title: A protein-conjugate approach to develop a monoclonal antibody-based antigen detection test for the diagnosis of human brucellosis.
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.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Kailash P
authors.initials: KP
authors.lastname: Patra
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Saito
authors.affiliation: Department of Medical Microbiology, University of California Davis, Davis, California, United States of America.
authors.forename: Vidya L
authors.initials: VL
authors.lastname: Atluri
authors.affiliation: Department of Medical Microbiology, University of California Davis, Davis, California, United States of America.
authors.forename: Hortensia G
authors.initials: HG
authors.lastname: Rolán
authors.affiliation: Department of Medical Microbiology, University of California Davis, Davis, California, United States of America.
authors.forename: Briana
authors.initials: B
authors.lastname: Young
authors.affiliation: Department of Medical Microbiology, University of California Davis, Davis, California, United States of America.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kerrinnes
authors.affiliation: Department of Biomedical Research, Royal Tropical Institute, Amsterdam, the Netherlands.
authors.forename: Henk
authors.initials: H
authors.lastname: Smits
authors.affiliation: Institute of Tropical Medicine Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Ricaldi
authors.affiliation: Institute of Tropical Medicine Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Gotuzzo
authors.affiliation: Department of International Health, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, United States of America; Laboratory of Research and Development, and Department of Cellular and Molecular Sciences, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Gilman
authors.affiliation: Department of Medical Microbiology, University of California Davis, Davis, California, United States of America.
authors.forename: Renee M
authors.initials: RM
authors.lastname: Tsolis
authors.affiliation: Division of Infectious Diseases, Department of Medicine, University of California San Diego, La Jolla, California, United States of America; Institute of Tropical Medicine Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru; Laboratory of Research and Development, and Department of Cellular and Molecular Sciences, Faculty of Sciences, Universidad Peruana Cayetano Heredia, Lima, Peru.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Vinetz
mesh_terms: D000328:Adult; D000818:Animals; D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000097:blood*; D002003:Brucella abortus / Q000737:chemistry / Q000302:isolation & purification*; D017347:Brucella melitensis / Q000737:chemistry / Q000302:isolation & purification*; D002006:Brucellosis / Q000175:diagnosis*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D008070:Lipopolysaccharides / Q000097:blood*; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D012680:Sensitivity and Specificity
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D008070:Lipopolysaccharides