Quantification of cibenzoline by enzyme-linked immunosorbent assay

We have developed an enzyme-linked immunosorbent assay (ELISA) suitable for routine monitoring of serum levels of cibenzoline. Anti-cibenzoline antibody was obtained by immunizing rabbits with cibenzoline conjugated with bovine serum albumin using N-(4-maleimidobutyryloxy)succinimide as a heterobifunctional coupling agent. An enzyme marker was prepared by coupling 2,2-diphenylethylamine with beta-D-galactosidase using glutaraldehyde. The detection limit of cibenzoline by ELISA was approximately 640 pg/ml with 50-microl samples. Cross-reactivity data showed that the antibody well recognizes both the diphenyl and cyclopropyl moieties, and is thus specific enough to the structure of cibenzoline. The values for the cibenzoline concentrations detected using this assay were comparable with those detected using HPLC. There was a good correlation between the values determined by the two methods. Moreover, ELISA was about 15-fold more sensitive in detecting cibenzoline at lower concentrations. Using this assay, drug levels were easily measured in the serum of rabbits after oral administration of cibenzoline at a single dose of 3 mg/kg.