An HLA class I peptide-binding assay based on competition for binding to class I molecules on intact human B cells

Autoreactive CD8 T cells associated with β cell destruction in type 1 diabetes

“…Peptides of 9-11 aa containing at least one anchor residue for HLA binding at position 2 or the N terminus were defined (28,29). In total, 15 peptides (five 9-mer, five 10-mer, and five 11-mer) were synthesized and tested to determine their actual binding affinity for HLA-A*0201 by using a cellular-free binding assay (30). Ten high-affinity binding peptides were identified (IC 50 Ͻ 6 M), and two peptides bound with intermediate affinity (6 M Ͻ IC 50 Ͻ 15 M), whereas the other peptides displayed a low (15 M Ͻ IC 50 Ͻ 100 M) or undetectable binding capacity (IC 50 Ͼ 100 M) ( Table 1).…”

A Human CTL Recognizes a Caspase-8-Derived Peptide on Autologous HLA-B*3503 Molecules and Two Unrelated Peptides on Allogeneic HLA-B*3501 Molecules

“…To determine whether the affinity of each of the three peptides for the two HLA-B35 subtypes influenced the recognition by the CTL, we designed a peptide binding assay similar to the assay on intact human B cells described by van der Burg and colleagues (23). Briefly, the natural peptides were stripped from the HLA class I molecules by mild acid treatment, after which the .221.B*3501 or .221.B*3503 cells were incubated overnight at 4°C with a FL-labeled reference peptide at a fixed concentration, together with decreasing concentrations of the three peptides of interest.…”

“…We designed a peptide binding assay similar to the assay on intact human B cells described by van der Burg and colleagues (23). Peptides were stripped by mild acid treatment (pH 2.9), after which cells were incubated overnight at 4°C with a fluorescein (FL) 3 -labeled reference peptide at a fixed concentration (250 nM), together with decreasing concentrations (50 M to 0.18 M) of the three peptides of interest.…”

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes

“…These peptides were synthesized and tested for their actual binding capacity in competition-based cellular-binding assays which we developed for these HLA molecules. 43,44 Ten peptides from the b3a2 and b2a2 breakpoint regions that contained an anchor residue at position 2 (Val, Thr or Ser) and/ or the C-terminal anchor (Arg or Lys) 41 were tested for binding to HLA-A68 (A*6801). Five peptides bound with high (IC 50 p5 mM) or intermediate affinity (5 mM oIC 50 p15 mM).…”