Detection of specific antigens for ten serogroups of Clostridium difficile

We previously described a serogrouping technique for Clostridium difficile based on slide agglutination with rabbit antisera raised against formol-treated cells. It allows the differentiation of ten serogroups, namely A, B, C, D, F, G, H, I, K and X. Each serogroup displays a specific profile with several distinctive bands by polyacrylamide gel electrophoresis (PAGE). In this study we investigated the common and specific antigenic determinants of the ten serogroups by immunoblotting. In a first experiment, whole cell proteins of the ten reference strains were separated by SDS-PAGE, transferred onto nitrocellulose membrane and immunoblotted against their homologous and heterologous antisera. Each serogroup was characterized by several common bands and one or two specific antigens which were proven to correspond to the lowest molecular weight distinctive band observed on PAGE profiles. New rabbit antisera were subsequently raised against the purified specific antigen obtained by electro-elution from polyacrylamide gels. Immunoblots were repeated with these new antisera: all reactions were serogroup specific except one minor cross reaction between C and F. The antisera still agglutinated the homologous strain without any cross agglutination, suggesting that the serogroup specific determinant is a surface antigen responsible for agglutination.