Purification of Borrelia burgdorferi outer surface protein A (OspA) and analysis of antibody binding domains.
The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.
Jiang
W
W
Department of Medicine, State University of New York, Stony Brook 11794, USA.
Gorevic
P D
PD
Dattwyler
R J
RJ
Dunn
J J
JJ
Luft
B J
BJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification; D001428:Bacterial Vaccines; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D015748:Borrelia burgdorferi Group / Q000737:chemistry / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D008074:Lipoproteins; D008969:Molecular Sequence Data; D019308:Palmitic Acid; D010169:Palmitic Acids; D010449:Peptide Mapping
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D001428:Bacterial Vaccines; D000939:Epitopes; D008074:Lipoproteins; C072045:OspA protein; D010169:Palmitic Acids; D019308:Palmitic Acid