Development of ELISA kits for antibodies against avian reovirus using the σC and σB proteins expressed in the methyltropic yeast Pichia pastoris

Both the σC and σB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 σC, 1071-1 σC, S1133 σB, and S1133 σC–σB fusion genes of ARV were cloned into a secreted vector pPICZαA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV σC, σB, and σC–σB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1σC and S1133 σB and S1133 σC–σB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1σC/S1133 σB and S1133 σC–σB ELISA kits were 0.24 and 0.12, respectively. Forty-four serum neutralization test-positive and twenty-eight serum neutralization-negative samples from vaccinated and commercial farm chickens were tested by the new ELISA kits and by the conventional ELISA. The new ELISA kits have higher positive rates than the conventional ELISA. The results revealed that the correlation rates for the serum neutralization titer and the absorbance values with the new ELISA kits and the conventional ELISA were 100% and 95.8%, respectively.