A novel, rapid and simple magnetic bead-based assay for the diagnosis of human and animal brucellosis.

CIOCCHINI, ANDRÉS E.; REY SERANTES, DIEGO; MELLI, LUCIANO; IWASHKIW, JEREMY; ELENA, SEBASTIAN; DEODATO, BETTINA; NICOLA, AM; WALLACH, JORGE; FELDMAN, MARIO F; UGALDE, JUAN E; COMERCI, DIEGO J

Buenos Aires

Congreso; Brucellosis 2011, International Research Conference. Including the 64th Brucellosis Research Conference; 2011

Asociación Argentina de Microbiología

Brucellosis is a highly contagious zoonosis affecting livestock and human beings. Laboratory testing is essential for diagnosis of human and animal brucellosis. However, most currently available diagnostic tools are difficult to implement in certain areas and countries where brucellosis is endemic. In the present work, we present an indirect immunoassay to detect antibodies against "smooth" Brucella spp. using as a novel antigen a recombinant O-polysaccharide-protein conjugate (AcrA-OAg) coupled to magnetic beads. AcrA-OAg was produced in Escherichia coli by co-expressing PglB, a Campylobacter jejuni oligosaccharyltransferase, the gene cluster encoding the enzymes required for the synthesis of Brucella abortus O-polysaccharide and the protein acceptor AcrA. Introduction of PglB in E. coli results in the transfer of B. abortus O-polysaccharide from its carrier to AcrA. This procedure is very advantageous because the AcrA-OAg glycoprotein, expressed as an histidine-tag fusion protein, can be purified from E. coli fermentations without the need for culturing the pathogenic bacteria. Furthermore, no chemical treatments are required for the isolation of the O-polysaccharide. AcrA-OAg was purified in one-step by affinity chromatography and immobilized on carboxi-modified magnetic beads. Functionalized micro-beads were incubated with the samples and the immune complexes were detected using anti-human or anti-bovine IgM/G Cy5 conjugate antibodies. To evaluate the usefulness of our immunoassay for the diagnosis of human brucellosis we have tested sera of patients with no brucellosis, culture-positive brucellosis patients, serologically-positive patients with clinical diagnosis of brucellosis, and healthy persons exposed to the pathogen. Additionally, to evaluate the utility of the assay to diagnose bovine brucellosis we have analyzed serum, whole blood and milk samples from non-infected, experimentally infected and vaccinated animals. Our data demonstrate that this immunoassay can clearly differentiate infected from non-infected patients as well as infected from non-infected and vaccinated animals. We propose the use of this technology for the development of a Point Of Care immune-diagnosis for human and bovine brucellosis.