Peptide-MHC class I interaction measured by radioassay and spun column chromatography A HLA-A*11:01-binding peptide, KVFPYALINK (nonnatural consensus sequence A3CON1 ), was radiolabeled with iodine ( 125 I) using a chloramine-T procedure (Hunter and Greenwood 1962) . Dose titrations of MHC-I heavy chains (HHH or HHP) were diluted into refolding buffer (Tris-maleate-PBS) and mixed with β 2 m (human or porcine) and radiolabeled peptide, and incubated at 18°C overnight. Then binding of radiolabeled peptide to MHC-I was determined in duplicate by Sephadex™ G50 spun column gel chromatography as previously described . MHC bound peptide eluted in the excluded volume, whereas free peptide was retained on the microcolumn. Both fractions were counted by gamma spectroscopy, and the fraction peptide bound was calculated as excluded radioactivity divided by total radioactivity.