1. Introduction
Immediate hypersensitivity reactions to peanuts, an IgE-mediated food allergy, have long been a major public health concern, particularly in Western countries where peanut allergies frequently persist into adulthood. For affected individuals, strict allergen avoidance currently remains the only viable management strategy [1]. Peanut allergens are class I, water-soluble glycoproteins that exhibit notable stability to heat, acidic conditions, and enzymatic digestion. Within the Allergome database—the most comprehensive collection of allergen data—and protein family classification systems, allergens are organized into distinct protein families. To date, eleven clinically relevant peanut allergens have been identified, among which Ara h 1, Ara h 2, Ara h 3, and Ara h 6 are considered the major allergens (Please refer to S1 below).
For allergy studies using animal models, the consistency and composition of allergen extracts are essential, especially for sensitization, yet these properties can vary depending on the batch, extraction method, or material condition (such as baked, roasted, or raw).
Chondrex, Inc. analyzed the composition of crude peanut extract (CPE) prepared from raw peanuts (Cat # 3069) by SDS-PAGE and allergen detection ELISA kits for Ara h 1, h 2, h 3, and h 6 (Catalog # 6045, 6043, 6047, and 6042, respectively) (Please refer to S2 below). Other allergy models are available here.
2. Results and Discussion
2-1. Identification of Peanut Allergens in SDS-PAGE
Non-reducing 12% SDS-PAGE of CPE showed overlapping bands for Ara h 1 and Ara h 3 between 60-70 kDa, consistent with their similar molecular weights. Under reducing conditions, these allergens were well-resolved, enabling identification of individual CPE components (Figure 1).
Figure 1. 12% SDS-PAGE of Crude Peanut Extract Under Non-reducing (Left) and Reducing (Right) Conditions
M: Molecular weight marker, CPE: Crude Peanut Extract
2-2. Peanut Allergen Determination by ELISA
ELISA analysis of 5000 µg/ml CPE showed significant differences in the levels of individual allergens. Ara h 3 and Ara h 1 were the dominant components (2641.7 and 1842.2 µg/ml, respectively), followed by Ara h 2 (219.6 µg/ml) and Ara h 6 (21.9 µg/ml). These composition ratios (52.8%, 36.8%, 4.4%, and 0.4%, respectively) closely correlate with the band intensities observed in SDS-PAGE, demonstrating internal consistency between the biochemical and immunological assays. Chondrex, Inc.’s ELISA kits employ recombinant proteins as standards and although antibody reactivity may differ between recombinant and native proteins, the use of validated standards ensures consistent results and excellent reproducibility across independent studies.
Table 2. Ratio of Major Peanut Allergens from Total Protein
| Ratio from Total Proteins (%) | |
| Ara h 1 | 36.8 |
| Ara h 2 | 4.4 |
| Ara h 3 | 52.8 |
| Ara h 6 | 0.4 |
3. Conclusion
The composition of CPE is influenced by preparation protocols and the condition of the material, such as whether it is raw or heated. Although peanut allergens themselves are heat-stable, co-extracted proteins may differ depending on the processing method used. Chondrex, Inc. provides CPE under native conditions to maintain consistency across batches, but minor batch-to-batch variations must be considered.
Supplementary Information
S1. Ara Allergens in CPE
Ara h 1 is a 66 kDa monomer and a bicupin that assembles into a stable, non-covalent homotrimer and forms additional large molecular-weight structures in the peanut cotyledon [2,3]. Its internal IgE-binding regions show relatively weak IgE binding in the native trimeric form, but markedly strong binding in the denatured monomeric form, facilitating IgE cross-linking [4].
Ara h 2 and Ara h 6, two closely related 2S albumins within the seed-storage protein family, play a particularly important role in eliciting allergic reactions [5]. Ara h 2 contains ten independent linear IgE-binding epitopes and two isoforms, Ara h 2.01 (17 kDa) and Ara h 2.02 (19 kDa) [6,7]. Ara h 6 (14.5 kDa) shares 59% sequence homology with Ara h 2 and possesses comparable heat resistance, digestive stability, protease-stable core structure, and allergenic potency [8,9].
Ara h 3 is an 11S globulin seed-storage protein composed of a 43 kDa acidic subunit and a 28 kDa basic subunit connected by a disulfide bond [10]. Several subfragments (14, 25, 42, and 45 kDa) are detectable even under extraction conditions designed to minimize proteolysis [11]. Ara h 3 displays strong IgE-binding activity in sensitized individuals and can trigger allergic reactions [12,13].
S2. CPE Preparation (14)
CPE was prepared according to a modified published protocol. Raw peanuts were manually ground using a coffee grinder. The peanut powder was then suspended in TBS buffer (65 mM Tris-HCl, 1 mM EDTA, 1 mM PMSF, 200 mM NaCl, pH 8.3) at a ratio of 2:100 (w/v) and stirred for 1 hour at room temperature (RT). The extract was then filtered through Grade GF/A filters, and centrifuged at 10,000 × g for 30 minutes at 4°C. The supernatant was dialyzed against distilled water and lyophilized. The lyophilized powder was dissolved in TBS buffer. This solution was taken to 40% ammonium sulfate saturation and centrifuged at 30,000 × g for 30 minutes at 4°C. The resulting supernatant was then taken to 70% ammonium sulfate saturation and centrifuged at 30,000 × g for 30 minutes at 4°C. The pellet was resuspended in TBS buffer. Batches of CPE were clarified by centrifugation and stored at −70°C. Protein concentrations were determined using the Pierce™ BCA Protein Assay Kit with BSA as the standard.
REFERENCES
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10. C. Palladino, H. Breiteneder. Peanut allergens. Mol Immunol. 100; 58070 (2018).