Peptide-HLA Stability Measurement by SPA

The stability of peptide-HLA class I complexes was measured using dissociation of ¹²⁵I radiolabelled β₂m in a scintillation proximity assay (SPA) as previously described (69 (link)). Briefly, recombinant, biotinylated HLA class I heavy chains were diluted into a refolding buffer containing the test peptide and trace amounts of ¹²⁵I radiolabeled β₂m, and allowed to refold at 18°C for 24 h in a Streptavidin-coated scintillation microplate (Flashplate PLUS, Perkin Elmer, Boston, MA). Dissociation was initiated by adding excess unlabeled β₂m and placing the microplate in a scintillation counter (TopCount NXT, Packard) adjusted to 37°C. Reading the microplate continuously for 24 h allowed determination of the dissociation of radiolabeled β₂m.