Multimer Preparation of HLA Proteins

DRB1*04:01 (DR0401) and DQB1*03:02 (DQ8) protein was purified from insect cell cultures as previously described (18 (link), 19 (link)). Briefly, DR0401 or DR0401-myc, and DQ0302 protein was purified from supernatants by affinity chromatography, biotinylated with biotin ligase, and dialyzed against phosphate buffer (0.0625 M monobasic sodium phosphate, 0.0375 M monobasic sodium phosphate, pH 6.0). To prepare multimers DR0401, DR0401-myc, or DQ8 monomer was incubated with 0.2 mg/mL peptide, 0.2% n-octyl-β-d-glucopyranoside (Sigma) and 1 mM Pefabloc SC (Sigma) at 37 °C for 72 h. After incubation PE labeled streptavidin (ThermoFisher), PE-CF594 labeled streptavidin (BD), or BV421 labeled streptavidin (BD) was added to conjugate monomers into tetramers. As summarized in Table 1, DR0401 restricted epitopes were labeled with PE, whereas DQ8 restricted epitopes were labeled with PE-CF594. The viral control epitope, influenza matrix protein (MP) 97–116, was prepared with myc tagged DR0401 and labeled with BV421.