Enzyme-linked immunosorbent assay for TA-2005-glucuronide in human plasma

Abstract

A sensitive enzyme-linked immunosorbent assay (ELISA) for TA-2005-glucuronide, a main metabolite of new adrenergic β-receptor agonist TA-2005, has been investigated without prior deconjugation. Coupling of the hapten with bovine serum albumin (BSA) or β- d-galactosidase was carried out by the N-hydoxysuccinimide ester method. An anti-TA-2005-glucuronide antiserum was obtained from guinea pig immunized with the hapten–BSA conjugate. The ELISA was based upon a competitive assay in which the separation of bound from free fraction was performed by the double antibody technique using rabbit anti guinea pig immunoglobulin antibody adsorbed to microtiter plates. A satisfactory standard curve for the ELISA of TA-2005-glucuronide was observed in the range of 30 pg–3 ng ml −1 using 25 μl of human plasma. Inter-day and intra-assay variations were 7.0–17.5% and 1.0–11.7% respectively. The recoveries of TA-2005-glucuronide spiked to plasma samples were 95.5–120% (inter-assay) and 96.0–123.3% (intra-assay). The cross-reactivities of the prepared 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 by a simple pretreatment of the plasma sample with a C 18 cartridge column. This method was applied to the determination of TA-2005-glucuronide in human plasma samples for the evaluation of the pharmacokinetics of TA-2005. From the results, it was demonstrated that the ELISA developed was useful for the determination of TA-2005-glucuronide in human plasma and that the method was applicable to pharmacokinetic studies in humans.