Abstract: In order to establish an immunoanalytical method for the detection of sibutramine hydrochloride, the hapten of N,N-didesmethyl sibutramine hydrochloride (M2) was synthesized from 4-chlorophenylacetonitrile and 1,3-dibromopropane and then conjugated to the carrier protein of bovine serum albumin (BSA) or ovalbumin (OVA) by active ester method, glutaraldehyde method or mixed anhydride method to prepare immunogens and coating antigens. The results indicated that the coupling ratios of M2-BSA and M2-OVA were 24.6:1 and 16.2:1 as indicated by UV spectroscopic analysis. The antisera displayed high-level affinity to each coating antigen with a titer of more than 1:8000 and an IC50 of 0.42 μg/mL. Therefore, M2 hapten and carrier proteins were successfully conjugated. The active ester method revealed the smallest damage on the reactive groups of the hapten and the highest specificity. These results suggested that the artificial antigens could be used for the preparation of monoclonal antibodies.

Key words: sibutramine hydrochloride, N, N-didesmethyl sibutramine hydrochloride (M2), prohibited additives, immunoassay