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 li poprotein which is of particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analyses of OspA f rom both European and North American strains have demonstrated antigen ic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochete s 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 [C-14] palmitic acid confirm ed that OspA was lipidated, and partial digestion established lipidati on at the amino-terminal end of the molecule. The reactivity of five a nti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Pu rified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic do mains. Further resolution of the epitope specificity to determine humo ral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagn ostic testing for Lyme borreliosis.