ANTIBODY-BASED APPROACHES TO COUMARIN ANALYSIS

7-Hydroxycoumarin (7-HC) was chemically conjugated by diazo coupling t o carrier proteins such as bovine serum albumin (BSA), thyroglobulin a nd ovalbumin. These conjugates were characterised by sodium dodecyl su lphate/polyacrylamide gel electrophoresis (SDS-PAGE) and high-performa nce liquid chromatography (HPLC). Rabbits were immunised using the 7-H C-BSA conjugate. The highest antibody titre achieved was 1:10 000, as determined by competitive enzyme-linked immunosorbent assay (ELISA). T he resulting antibodies were purified by ammonium sulphate precipitati on, followed by protein A affinity chromatography. Their purity was as sessed by SDS-PAGE and HPLC. These antibodies have been used in the de velopment of a competitive ELISA, an amperometric biosensor and an ele ctrochemical immunoassay. Both the ELISA and amperometric biosensor ha ve been successfully applied to the analysis of 7-HC and its glucuroni de conjugate in human urine samples. Each of these antibody-based meth ods provides a novel approach to the analysis of the main metabolites of coumarin.