Anti hla dr clone l243

Candidate peptides were synthesized with N-terminal 2,4-dinitrophenyl (DNP) tags joined by a 6-aminohexanoic acid linker (Sigma). Biotinylated HLA-DR recombinant proteins (HLA-DRB1*04:03 and HLA-DRB1*07:01) molecules were provided by the NIH tetramer core. Intrinsic CLIP peptide was cleaved from the HLA-DR molecules with human rhinovirus 3C protease. DNP-tagged peptides were supplied in molar excess to encourage efficient exchange of binders and incubated overnight at 32°C or 37°C (pH 4.5). Exchange reactions were then neutralized with 1 M Tris, pH 8.0 and biotinylated HLA-DR molecules were bound to streptavidin microspheres (Polyscience). Microspheres were washed and stained with allophycocyanin (APC)-labeled anti-HLA-DR (clone L243; BD Biosciences, 340549) and anti-DNP (clone 2–9(4); Abcam, ab6306) followed by rat anti-mouse IgE FITC secondary antibody (clone R35-72; BD Biosciences, 553415). Microspheres that were positive for HLA-DR and DNP-tagged peptide were detected by flow cytometry. Peptides were considered to be binders if both HLA-DR and DNP signals were detectable above an HLA-DR unexchanged control (Supplementary Fig. 1). Supplementary Fig. 1d shows full benchmarking with reported binders and non-binders.