Enzyme-linked immunosorbent assay of CS-518, a thromboxane synthetase inhibitor, in rabbit plasma and platelets.
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.
Takasaki
W
W
Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan.
Nakagawa
A
A
Shigeta
A
A
Tanaka
Y
Y
D000284:Administration, Oral; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D008297:Male; D010949:Plasma / Q000378:metabolism; D011817:Rabbits; D015203:Reproducibility of Results; D013876:Thiophenes / Q000008:administration & dosage / Q000097:blood*; D013930:Thromboxane-A Synthase / Q000037:antagonists & inhibitors*
D013876:Thiophenes; C055806:RS 5186; D013930:Thromboxane-A Synthase