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.
Fitzpatrick
Brian
B
School of Biotechnology and National Centre for Sensor Research, Dublin City University, Glasnevin, Dublin 9, Ireland.
O'Kennedy
Richard
R
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000936:Antigen-Antibody Complex / Q000187:drug effects; D000941:Antigens / Q000276:immunology; D002138:Calibration; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D006440:Hemofiltration; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008024:Ligands; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*; D014462:Ultrafiltration; D014859:Warfarin / Q000097:blood*
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000941:Antigens; D016326:Extracellular Matrix Proteins; D008024:Ligands; D012710:Serum Albumin, Bovine; D012965:Sodium Chloride; D014859:Warfarin