MAGNETIC BEAD PROTAMINE-LINKED MICROTITER ASSAY FOR DETECTION OF HEPARIN USING IODINATED LOW-MOLECULAR-MASS HEPARIN-TYRAMINE

We have developed a competitive heparin binding assay employing protam ine-coated magnetic beads for detection and measurement of heparin. Th e assay utilizes 125-iodine specifically bound to newly synthesized lo w-molecular-mass (LMM) heparin-tyramine. The tracer was stable over a period of 3 weeks, as demonstrated by gel filtration chromatography. T he protamine-coated beads were found to be stable over at least two mo nths. The heparin-tyramine bead assay had in buffer a lower detection limit of 0.04 mu g/ml and in plasma of 0.23 mu g heparin/ml. 50 % bind ing was obtained at 0.7 mu g/ml and 20 % binding at 4 mu g/ml in plasm a. 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 frac tions displaced the tracer from the protamine-coated magnetic beads to different extents. The validity of the assay was proven after intrave nous administration of unfractionated and LMM-heparin in man. The elim ination rate was similar using the heparin-tyramine bead assay compare d with the anti-factor Xa coagulation assay. After intravenous dosing of LMM-heparin the maximal concentration was lower using the heparin-t yramine bead assay compared with the anti-factor Xa coagulation assay. The bead assay was found to be reproducible, valid, and rapid for mea surement of the concentration of heparin preparations in purified syst ems and for HMM-heparin in plasma. Measurement of the concentration of LMM-heparin in plasma has a high coefficient of variation using the b inding assay.