Francis Blankenberg

Publication Details

  • Improved detection of cell death in vivo with annexin V radiolabeled by site-specific methods JOURNAL OF NUCLEAR MEDICINE Tait, J. F., Smith, C., Levashova, Z., Patel, B., Blankenberg, F. G., Vanderheyden, J. 2006; 47 (9): 1546-1553

    Abstract:

    Labeled annexin V is widely used to detect cell death in vitro and in vivo. Nearly all studies have been done with annexin V derivatized via amine-directed bifunctional agents; it was thought that these molecules retained full bioactivity compared with unmodified protein. We now show that this assumption is incorrect by measuring the affinity of annexin V for cells in vitro by quantitative calcium titration under conditions of low membrane occupancy.Annexin V was modified with 4 different amine-directed agents: the N-hydroxysuccinimide esters of hydrazinonicotinic acid, mercaptoacetyltriglycine, and biotin; and with fluorescein isothiocyanate.In all cases, the membrane-binding affinity was decreased by derivatization, even at very low average stoichiometries. A statistical model based on the Poisson distribution accurately predicted the observed heterogeneity of derivatization as a function of average derivatization stoichiometry. This model also showed that multiply derivatized forms, which are the ones most likely to have compromised bioactivity, contributed disproportionately to the binding and imaging signals. The in vitro binding assay correctly predicted in vivo uptake in a mouse liver model of apoptosis for all proteins tested. The annexin V-128 protein, labeled at a single specific site at the N terminus, showed twice as much apoptosis-specific liver uptake as did all forms of annexin V derivatized randomly via amino groups.The membrane-binding activity of annexin V is much more sensitive to amine-directed chemical modification than previously realized. New annexin V molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo.

    View details for Web of Science ID 000240460600026

    View details for PubMedID 16954565

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