Cloning and expression of immunoreactive Brucella melitensis 28 kDa outer membrane protein (Omp28) gene and evaluation of its potential for clinical diagnosis of brucellosis.

J Med Microbiol. 2010 Jan 14;

Authors: Thavaselvam D, Kumar A, Tiwari S, Mishra M, Prakash A

Brucellosis is a disease caused by bacteria belonging to the genus Brucella that are gram negative, facultative and intracellular. It is an emerging zoonosis and economically important infection of human and livestock with worldwide distribution. The human infection is known to occur through consumption of infected raw milk, milk products and undercooked or raw meat. The serodiagnosis of brucellosis is done by detection of antibodies generated against the LPS or whole cell bacterial extracts by ELISA or by agglutination tests using coloured bacterial antigens. The present study was designed to develop a highly sensitive and specific indirect ELISA in microtitre plate and dot blot format employing the recombinant Omp28 protein. The cloning and expression of Brucella melitensis Omp28 protein that belong to the group 3 antigen was accomplished as the gene encoding for this protein was PCR amplified and cloned in pET 28a expression system and the histidine tagged recombinant protein was purified by Ni-NTA affinity chromatography. The indirect ELISA in microtitre plate and dot blot format was optimized with sera collected from three groups namely culture confirmed, clinically suspected and from healthy individuals. The rOmp28 protein reacted only with the culture confirmed positive samples and no reaction was observed with the culture negative samples confirming the immuno-reactivity of the recombinant protein. The test in both the formats had a correlation around 90% with RBPT and STAT, the agglutination assays that are routinely performed for the serodiagnosis of brucellosis. The sensitivity and specificity of the assay in plate format was of 97.50 and 85.59% and in dot blot format it was 82.05% and 92.41% respectively in comparison with RBPT. Similarly, very high sensitivity and specificity was also observed in the dot blot format. The specificity of this assay was further confirmed by testing on samples that are positive for malaria and typhoid. This ELISA system on microtitre plate and dot format will be highly useful in rapid screening of large number of samples for the diagnosis of human brucellosis in the endemic areas.

PMID: 20075115 [PubMed - as supplied by publisher]