Development of ELISA kits for antibodies against avian reovirus using the sigmaC and sigmaB proteins expressed in the methyltropic yeast Pichia pastoris.
Both the sigmaC and sigmaB proteins of avian reovirus (ARV) can induce type- and group-specific neutralizing antibodies, respectively. In this study, the full-length of S1133 sigmaC, 1071-1 sigmaC, S1133 sigmaB, and S1133 sigmaC-sigmaB fusion genes of ARV were cloned into a secreted vector pPICZalphaA and then integrated into the chromosome of Pichia pastoris for induced expression. Western blot assay showed that ARV sigmaC, sigmaB, and sigmaC-sigmaB fusion proteins were expressed and secreted into the medium. Two types of ELISA kits using equal mixtures of 1071-1sigmaC and S1133 sigmaB and S1133 sigmaC-sigmaB fusion proteins as antigens were developed. After a checker board titration for optimal conditions, the cut-off values of positive results for the 1071-1sigmaC/S1133 sigmaB and S1133 sigmaC-sigmaB 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.
Yang
Zi-Jiang
ZJ
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Neipu, Pingtung 912, Taiwan.
Wang
Chi-Young
CY
Lee
Long-Huw
LH
Chuang
Kuo-Pin
KP
Lien
Yi-Yang
YY
Yin
Hsien-Sheng
HS
Tong
De-Wen
DW
Xu
Xin-Gang
XG
Liu
Hung-Jen
HJ
D000818:Animals; D000914:Antibodies, Viral / Q000097:blood*; D000956:Antigens, Viral / Q000235:genetics; D001715:Bird Diseases / Q000175:diagnosis* / Q000821:virology; D002645:Chickens; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015870:Gene Expression; D030101:Orthoreovirus, Avian / Q000276:immunology*; D010843:Pichia / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012088:Reoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D012680:Sensitivity and Specificity
D000914:Antibodies, Viral; D000956:Antigens, Viral; D011993:Recombinant Fusion Proteins