Selection and screening of antibody libraries. (A) Overview of the selection strategy. Libraries were packaged with either M13K07 or DeltaPhage helper phages to achieve LV and HV display, respectively. After R1, the libraries were selected in a competition branch and a thermostability branch in parallel. (B) Polyclonal phage ELISA to assess enrichment of binders against HLA-DQ2.5:DQ2.5-glia-α1a in the phage outputs after R1-R3. HLA-DQ2.5:CLIP2, which was used for negative selection, was used to monitor HLA-DQ2.5 binding irrespective of peptide. (C and D) The selection outputs after three rounds of panning were screened in HV format (C) and scFv format (D) to assess binding to target pHLA complexes and HLA-DQ2.5:CLIP2 (background) in ELISA and signal/noise ratios were calculated. Each dot represents one clone. Gray dots denote unknown sequences, black dots denote unique amino acid sequences and colors represent enriched sequences. Both libraries were selected in the thermostability branch. (E) Binding of purified Fab fragments (5 μg/mL) to different HLA-DQ2.5:peptide complexes was assessed by ELISA. Error bars illustrate mean ± SD of duplicates (n = 2). Alignment of 9mer core peptide sequences is shown on the right.