Iodine-124 labelled Annexin-V as a potential radiotracer to study apoptosis using positron emission tomography

Annexin-V is a calcium-dependent protein that binds with high affinity to phosphaditylserine exposed during apoptosis. The aim of this study was to radiolabel annexin-V with iodine-124 for use as a potential probe of apoptosis by positron emission tomography. Annexin-V was radioiodinated directly using the cyclotron-produced positron emitter iodine-124 by the chloramine-T (CAT) method and indirectly by the pre-labelled reagent N-succinimidyl 3-[124I]iodobenzoate ([124I]m-SIB). Some reaction parameters of the CAT method such as reaction time and pH were optimised to give radiochemical yields of 22.3±2.6% (n=3, gel-filtration). After incubation with [124I]m-SIB, radiolabelled annexin-V was obtained in 14% and 25% yield by FPLC and gel-filtration, respectively. The radiochemical purities from direct and indirect labelling were 97.7±1.0% (n=3) and 96.7±2.1% (n=3), respectively. The new radiotracers could be stored for up to four days without significant de-iodination. The biological activity of radiolabelled annexin-V was tested in control and camptothecin-treated (i.e. apoptotic) human leukaemic HL60 cells. A significantly higher (21%) binding in treated cells was observed with [125I]m-SIB-annexin-V. The binding of [125I]m-SIB labelled annexin-V to camptothecin treated cells was blocked (68%) by a 100-fold excess of unlabelled annexin-V. Abbreviations: Fast protein liquid chromatography (FPLC), Instant thin layer chromatography (ITLC), Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE), 3-iodobenzoate (m-IBA), N-succinimidyl 3-(trimethylstannyl)benzoate (m-MeATE)