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.