Development of ELISA kits for antibodies against avian reovirus using the sigma C and sigma B proteins expressed in the methyltropic yeast Pichia pastoris

Abstract

Both the sigma C and sigma B proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigma C, 1071-1 sigma C, S1133 sigma B, and S1133 sigma C-sigma B fusion genes of ARV were cloned intoa secreted vector pPICZ alpha LA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigma C, sigma B, and sigma C-sigma B fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1 sigma C and S1133 sigma B and S1133 sigma C-sigma B fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1 sigma C/S1133 sigma B and S1133 sigma C-sigma B 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. (C) 2009 Elsevier B.V. All rights reserved.