Development and application of a Saccharomyces cerevisiae-expressed nucleocapsid protein-based enzyme-linked immunosorbent assay for detection of antibodies against infectious bronchitis virus

Abstract

A Saccharomyces cerevisiae-expressed nucleocapsid (N) polypeptide of the M41 strain of infectious bronchitis virus (IBV) was used as antigen in a recombinant yeast-expressed N protein-based enzyme-linked immunosorbent assay (Y-N-ELISA). The Y-N-ELISA was rapid, sensitive, and specific for detecting chicken serum antibodies to IBV, and it compared favorably with a commercial ELISA.

FIG. 1.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (A) and Western blot analysis (B) of recombinant nucleocapsid protein of M41 IBV expressed in S. cerevisiae. Lanes: 1, molecular size marker; 2, proteins of M41 IBV; 3, recombinant IBV N protein purified by nickel-agarose column chromatography; 4, crude protein extract of transformed S. cerevisiae. Arrows indicate the natural IBV N protein (a) and yeast-expressed recombinant N protein (b).

FIG. 2.

Detection of IBV-specific antibodies in hyperimmune chicken serum by Y-N-ELISA. Microplates were coated with different concentrations of the recombinant IBV N protein and reacted with serial twofold dilutions of M41 IBV-positive and -negative chicken serum.

FIG. 3.

IBV antibody levels (mean S/P values) detected by Y-N-ELISA in sera of chickens infected with M41 IBV and infected 7 weeks later (arrow) with homologous virus.

FIG. 4.

Correlation between antibody levels (S/P values) measured by Y-N-ELISA and a commercial ELISA kit.