Magnetic bead protamine-linked microtiter assay for detection of heparin using iodinated low-molecular-mass...

We have developed a competitive heparin binding assay employing protamine-coated magnetic beads for detection and measurement of heparin. The assay utilizes 125-iodine specifically bound to newly synthesized low-molecular-mass (LMM) heparin-tyramine. The tracer was stable over a period of 3 weeks, as demonstrated by gel filtration chromatography. The protamine-coated beads were found to be stable over at least two months. The heparin-tyramine bead assay had in buffer a lower detection limit of 0.04 microgram/ml and in plasma of 0.23 microgram heparin/ml. 50% binding was obtained at 0.7 microgram/ml and 20% binding at 4 micrograms/ml in plasma. The within assay coefficient of variation ranged from 9 to 28% for unfractionated, high molecular mass (HMM) heparin and from 12 to 15% for LMM-heparins in buffer system and in plasma. Various heparin fractions displaced the tracer from the protamine-coated magnetic beads to different extents. The validity of the assay was proven after intravenous administration of unfractionated and LMM-heparin in man. The elimination rate was similar using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. After intravenous dosing of LMM-heparin the maximal concentration was lower using the heparin-tyramine bead assay compared with the anti-factor Xa coagulation assay. The bead assay was found to be reproducible, valid, and rapid for measurement of the concentration of heparin preparations in purified systems and for HMM-heparin in plasma. Measurement of the concentration of LMM-heparin in plasma has a high coefficient of variation using the binding assay.