Monoclonal antibody and diagnostic kit development studies for pathogen Clostridium perfringens neuraminidase enzyme

Clostridium perfringens is an important pathogen that is a natural part of the enteric system of humans and animals, which can divide rapidly and produces a large array of toxins under permissive conditions. The type and severity of its emerging pathogenicity are closely related to these toxins produced by the bacteria. The rapid progression of their pathologies, which may cause serious economic loss and mortality makes rapid diagnosis of C. perfringens crucial. Neuraminidases are a group of toxins produced by all C. perfringens strains, involved in both bacterial metabolism and virulence, and whose production in pathogenic conditions increases. The aim of this doctoral thesis is to try to develop monoclonal antibodies, through hybridoma technique, that can recognize C. perfringens neuraminidases, and to examine the neuraminidase-mediated diagnosis of C. perfringens with these antibodies. In addition, in case of successful production of monoclonal antibodies, it is aimed to create ELISA diagnostic kit prototypes for C. perfringens. As a result of this study, 26 hybrid cells were obtained, of which prototype candidates were selected, antibodies produced in large-scale, purified, and isotyped. However, attempts to diagnose C. perfringens from diseased-animal specimens using these antibodies, and production of a C. perfringens diagnostic kit prototype based on sandwich ELISA technique have not been successful. Further investigations using western blot showed that the 1C7, 3C5 and 5A9 monoclonal antibodies obtained were specific to NanI, the commercial neuraminidase product used during the studies contained impurities and antibody responses developed against these impurities. In addition, an IgM type monoclonal antibody, 4F3, with high affinity for bovine serum albumin was incidentally obtained. These results also demonstrate the importance of multi-faceted validations for antibody development studies.