*: p < 0.05, **: p < 0.01, Student’s t -test.
†Allergic subjects were divided into two sub-groups
based on previous exposure to peanut: those at risk for anaphylaxis and
those who experience only oral symptoms.
Figure legends:
Figure 1 1H Nuclear Magnetic Resonance (NMR)
spectra of raw (A), roasted (B) and autoclaved (C) peanut-soaked
solutions. Horizontal axis represents 1H chemical
shifts in parts per million (ppm) referenced using an internal standard
(TSP-d 4) set to 0.0 ppm. Vertical axis represents
relative intensity. Framed regions correspond to methyl region (right)
and amide/aromatic regions (left) of peptide fragments and amino acid
side chains.
Figure 2 Western blot following SDS PAGE using antibodies
specific for Ara h 1 (A), Ara h 2 (B) and Ara h 8 (C). Lanes correspond
to the processing conditions as follows: M = Molecular weight marker, 1
= Raw, 2 = Roast, 3 = Autoclave, 4 = Autoclave then Roasted, 5 = Roasted
then Autoclaved.
Figure 3 Relative Ara h 2 (A) and Ara h 8 (B) quantification by
ELISA. Optical density (OD) values were measured at 450 nm and
referenced at 570 nm. Plates were coated with a maximum concentration of
1 μg/mL and 1 mg/mL peanut protein in A and B, respectively.
Figure 4 Peanut-Specific IgE ELISA using the serum of 4 highly
allergic subjects. Optical density (OD) values measured at 450 nm,
referenced at 570 nm, were normalized to corresponding raw values.
Auto-Roast: autoclaved, then roasted. Roast-Auto: roasted, then
autoclaved. n = 4 patients. ****: p < 0.0001, one-way ANOVA,
Tukey’s multiple comparisons test. ns: not significant.
Figure 5 Summary figure of hypothesized effect of
high-temperature and pressure autoclaving on peanut protein allergens
Ara h 2 and Ara h 8 when compared to raw or roasted allergens and
expected outcomes of proposed diagnostic method. 50% and 0% of intact
Ara h 2 and Ara h 8, respectively, can be detected in autoclaved peanut
extracts when compared to raw or roasted extracts. Highlighted areas in
red represent epitope regions in protein structures. When using the
whole protein extract made from raw or roasted peanuts for Skin Prick
Testing (SPT) as currently done in the clinic, patients who have IgE
specific for any combination of peanut allergens will experience a
positive SPT result. However, when using the autoclaved extract,
patients at risk for anaphylaxis (specific IgE primarily for Ara h 2)
will experience a positive SPT result while patients who experience only
oral symptoms to peanut (specific IgE primarily for Ara h 8) will
experience a negative SPT result. Patients tolerant to peanut will
experience a negative SPT result to both extracts.