Enzyme-Linked Immunosorbent Assay of CS-518, a Thromboxane Synthetase Inhibitor, in Rabbit Plasma and Platelets

Abstract

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CS-518, a novel thromboxane synthetase inhibitor. Antisera against CS-518 were obtained from rabbits immunized with bovine serum albumin linked to CS-518 via carboxylic acid introduced into the imidazolyl ring (for ELISA-1) or via 6-carboxylic acid directly (for ELISA-2). Each of two CS-518 derivatives was conjugated to horseradish peroxidase by a N-succinimidyl ester method, and it was used as a labeled-antigen in homogeneous combination with antisera. In ELISA-1, CS-518 was detectable in a range of 5pg-1ng, and all cross-reactivities with main metabolites were less than 5%, in contrast to high affinity to the taurine and glucuronic acid conjugates of CS-518 in ELISA-2. Validity of ELISA-1 was confirmed by a high-performance liquid chromatography and ELISA-1 enabled specific determination of CS-518 in plasma samples deproteinized by methanol. When ELISA-1 was applied to determine CS-518 in platelets after oral administration to rabbits, CS-518 uptake up to maximum capacity in platelets (4.2-5.4 x 10(6) M) and slow elimination of CS-518 from platelets (T1/2 = 36-41 hr) were observed independent of CS-518 doses. These results confirm that CS-518 binds to thromboxane synthetase in platelets with high affinity.