title: Purification and characterization of lipopolysaccharides from six strains of non-O157 Shiga toxin-producing Escherichia coli.
abstract: Certain Shiga toxin-producing Escherichia coli (STEC) are virulent human pathogens that are most often acquired through contaminated food. The United States Department of Agriculture, Food Safety and Inspection Service has declared several serogroups of STEC as adulterants in non-intact raw beef products. Hence, sensitive and specific tests for the detection of these STEC are a necessity for implementation in food safety programs. E. coli serogroups are identified by their respective O-antigen moiety on the lipopolysaccharide (LPS) macromolecule. We propose that the development of O-antigen-specific immunological assays can facilitate simple and rapid discriminatory detection of STEC in beef. However, the resources (antigens and antibodies) required for such development are not readily available. To overcome this, we extracted and characterized LPS and O-antigen from six STEC strains. Using hot phenol extraction, we isolated the LPS component from each strain and purified it using a series of steps to eliminate proteins, nucleic acids, and lipid A antigens. Antigens and crude LPS extracts were characterized using gel electrophoresis, immunoblotting, and modified Western blotting with commercially available antibodies, thus assessing the serogroup specificity and sensitivity of available ligands as well. The results indicate that, while many commercially available antibodies bind LPS, their activities and specificities are highly variable, and often not as specific as those required for serogroup discrimination. This variability could be minimized by the production of antibodies specific for the O-antigen. Additionally, the antigens generated from this study provide a source of characterized LPS and O-antigen standards for six serogroups of STEC.
authors.affiliation: The University of New Mexico, Center for Biomedical Engineering, MSC01 1141 1 University of New Mexico, Albuquerque, NM 87131, USA; Chemistry Division, Los Alamos National Laboratory, MS J567, C-PCS, Los Alamos, NM 87545, USA; The New Mexico Consortium 4200 West Jemez Rd., Suite 301, Los Alamos, NM 87544, USA.
authors.forename: Loreen R
authors.initials: LR
authors.lastname: Stromberg
authors.affiliation: University of Nebraska-Lincoln, School of Veterinary Medicine and Biomedical Sciences, P.O. Box 830905, East Campus Loop and Fair Street Lincoln, NE 68583, USA.
authors.forename: Zachary R
authors.initials: ZR
authors.lastname: Stromberg
authors.affiliation: Chemistry Division, Los Alamos National Laboratory, MS J567, C-PCS, Los Alamos, NM 87545, USA.
authors.forename: Afsheen
authors.initials: A
authors.lastname: Banisadr
authors.affiliation: The University of New Mexico, Center for Biomedical Engineering, MSC01 1141 1 University of New Mexico, Albuquerque, NM 87131, USA; The New Mexico Consortium 4200 West Jemez Rd., Suite 301, Los Alamos, NM 87544, USA.
authors.forename: Steven W
authors.initials: SW
authors.lastname: Graves
authors.affiliation: University of Nebraska-Lincoln, School of Veterinary Medicine and Biomedical Sciences, P.O. Box 830905, East Campus Loop and Fair Street Lincoln, NE 68583, USA.
authors.forename: Rodney A
authors.initials: RA
authors.lastname: Moxley
authors.affiliation: Chemistry Division, Los Alamos National Laboratory, MS J567, C-PCS, Los Alamos, NM 87545, USA; The New Mexico Consortium 4200 West Jemez Rd., Suite 301, Los Alamos, NM 87544, USA. Electronic address: harshini@lanl.gov.
authors.forename: Harshini
authors.initials: H
authors.lastname: Mukundan
mesh_terms: D000818:Animals; D002417:Cattle; D005516:Food Microbiology / Q000379:methods; D059022:Food Safety; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D019081:O Antigens / Q000276:immunology / Q000302:isolation & purification*; D000069466:Red Meat / Q000382:microbiology; D012703:Serotyping; D054323:Shiga-Toxigenic Escherichia coli / Q000276:immunology*; D014481:United States
chemical_list: D008070:Lipopolysaccharides; D019081:O Antigens
keywords: Antibody specificity; E. coli; Lipopolysaccharide (LPS); O-antigen (O-ag); Serotyping; Shiga toxin-producing E. coli (STEC)