecting Antibodies Against a Novel Variant Avian Reovirus Based on Dual σC and σB Antigens

This study developed a dual-antigen enzyme-linked immunosorbent assay (ELISA) based on σB protein and genotype 5-specific σC protein of avian reovirus (ARV). First, σB and σC proteins were expressed and purified using recombinant technology. Through optimization of coating conditions, the optimal antigen combination was determined to be a mixture of the two proteins at a 1:3 molecular ratio (total concentration: 0.8 μg/mL). Key parameters of the indirect ELISA were optimized via checkerboard titration. Validation confirmed that the dual-antigen ELISA exhibited a sensitivity of 1:3200 against genotype 5 ARV-positive sera, with no cross-reactivity and a coefficient of variation of 2.9-8.6%, demonstrating excellent reproducibility. In application testing, the method specifically detected serum antibodies against genotype 5 ARV variant strains, achieving a 100% positive detection rate in experimental chickens within the first week post-challenge and effectively monitoring dynamic antibody changes in infected flocks. Furthermore, the detection rate for genotype 5-positive serum samples (100%) was significantly higher than that of a commercial kit (75%). This dual-antigen indirect ELISA overcomes the sensitivity limitations associated with conventional genotype 5 ARV detection methods and provides a reliable tool for epidemiological surveillance and infection monitoring.