Abstract
Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases.
Highlights
Human brucellosis is most commonly caused by two species of the genus Brucella, typically B. abortus from cattle and B. melitensis from goats and sheep
Brucellosis is a OneHealth disease reflecting the risk for human infection by interaction with and relation to affected animal populations
We have developed the basis for developing a new test based on the detection of the B. melitensis lipopolysaccharide, which provides rapid and definitive identification of the presence of the organism in clinically obtainable body fluids
Summary
Human brucellosis is most commonly caused by two species of the genus Brucella, typically B. abortus from cattle and B. melitensis from goats and sheep. Because culture is technically challenging and hazardous in many clinical laboratories, brucellosis is most commonly diagnosed using serological methods that use fixed, whole Brucella abortus as antigen [17,18,19,20,21]. Such methods include the Rose Bengal, slide agglutination, and tube agglutination tests, sometimes accompanied with the use of 2-mercaptoethanol to distinguish IgG from IgM antibodies when determining the presence of active infection requiring antibiotic therapy; newer data obtained using genomelevel screens suggest the potential utility of recombinant B. melitensis proteins for characterization of human infection [22,23,24]. False positive serological results may be found with other
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