Fluorescence immunoassay for cyclosporin A

Monodansylcadavarine (MDC) was used to synthesise a fluorescent derivative of cyclosporin A and the product of the reaction was isolated by preparative thin layer chromatography (TLC) and purified by high performance liquid chromatography (HPLC). The fluorescent derivative was shown to bind with a polyclonal antibody to cyclosporin A by submitting the derivative for analysis by cyclosporin radioimmunoassay (RIA). However this derivative did not bind with a monoclonal antibody used in a RIA specific for the parent compound. To achieve this fluorescent derivatives were synthesised using cyclosporin-C-hemisuccinate as the staring material with MDC, 4-bromomethyl-7-methoxycoumarin (BMMC), 4-bromomethyl-6,7-dimethoxycoumarin (BMDC) and tetramethyl rhodaminecadavarine (TRC) as the labels. All derivatives were isolated and purified by TLC and HPLC and shown to have antibody binding in the parent compound specific RIA. The fluorescent properties of the derivatives were investigated and the most promising, BMMC and TRC used in the immunoassay development. [Continues.]