Methods
Blood samples (100 ml) were drawn from allergic (n=6) and tolerant (n=6) adults (≥18 years) sensitized to Ara h2 and/or 6 (≥0.1 kU/l10). Classification was achieved by DBPCFC or convincing history (n=2) of tolerated peanut ingestion. Non-atopic donors (n=5) served as reference group. The present study was approved by the ethical committee of the University Medical Center Utrecht (No. 17-945) and informed consent was given by all participants.
2S albumins isolated from roasted peanuts were used for tetramer formation with Streptavidin-APC or -PE11, 12. B-cells double positive for tetramer-PE/APC staining were single cell sorted and the V(D)J gene transcript of the heavy and corresponding light chain were amplified as described previously11, 13, 14. Quality checked sequences of successfully amplified gene transcripts were evaluated with the IgBlast web interface15. pFUSEss vectors (Invivogen), containing the IgE or IgG1 backbone, were used for antibody cloning and expression in HEK293 cells. Generated mAbs were tested for their specificity to nAra h2 and/or 616 in comparison with their binding to transferrin. The ability of generated mAbs to induce degranulation was determined by CD63 upregulation of re-sensitized and stimulated human basophils.
Descriptive gene lineage analysis consisted of isotype distribution, mutational status and VH-family gene usage17, 18. Sequence motifs were identified by calculating Levenshtein distances of HCDR3 regions combined with hierarchically clustering. HCDR3 sequences with five or less differences were defined as one motif. Detailed descriptions are shown in Supplementary File 1. These sequence data have been submitted to the GenBank database under submission number 2395667.