Mini scan radio tlc strip scanner

The radiochemical purity of the radiolabelled antibodies was determined by size-exclusion high-performance liquid chromatography (SE-HPLC) and radio-thin-layer chromatography (Mini-Scan radio-TLC Strip Scanner, Bioscan, Washington, DC, USA).
The HPLC system (Dionex UltiMate 3000, Sunnyvale, California, USA) used consisted of a manual injector with a 20-μl injection loop (7725i injector, Rheodune LLC, Rohnert Park, CA, USA), an HPLC pump, a variable wavelength detector and an in-line radioactivity detector coupled to a multichannel analyser. Chromatograms were analysed using the Chromeleon software (Dionex). A MAbPac™ SEC-1 size-exclusion column (Dionex) was used. The mobile phase consisted of 50 mM sodium phosphate, pH 6.8, and 300 mM NaCl. The flow rate was 0.20 ml/min., and the UV-detector wavelengths were set to 280 nm at 25°C. The retention time of the anti-TLR5 mAb was 10.9 min. Radioactivity was determined by thin-layer (Mini-Scan radio-TLC Strip Scanner; Bioscan) and paper chromatography.

Sodium [¹³¹I] iodide was obtained from the China Institute of Atomic Energy (Beijing, China). Anti-TLR5 mAb and control IgG were labeled with ¹³¹I-NaI by the Iodogen method. Briefly, 100 μl 0.05 M phosphate buffer (PB) and 22.8 MBq ¹³¹I-NaI were added into the prepared Iodogen-coated tubes (Pierce Biotechnology Ltd., Rockford, IL, USA) and then 20 μg of anti-TLR5 mAb or IgG were added respectively. Subsequently, the mixture was incubated at room temperature for 15 min with occasional shaking. The reaction was quenched by incubation with 150 μl 0.05 M PB for 15 min at room temperature. Radiolabeled antibodies were then purified by size-exclusion chromatography using a PD-10 Sephadex G-25 column (GE-Healthcare, Diegem, Belgium). The radiochemical purity was measured by a Wipe Test/Well Counter (Caprac; Capintec, Inc., Ramsey, NJ, USA). The in vitro stability of the radiotracer was determined in a serial time in human serum (Fame Ltd., Beijing, China) or PBS (0.05 mol/l; pH 7.4) at 37°C, and analyzed by radio-thin-layer chromatography (TLC) Strip Scanner (Mini-Scan radio-TLC Strip Scanner; Bioscan, Inc., Washington, DC, USA).