Mapping of a Protective Epitope of Pertussis Toxin by In Vitro Refolding of Recombinant Fragments

Abstract

Antigenic determinants are often composed of discontiguous polypeptide segments, adjacent on protein surfaces, which are difficult to map by conventional techniques. Here we show that although monoclonal antibodies did not recognize recombinant molecules containing only the aminoterminal or only the carboxyterminal part of a protein, after in vitro refolding they are able to recognize a mixture of the two molecules. This suggests that two molecules, each containing part of a discontiguous epitope, are able to reconstitute in vitro the conformation recognized by a monoclonal antibody. Mapping of the regions required for antibody binding can therefore be achieved by in vitro refolding of recombinant molecules. Site-directed mutagenesis is then used to further define the sequences involved in antibody recognition. This new approach was used to map a discontiguous, conformational epitope on the S1 subunit of pertussis toxin. The analysis revealed that pertussis toxin is likely to contain only one immunodominant epitope, which is recognized by all seven protective monoclonal antibodies available to us. In theory, the method described can be used to map conformational epitopes in any protein.