Figure 3

Competitive peptide-HLA-DR1 binding assay. Titration of peptides and proteins on DR1 using radioactive spun column assay. (A) X-axis: Log10 to peptide concentrations in nanomolar, Y-axis: percent of offered ¹²⁵I labelled (Y)Ha_306–318 peptide incorporated into DR1 complexes. 80 nM of urea denatured DR1 α and β chain was diluted into refolding buffer supplemented with titrations of peptide or protein and 3 nM ¹²⁵I labelled YHa_306–318 peptide. The following competitors were used: a recombinant version of the extracellular part of human invariant chain (positions 73–208), the CLIP peptide fragment of the invariant chain (positions 97–120), a C-terminal fragment of the invariant chain (positions 118–208), and the HA_306–318 peptide. After 24 h of incubation, fractions were analyzed using the spun column assay and IC₅₀ values (B) determined as described in Materials and Methods. All curves fitted with a regression coefficient better than 0.99. Note that the MHC concentration used (80 nM) bound approximately 70–80% of reference peptide (see Figure 2A), hence some degree of ligand depletion could be expected.