INDIRECT ENZYME-LINKED IMMUNOASSAY SYSTEM FOR THE DETECTION OF AMINO-TRIAZOLES

In the scope of the development of an enzyme-linked immunosorbent assay (ELISA) for the detection of aminotriazole compounds i.e. the herbicide 3-amino-1,2,4-triazole (amitrole), a series of new aminotriazole haptenic derivatives and protein conjugates have been synthesized. The conjugates were used for immunization of rabbits and mice, and the antibodies obtained from the sera were studied in a competitive ELISA system. Spacer arms were attached on the N1 and C5 atoms of the triazole ring. The free amino groups of the haptenic compounds were protected via sulfenylation during conjugation to proteins: the chromophore protecting group was also utilized for monitoring epitope densities. Due to the low molecular weight and zwitterionic structure of the analyte as well as the presence of reactive functional groups to be protected, presentation of the compounds to the immune system appeared to be problematic and resulted in low natural immunogenicity. Competitive inhibition of the specific polyclonal antibodies in an indirect ELISA system by amitrole and related compounds appeared in the range of 0.1-100 muM concentration, the I50 values ranged around 10 muM. Imidazole and similar five-membered ring systems did not show cross-reactivity.