title: Development of Immunoassays forBurkholderia pseudomalleiTypical and Atypical Lipopolysaccharide Strain Typing.
abstract: Burkholderia pseudomalleiis the causative agent of melioidosis, a severe infection endemic to many tropical regions. Lipopolysaccharide (LPS) is recognized as an important virulence factor used byB. pseudomalleiIsolates ofB. pseudomalleihave been shown to express one of four different types of LPS (typical LPS, atypical LPS types B and B2, and rough LPS) and in vitro studies have demonstrated that LPS types may impact disease severity. The association between LPS types and clinical manifestations, however, is still unknown, in part because an effective method for LPS type identification is not available. Thus, we developed antigen capture immunoassays capable of distinguishing between the LPS types. Mice were injected with B or B2 LPS for atypical LPS-specific monoclonal antibody (mAb) isolation; only two mAbs (3A2 and 5B4) were isolated from mice immunized with B2 LPS. Immunoblot analysis and surface plasmon resonance demonstrated that 3A2 and 5B4 are reactive with both B2 and B LPS where 3A2 was shown to possess higher affinity. Assays were then developed using capsular polysaccharide-specific mAb 4C4 for bacterial capture and 4C7 (previously shown to bind typical LPS) or 3A2 mAbs for typical or atypical LPS strain detection, respectively. The evaluations performed with 197 strains ofBurkholderiaand non-Burkholderiaspecies showed that the assays are reactive toB. pseudomalleiandBurkholderia malleistrains and have an accuracy of 98.8% (zero false positives and two false negatives) for LPS typing. The results suggest that the assays are effective and applicable forB. pseudomalleiLPS typing.
authors.affiliation: Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: Teerapat
authors.initials: T
authors.lastname: Nualnoi
authors.affiliation: Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida.
authors.forename: Michael H
authors.initials: MH
authors.lastname: Norris
authors.affiliation: Department of Infectious Diseases and Pathology, University of Florida, Gainesville, Florida.
authors.forename: Apichai
authors.initials: A
authors.lastname: Tuanyok
authors.affiliation: Department of Microbiology and Immunology, University of South Alabama, Mobile, Alabama.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Brett
authors.affiliation: Department of Microbiology and Immunology, University of South Alabama, Mobile, Alabama.
authors.forename: Mary N
authors.initials: MN
authors.lastname: Burtnick
authors.affiliation: Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Keim
authors.affiliation: Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona.
authors.forename: Erik W
authors.initials: EW
authors.lastname: Settles
authors.affiliation: Department of Biological Sciences, Northern Arizona University, Flagstaff, Arizona.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Allender
authors.affiliation: Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, Nevada.
authors.forename: David P
authors.initials: DP
authors.lastname: AuCoin
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D016957:Burkholderia pseudomallei / Q000276:immunology; D004396:Coloring Agents / Q000627:therapeutic use; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D007104:Immune Adherence Reaction / Q000379:methods; D007118:Immunoassay / Q000379:methods*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008554:Melioidosis / Q000175:diagnosis* / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D012680:Sensitivity and Specificity
chemical_list: D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D004396:Coloring Agents; D008070:Lipopolysaccharides