Development of Monoclonal Antibodies to Gizzerosine,A Toxic Component Present in Fish Meal

This study is the first report of the development of monoclonal antibodies (MAbs) against gizzerosine (GZ), one of the causative agents of black vomit, a serious poultry disease. Balb/c mice were immunized with different GZ conjugates; the most immunogenic conjugate in experimental animals was determined by enzymelinked immunoadsorbent assays (ELISA). Somatic fusions were carried out using splenic lymphocytes from GZ-immune mice and the NSO/2 myeloid cell line. Primary selection of hybridomas secreting antibodies to GZ was done using a direct ELISA, with GZ bound to bovine serum albumin (BSA), GZ directly bound to maleinimide preactivated plates and histamine bound to BSA, a GZ related biogenic amine present in fish meal. Four MAbs—3H4, 3H10, and 5B1 of the IgG₁ isotype, and 8G7 of the Ig_2a isotype—were specific to GZ and did not cross-react with histamine. Only monoclonals 3H4 and 8G7 bound GZ in solution by means of a competitive ELISA. Finally, to determine the performance of the competitive ELISA developed with the MAbs, experiments were conducted with GZ in solution (0 to 10 μg/ml) and with GZ labeled with horseradish peroxidase (HRP) as the tracer; the antibody complex was captured by using rabbit anti-mouse IgG preactivated ELISA plates. These experiments showed that monoclonal anti-GZ-3H4 generates a more sensitive assay close to linearity in the range about of 0.1 to 10 μg/ml of GZ. No cross-reaction was observed with histamine, histidine, or lysine at all concentrations tested.