Development of recombinant SpaA-based ELISA for detection of antibodies against swine erysipelas

Abstract:

[Objective] In the present study, an indirect ELISA was developed using the purified recombinant SpaA protein for detection of the antibodies against Erysipelothrix rhusiopathiae. [Methods] Using gene recombination technology, the SpaA gene was cloned and inserted into prokaryotic expression vector pGEX-6P-1 and the recombinant expression vector was identified with PCR, restriction enzyme digestion and sequencing. The positive recombinant plasmid was then transformed into E. coli Rosetta(DE3) and induced by IPTG. The recombinant protein was detected by SDS-PAGE and Western-blot. An indirect ELISA was developed with the purified protein at different concentrations and the optimal antigen concentration and serum dilution were determined by phalanx titration. The other assay conditions were also optimized. [Results] The optimal coating concentration of SpaA and serum dilution were 1.0 mg/L and 1:100, respectively. Both the intra- and inter-coefficient of variation were lower than 10%, suggesting its reproducibility and repeatability. The developed indirect ELISA method was compared with the ELISA kit supplied by TSZ and Western blot, the coincidence rate was 92.20% and 92.59% respectively. [Conclusion] The developed ELISA had good specificity, reproducibility and sensitivity. It could provide a reliable method for the clinical detection of Erysipelothrix rhusiopathiae and epidemiological investigations．

Key words:Erysipelothrix rhusiopathiae, Recombinant surface protect antigen A Protein, Clone expression, Indirect ELISA, establish