Improved Methods for Conjugating Selected Mycotoxins to Carrier Proteins and Dextran for Immunoassays

Simplified protein-coupling chemistries were utilized for the mycotoxins, T-2 toxin (T-2), ochratoxin A (OA), citrinin (CTN), and aflatoxin B1 (AFB1). Iodoacetate (IAc) and 1,1'-carbonyldiimidazole (CDI) were found to be excellent activators of hydroxyl and carboxyl groups of these mycotoxins. Iodoacetate reacted with the C-3 hydroxyl groups of T-2 toxin to form methylcarboxylated T-2 (T-2-metCOOH) containing a two-carbon spacer and a stable ether-based bond. 1,1'-Carbonyldiimidazole directly activated the carboxyl groups of OA, T-2-metCOOH, the C-3 hydroxyl group of T-2, succinic anhydride modified T-2, the C-8 hydroxyl group of citrinin, and carboxymethoxylamine hemihydrochloride modified AFB1. The resulting acylimidazole and imidazole carbamate intermediates reacted readily with the primary amine groups in proteins and ethylenediamine modified dextran to form stable amide or carbamate linkages. These coupling chemistries minimized the formation of undesired immunodeterminants in the conjugates. Toxin-protein coupling ratios could be readily controlled by varying the amount of toxin relative to that of protein in the CDI coupling reaction. These results demonstrate that IAc and CDI are excellent and versatile chemical couplers of hydroxyl- and carboxyl-containing mycotoxins and complement other chemistries in mycotoxin ELISA development