title: Magnetic bead protamine-linked microtiter assay for detection of heparin using iodinated low-molecular-mass heparin-tyramine.
abstract: 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.
authors.affiliation: 1st Department of Medicine, Faculty for Clinical Medicine Mannheim, University of Heidelberg, Klinikum Mannheim, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Harenberg
authors.forename: G
authors.initials: G
authors.lastname: Löhr
authors.forename: R
authors.initials: R
authors.lastname: Malsch
authors.forename: M
authors.initials: M
authors.lastname: Guerrini
authors.forename: G
authors.initials: G
authors.lastname: Torri
authors.forename: B
authors.initials: B
authors.lastname: Casu
authors.forename: D L
authors.initials: DL
authors.lastname: Heene
mesh_terms: D001667:Binding, Competitive; D006493:Heparin / Q000032:analysis*; D006495:Heparin, Low-Molecular-Weight; D006801:Humans; D007457:Iodine Radioisotopes; D008280:Magnetics; D008832:Microchemistry; D008863:Microspheres; D015394:Molecular Structure; D011479:Protamines; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017186:Titrimetry; D014439:Tyramine
chemical_list: D006495:Heparin, Low-Molecular-Weight; D007457:Iodine Radioisotopes; D011479:Protamines; D006493:Heparin; D014439:Tyramine