ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR TA-2005-GLUCURONIDE IN HUMAN PLASMA

A sensitive enzyme-linked immunosorbent assay (ELISA) for TA-2005-gluc uronide, a main metabolite of new adrenergic beta-receptor agonist TA- 2005, has been investigated without prior deconjugation. Coupling of t he hapten with bovine serum albumin (BSA) or beta-D-galactosidase was carried out by the N-hydoxysuccinimide ester method. An anti-TA-2005-g lucuronide antiserum was obtained from guinea pig immunized with the h apten-BSA conjugate. The ELISA was based upon a competitive assay in w hich the separation of bound from free fraction was performed by the d ouble antibody technique using rabbit anti guinea pig immunoglobulin a ntibody adsorbed to microtiter plates. A satisfactory standard curve f or the ELISA of TA-2005-glucuronide was observed in the range of 30 pg -3 ng ml(-1) using 25 mu l of human plasma. Inter-day and intra-assay variations were 7.0-17.5% and 1.0-11.7% respectively. The recoveries o f TA-2005-glucuronide spiked to plasma samples were 95.5-120% (inter-a ssay) and 96.0-123.3% (intra-assay). The cross-reactivities of the pre pared antiserum with the related compound of TA-2005-glucuronide were quite low though there was a considerable cross-reaction with TA-2005. However, TA-2005-glucuronide could be easily separated from TA-2005 b y a simple pretreatment of the plasma sample with a C-18 cartridge col umn. This method was applied to the determination of TA-2005-glucuroni de in human plasma samples for the evaluation of the pharmacokinetics of TA-2005. From the results, it was demonstrated that the ELISA devel oped was useful for the determination of TA-2005-glucuronide in human plasma and that the method was applicable to pharmacokinetic studies i n humans. (C) 1998 Elsevier Science B.V. All rights reserved.