Peptide competition assay by capture ELISA

Soluble recombinant MHC-II proteins were incubated with a biotinylated indicator peptide with or without competitor peptides in the presence of sDM at 37 °C for 20 h. The pH 4.7 reaction buffer contained 100 mM acetate buffer (acetic acid and sodium acetate), 150 mM NaCl, 1% (w/v) BSA, 0.5% (v/v) IGEPAL CA-630 (Sigma), and 0.1% (w/v) NaN₃. After incubation, the peptide exchange reaction was stopped by the addition of two volumes of neutralization buffer containing 100 mM Tris-Cl (pH 8.5), 150 mM NaCl, 1% (w/v) BSA, 0.5% (v/v) IGEPAL CA-630, and 0.1% (w/v) NaN₃. The mixture was transferred to a precoated ELISA plate for capture of biotinylated peptide-loaded MHC-II by anti-MHC-II antibodies (L243 or SPV-L3). A DELFIA Eu-N1 Streptavidin System (PerkinElmer) was used to quantify the time-resolved fluorescence, as previously described [5, 24]. Peptide–MHC binding was quantified as background-subtracted europium fluorescence: [pepMHC] = Eu-SA_indicator − BG. To calculate %Binding and %Competition and fit data the same equations described above and elsewhere were used [24].