The development and application of a surface plasmon resonance-based inhibition immunoassay for the determination of warfarin in plasma ultrafiltrate.

Warfarin is the most widely prescribed oral anticoagulant for the management of a wide variety of thromboembolic disorders such as atrial fibrillation and deep vein thrombosis. A panel of warfarin-protein conjugates were produced and characterised and subsequently used for the production of monoclonal antibodies to warfarin. Following characterisation, the monoclonal antibodies were used in the development of a surface plasmon resonance-based inhibition immunoassay for the determination of the physiologically active 'nonprotein'-bound fraction of the drug in plasma ultrafiltrate. The inhibition immunoassay was compared with an existing high-performance liquid chromatography (HPLC) chromatographic technique for the determination of warfarin in plasma ultrafiltrate, and an excellent correlation was achieved between the two independent analytical techniques. The ligand-binding capacity and stability of various immobilised ligands were also compared. The BIACore-based inhibition immunoassay demonstrated an assay precision range of approximately 4-250 ng/ml, which is within the clinical range and demonstrated good reproducibility and robustness.