title: The ApoCorrect assay: a novel, rapid method to determine the biological functionality of radiolabeled and fluorescent Annexin A5.
abstract: We have demonstrated that imaging of programmed cell death (PCD) in patients is possible using 99mTc-Annexin A5. Because of the short half-life of the technetium label it is important to limit the time span between the preparation of 99mTc-Annexin A5 and its administration into the patient. Therefore methods of quality control that determine the biological active fraction in the 99mTc-Annexin A5 should be not only accurate and precise but also rapid. We report the development and validation of a rapid, simple assay measuring the biological active fraction of 99mTc-Annexin A5. The assay is based on a solid phase of paramagnetic beads which are coated with phospholipids. Annexin A5 binds to these beads with high affinity if phosphatidyl serine is present within the phospholipid coat. Furthermore the binding depends on Ca2+ ions and functional Ca2+/phospholipid binding sites of Annexin A5. The bead assay is specific, stability-indicating, repeatable, and reproducible. It allows one to determine within 25 min the biological active fraction of a 99mTc-Annexin A5 preparation. We dubbed this assay the ApoCorrect assay.
authors.affiliation: Department of Clinical Pharmacy and Toxicology, University Hospital Maastricht, P.O. Box 5800, NL-6202 AZ Maastricht, The Netherlands. hboe@kfls.azm.nl
authors.forename: Hendrikus H
authors.initials: HH
authors.lastname: Boersma
authors.forename: Leo M L
authors.initials: LM
authors.lastname: Stolk
authors.forename: Heidi
authors.initials: H
authors.lastname: Kenis
authors.forename: Niko M
authors.initials: NM
authors.lastname: Deckers
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Vanderheyden
authors.forename: Leo
authors.initials: L
authors.lastname: Hofstra
authors.forename: Guido A K
authors.initials: GA
authors.lastname: Heidendal
authors.forename: Chris P M
authors.initials: CP
authors.lastname: Reutelingsperger
mesh_terms: D017304:Annexin A5 / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D017209:Apoptosis / Q000502:physiology*; D001667:Binding, Competitive; D001681:Biological Assay; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D006361:Heating; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D019169:Jurkat Cells; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008863:Microspheres; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000737:chemistry; D015609:Organotechnetium Compounds / Q000032:analysis* / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry; D019275:Radiopharmaceuticals / Q000032:analysis*; D015203:Reproducibility of Results; D013667:Technetium / Q000032:analysis / Q000737:chemistry
chemical_list: D017304:Annexin A5; C492760:FITC-annexin A5; D005456:Fluorescent Dyes; D006834:Hydrazines; D015609:Organotechnetium Compounds; D010743:Phospholipids; D019275:Radiopharmaceuticals; C117288:hydrazino nicotinamide; C117287:technetium Tc 99m HYNIC annexin V; D009536:Niacinamide; D013667:Technetium; D016650:Fluorescein-5-isothiocyanate