*: 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.