Preparation of a highly specific polyclonal antibody against fluvastatin and its use in development of ELISA for determination of fluvastatin in plasma

Abstract

For therapeutic monitoring and pharmacokinetic studies of the potent hypocholesterolaemic agent fluvastatin (FLV), a specific antibody was required for the development of a sensitive enzyme-linked immunosorbent assay (ELISA) for the accurate determination of FLV in plasma. In this study, a highly specific polyclonal antibody against FLV has been prepared. FLV was coupled to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) using carbodiimide reagent. FLV-KLH conjugate was used as an immunogen. Female 8-weeks old New Zealand white rabbits were immunized with an emulsion of FLV-KLH with Freund's adjuvant. The immune response of the rabbits was monitored by direct ELISA using FLV-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to FLV was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on a protein A column. The specificity of the purified antibody for FLV was evaluated by indirect competitive ELISA using various competitors from the FLV-structural analogues and therapeutic agents used with FLV in a combination therapy. The high affinity of the antibody (IC₅₀ = 150 pg ml⁻¹) enabled the determination of FLV in plasma at concentrations as low as 20 pg ml⁻¹.