Development of indirect ELISA for detecting antibodies against multi-serotypes of Riemerella anatipestife

Abstract:

[Objective] Riemerella anatipestifer (RA) is an important poultry pathogen. Considering the twenty-one serotypes, it still lacked a method for detecting multi-serotypes antibodies. Previous studies showed that the Outer membrane protein A (OmpA), widely existing in multi-serotypes of RA strains, is a protein with immunogenicity. Its encoding gene is highly conserved among the genomes of RA of different serotypes, which indicates that OmpA can be used as a target molecule for serum antibody detection of RA infection. An indirect ELISA method to detect the antibody caused by RA infection was developed with the recombinant OmpA as the coating antigen. [Methods] The recombinant OmpA was purified after optimizing the prokaryotic expression conditions, and its immuno reactivity was tested by Western-blot with sera obtained from ducks infected by different serotypes of RA. Furthermore, the optimum testing conditions of ELISA were determined by phalanx test. Reproducibility, specificity and sensitivity tests were performed to estimate the practicability. [Results] The recombinant protein would be more soluble by adding 1% of ethanol in the medium. As analysed by Western-blot, the purified protein is immunogenic among 1, 2, 6, 10, 11, 13, 14, 17 serotypes of RA. Phalanx test results showed the optimal concentration of coated antigen is 8 mg/L, and the optimal dilution degree of serum is 1:160. Also, the developed assay was proved to be quite repetitive, specific and sensitive. [Conclusion] An indirect ELISA method for detecting the infection of multi-serotypes of Riemerella anatipestifer was established in this study and could be used for evaluating the immune effects of RA vaccine and for diagnosing RA infections with different serotypes of RA.

Key words:Riemerella anatipestifer, OmpA, multi-serotypes, indirect ELISA