Development of a hybridoma secreting monoclonal antibody to citrinin

Citrinin Is a highly potent nephrotoxin produced as a secondary metabolite during the growth of numerous fungi. Though citrinin may be detected in a variety of food products, it is most commonly found as a contaminant of both the large and small grains. It has been implicated in the production of renal disease and death among livestock and is implicated in a fatal renal disease in humans.

Current methods used to detect citrinin such as high performance liquid chromatography (HPLC) and thin layer chromatography are either expensive, require extensive research equipment or are not satisfactory for the detection of the small, but toxic, levels of mycotoxin which may be present in a food, feed or blood sample. The objective of this study was to develop a hybridoma producing monoclonal antibody to citrinin for use in an enzyme-linked immunosorbent assay (ELISA) for the mycotoxin.

Citrinin was coupled to bovine serum albumin (BSA). Analysis of buffers by HPLC following equilibrium dialysis revealed a molar binding ratio of 19 moles of citrinin per mole BSA. This citrinin/BSA conjugate was used to immunize 8-week-old male RBF mice over a period of 6-8 weeks. Analysis of sera from the Immunized mice by Indirect ELISA showed that citrinin specific antibody was formed to the Immunogen. Spleen cells from the Immunized mice were fused to FOX-NY myeloma cells In the presence of polyethylene glycol. Actively growing hybridomas were expanded and screened by ELISA. Hybridomas positive for the production of antl-citrinin antibody were cloned by a limiting dilution technique and the clones then expanded and rescreened by ELISA. Positive clones were expanded and stored In liquid nitrogen as appropriate. It Is anticipated that a monoclonal antibody developed by this method may be used In the rapid screening of agricultural commodities or In diagnosis of animals suspected to be suffering from citrinin toxicosis.