Magnetizable solid-phase fluoroimmunoassay of phenytoin in disposable test tubes

We developed a fluoroimmunoassay for phenytoin in serum or plasma, based on the magnetic separation technique. The method involves sheep anti-phenytoin serum coupled to magnetizable cellulose/iron oxide particles, with a fluorescein-labeled phenytoin analog as tracer. After magnetic sedimentation of the solid phase from assay mixtures, the free fraction of the tracer is aspirated, removing endogenous fluorophores and other interfering components of the sample. The antibody-bound tracer is then eluted from the solid phase into a methanolic buffer medium and quantitated fluorometrically. The entire procedure, including fluorometry, is performed within disposable polystyrene test tubes. The assay involves only simple reagents and equipment, and correlates closely with established radioimmunoassay (r = 0.97) and gas-liquid chromatographic (r = 0.98) techniques.