Varian silica gel strips itlc sg

Briefly, [¹¹¹In]InCl₃ (Curium, Petten, The Netherlands) was added to DTPA-hMN-14-IRDye700DX in 3 V of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5. After 30 min of incubation at room temperature, 50 mM EDTA was added to the labeling reaction to a final concentration of 5 mM to chelate unincorporated [¹¹¹In]InCl₃. Labeling efficiency was determined by instant thin-layer chromatography on Varian silicagel strips (ITLC-SG; Agilent Technologies, Amstelveen, The Netherlands) using 0.1 mM ammonium acetate (NH₄Ac) buffer with 0.1 M EDTA, pH 5.5 as the mobile phase and labeling efficiency reached > 95%.
For the in vitro binding assay, DTPA-hMN-14-IRDye700DX was radiolabeled with 0.5 MBq/μg of [¹¹¹In]InCl₃. For the biodistribution studies, in two mice with s.c. LoVo tumors, DTPA-hMN-14-IRDye700DX was radiolabeled with 0.23 MBq/μg of [¹¹¹In]InCl₃.

All labeling procedures were performed under metal-free conditions. Briefly, [¹¹¹In]InCl₃ (Mallinckrodt Medical BV/Curium, Petten, the Netherlands) was added to IMP-288 or RDC018 in two volumes of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5. After 20 min of incubation at 95 °C, 50 mM ethylenediaminetetraacetic acid (EDTA) was added to the labeling reaction to a final concentration of 1 mM EDTA to chelate unincorporated ¹¹¹In. Lastly, Tween80 (Sigma-Aldrich, Saint Louis, MO, USA) was added to the labeling product in a final concentration of 0.01%. The labeling efficiency was determined by instant thin-layer chromatography on Varian silica gel strips (ITLC-SG; Agilent Technologies, Amstelveen, the Netherlands) using 0.1 mM ammonium acetate (NH₄Ac) buffer with 0.1 M EDTA (pH 5.5) as the mobile phase. If labeling efficiency was below 95%, labeled products were purified using solid-phase extraction on an HLB cartridge (Waters Chromatography B.V., Etten-Leur, the Netherlands) with 100% EtOH as mobile phase. Final radiochemical purity was > 95% for all compounds.
The antibody hMN-14 was conjugated to IRDye800CW (fluorophore:antibody substitution ratio 1.4) and diethylenetriaminepentaacetic acid (DTPA) which was labeled with [¹¹¹In]InCl₃ at a specific activity of 0.78 MBq μg⁻¹, as previously described [7 (link)].

Briefly, [¹¹¹In]InCl₃ (Curium, Petten, The Netherlands) was added to dual-conjugated hIgG or hMN-14 in two volumes of 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.5. After 45 min of incubation at 40 °C, 50 mM ethylenediaminetetraacetic acid (EDTA) was added to the labeling reaction in a final concentration of 5 mM to chelate unincorporated [¹¹¹In]InCl₃. Labeling efficiency was determined by instant thin-layer chromatography (ITLC) on Varian silica gel strips (ITLC-SG; Agilent Technologies, Amstelveen, The Netherlands), using 0.1 mM ammonium acetate (NH₄Ac) buffer with 0.1 M EDTA, pH 5.5, as the mobile phase. Antibody-conjugates were purified by gel filtration on a PD-10 column, and the radiochemical purity of the final dual-labeled conjugates reached >95% as determined by ITLC.