Immunochemical Approach to the Detection of Aminotriazoles Using Selective Amino Group Protection by Chromophores

A selective approach for amino masking and hapten design for aminotriazole conjugates is described. Haptenic 1,2,4-triazole and 3-amino-1,2,4-triazole compounds were conjugated to proteins through spacer arms on the C5 ring carbon, containing hydroxyl or mercapto groups. The amino groups present in several haptens were protected via sulfenylation, using a chromophoric protecting reagent, 2-nitro- phenylsulfenyl chloride. Protection of haptens, conjugation to proteins, and deprotection on the proteins were followed by UV spectroscopy, monitoring the specific absorbance of the protecting group at 349 nm. Homo- and heterobifunctional spacers allowing spacer, hapten, and multiple heterology were used for the protein conjugates for immunizing and coating. Antisera raised against aminotriazole conjugates in New Zealand white rabbits and Swiss Webster mice were found to be specific for amitrole. Inhibition tests resulted in 60% inhibition by amitrole, indicating low-affinity antibodies. Antibody binding to the various antigens synthesized was characterized in a qualitative immunoassay.