Describing the peptide binding specificity of HLA-C molecules

Peptide-HLA-I (pHLA-I) complexes were formed by refolding of recombinant, biotinylated HLA-I molecules in the presence of increasing concentrations of peptide. pHLA-I complexes were detected, via the formation of a conformational epitope recognized by the W6/32 pan-specific MHC-I antibody, using a proximity based Luminescence Oxygen Channeling Immunoassay (LOCI). The signal obtained was converted into nM MHC-I complex using a prefolded pHLA-A*02:01 standard. The concentration needed for half-saturation of MHC-I (the EC50) was calculated by non-linear regression. At low concentrations of receptor (i.e. receptor concentration < equilibrium dissociation constant observed), the EC50 is an approximation of the true equilibrium dissociation constant.