Mapping the major antigenic domains of the native flagellar antigen of Borrelia burgdorferi.
Purified flagellar protein (p41) of Borrelia burgdorferi (strain B31) was subjected to chemical cleavage with hydroxylamine or proteolysis with V8 protease, endoproteinase Asp-N, or alpha-chymotrypsin. The resulting polypeptides were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their positions in the published DNA sequence of the p41 protein were determined by amino-terminal sequencing and amino acid analysis. Epitope specificities of antibody binding by a monoclonal antibody raised by immunization of mice with purified flagella and pooled sera from patients with multiple erythema migrans, late Lyme borreliosis, or secondary syphilis were analyzed by Western blots (immunoblots) of peptides transferred to Immobilon polyvinylidene difluoride filters. The major epitope binding one murine monoclonal antibody (158) was localized to a carboxy-terminal domain that includes residues 300 to 336. The dominant epitopes binding human polyclonal antibodies are in the central portion of the molecule (residues 182 to 218) that is not conserved compared with other bacterial flagellins. Additional reactive epitopes were identified in the amino-terminal domain of the protein. Sera from patients with syphilis bound strongly to the amino-terminal conserved domain, providing a structural basis for cross-reactivity seen in standard enzyme-linked immunosorbent assays, but not to the central part of the molecule. Specific and cross-reactive antigenic determinants need to be considered in the design of improved immunodiagnostics for spirochetal diseases.
Jiang
W
W
Department of Medicine, State University of New York, Stony Brook 11794-8161.
Luft
B J
BJ
Schubach
W
W
Dattwyler
R J
RJ
Gorevic
P D
PD
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000276:immunology*; D015153:Blotting, Western; D015748:Borrelia burgdorferi Group / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D005407:Flagella / Q000276:immunology*; D005408:Flagellin; D006801:Humans; D008969:Molecular Sequence Data; D010449:Peptide Mapping
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; C075511:flagellin p41; D005408:Flagellin