Methods:
Methods related to the constructs, purification, biophysical
characterisation, NMR, and microarray statistical analysis are located
in the supplemental information.
Patient
sera
Sera from 40 individuals with walnut (12 patients), peanut (12
patients), and peanut/walnut allergy (16 patients) were collected in
accordance with rules and regulations of the institutional review board
of their respective institutions (Tulane University Biomedical IRB
09-00231, REF #: 140613; University of California at Davis IRB protocol
No. 200210194-6) and in accordance with U.S. federal policy for the
protection of human subjects (Table 1).
Patients were over the age of 18
and had experienced recurrent severe, systemic allergic reactions to
peanut, walnut or both. These patients were included in this study based
on their convincing clinical history of food allergy and did not undergo
food challenge due to the potential severity of reactions.
Microarray
The entire amino acid sequences of Ara h 1 and Jug r 2 were printed onto
microarray slides (JPT Peptide Technologies GmbH, Berlin, Germany) as
sequential overlapping 15 amino acid spots, offset by 5 amino acids.
Slides were placed into a HS400 Pro (Tecan, San Jose, CA), blocked in
filtered SuperBlock TBS (Thermo Fisher Scientific, Waltham, MA) for 30
minutes at room temperature under agitation and then washed with
Tris-buffered saline containing 0.5% Tween-20 (TBST) (Bio-Rad,
Hercules, California). After centrifugation, 200µL of each patient’s
undiluted sera was injected into individual chambers containing
microarray slides and incubated at 4°C for 16 hours with agitation.
Microarray slides were then washed and injected with 170 µL of mouse
α-human IgE (Life Technologies, Grand Island, NY) diluted into filtered
Superblock at a dilution of 1:5000 for 30 minutes at room temperature.
The slides were washed and dried before scanning on a GenePix-4000B
(Molecular Devices, San Jose, CA). IgE binding was measured by Cy3 green
fluorescence at 532nm. The data was analyzed by GenePix Pro 7.2
software.
Statistics
Statistical analyses were performed using R (version 3.6.3). Modified z
values were calculated from the microarray median signal-to-noise ratios
(SNRs) and used to determine the percent of leader sequence peptides
bound by IgE for each allergy group. Median SNR values were converted
into modified z values, calculated using median and median absolute
deviation (MAD) of a patient’s SNRs to an entire leader sequence or
leader sequence fragment. To approximate standard deviation, MAD was
calculated using the constant of 1.4826. We defined a positive binding
event as a z value >= 3. Using the fisher.testfunction, Fisher’s exact tests were used to compare the proportion of
positive binding events out of the total possible binding events for a
particular fragment among allergy groups. When Fisher’s tests indicated
a significant difference between one or more allergy groups (p-value
< 0.05), pairwise post hoc comparisons were made to determine
which allergy groups differed from one another. We defined major
epitopes as peptides positively bound by 50% or more or patients within
an allergy group. Average z values which directly relate to IgE binding
intensity are presented as low (3 – 6), medium (6.1 – 9) and high
(>9). See supplemental methods for more details.