Cloning and expression of the immunoreactive Brucella melitensis 28 kDa outer-membrane protein (Omp28) encoding geneand evaluation of the potential of Omp28 for clinical diagnosis of brucellosis

Brucellosis is a disease caused by Gram-negative, facultative, intracellular bacteria belonging to the genus Brucella. It is an emerging zoonosis, and an economically important infection of humans and livestock with a worldwide distribution. Human infection is known to occur through consumption of infected raw milk, milk products and undercooked or raw meat. Serodiagnosis of brucellosis is carried out by detection of antibodies generated against LPS or whole-cell bacterial extracts by ELISA or agglutination tests using colorimetry. The present study was designed to develop a highly sensitive and specific indirect ELISA in both a microtitre plate and dot-blot format employing the recombinant outer-membrane protein 28 (rOmp28). Cloning and expression of Brucella melitensis Omp28 protein, which is a group 3 antigen, was accomplished by PCR amplification and cloning of the gene in a pET-28a expression system, followed by Ni-NTA affinity chromatography purification of the His-tagged recombinant protein. An indirect ELISA in both a microtitre plate and dot-blot format was optimized with sera collected from three groups: culture-confirmed cases, clinically suspected cases and healthy individuals. The rOmp28 protein reacted only with the culture-confirmed positive samples and no reaction was observed with culture-negative samples, confirming the immunoreactivity of the recombinant protein. The test in both formats had a correlation of approximately 90 % with the Rose Bengal plate agglutination test (RBPT) and a standard tube agglutination test, assays that are routinely performed for the serodiagnosis of brucellosis. The sensitivity and specificity of the assay in the plate format were 97.50 and 85.59 %, and in the dot-blot format were 82.05 and 92.43%, respectively, in comparison with RBPT. The specificity of this assay was further confirmed by testing samples that were positive for malaria and typhoid, which gave negative results. This ELISA system in microtitre plates and a dot-blot format will be useful for the rapid screening of large numbers of samples for the diagnosis of human brucellosis in endemic areas.