Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae

Document Type: Full paper (Original article)

Authors

1 College of Life Science, Inner Mongolia University for Nationalities, Tongliao 028043, China

2 Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, Shandong Province, China; E. coli Reference Center, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park 16803, Pennsylvania, USA

3 E. coli Reference Center, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park 16803, Pennsylvania, USA

4 Shandong Binzhou Animal Science and Veterinary Medicine Academy, Binzhou 256600, Shandong Province, China

Abstract

The sip gene encoding for a conserved highly immunogenic surface protein of Streptococcus agalactiae was amplified using polymerase chain reaction (PCR) and subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in E. coli BL21 (DE3). An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified Sip protein as a coating antigen, which could identify S. agalactiae specific antibody in sera. The coating antigen at a concentration of 3.125 μg/ml, serum diluted to 1:160, and HRP-conjugated secondary antibody concentration at 1:4000 was found to be most effective
in exhibiting positive result. The ELISA was found to be highly specific for S. agalactiae that may be used for the detection of the pathogen in mastitis cases, for epidemiological studies and for surveillance.

Keywords