Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative <i>in vitro</i> study and partial characterization of digestion‐resistant peptides

Background: Peanut flour is a high-protein, low-oil, powdered material prepared from roasted peanut seed. In addition to being a well-established food ingredient, peanut flour is also the active ingredient in peanut oral immunotherapy trials. Enzymatic hydrolysis was evaluated as a processing strategy to generate hydrolysates from peanut flour with reduced allergenicity. Methods: Soluble fractions of 10% (w/v) light roasted peanut flour dispersions were hydrolyzed with the following proteases: Alcalase (pH 8.0, 60°C), pepsin (pH 2.0, 37°C) or Flavourzyme (pH 7.0, 50°C) for 60 min. Western blotting, inhibition ELISA and basophil activation tests were used to examine IgE reactivity. Results: Western blotting experiments revealed the hydrolysates retained IgE binding reactivity and these IgE-reactive peptides were primarily Ara h 2 fragments regardless of the protease tested. Inhibition ELISA assays demonstrated that each of the hydrolysates had decreased capacity to bind peanut-specific IgE compared with nonhydrolyzed controls. Basophil activation tests revealed that all hydrolysates were comparable (p > 0.05) to nonhydrolyzed controls in IgE cross-linking capacity. Conclusions: These results indicate that hydrolysis of peanut flour reduced IgE binding capacity; however, IgE cross-linking capacity during hydrolysis was retained, thus suggesting such hydrolysates are not hypoallergenic.

Section: Discussionmentioning

confidence: 99%

Allergenic Properties of Enzymatically Hydrolyzed Peanut Flour Extracts

Shi

Guo

White

et al. 2013

“…According to the literature [summarized in [30]], Ara h 2 and Ara h 6 are described to be present in approximately the same concentration of 6-9% in peanuts. Also, both allergens are described to survive digestion as relatively large digestion-resistant peptides [31] that can be taken up into the circulation. Therefore, one would expect that both 2S albumins are transferred in similar concentrations if following the same absorption mechanisms and secreted via the same pathways.…”

Section: Discussionmentioning

confidence: 99%

Detection of the Peanut Allergens Ara h 2 and Ara h 6 in Human Breast Milk: Development of 2 Sensitive and Specific Sandwich ELISA Assays

Schocker

Scharf

Kull

et al. 2017

Background: Little is known about breast milk as a vehicle for tolerance development or sensitization to peanuts very early in life. Thus, well-characterized and highly sensitive detection systems for the reliable determination of peanut allergens in breast milk are mandatory. Methods: For the quantification of the marker allergens Ara h 2 and Ara h 6 in the low nanogram per milliliter range in breast milk samples of a German cohort, sensitive and highly specific sandwich ELISAs were optimized and validated. Results: The Ara h 2 ELISA revealed a limit of detection (LOD) of 1.3 ng Ara h 2/mL and a quantification range of 2.3-250 ng/mL, the Ara h 6 ELISA showed an LOD of 0.7 ng/mL and a working range of 1.1-14.4 ng/mL. The assays showed no relevant cross-reactivity against other potentially cross-reactive legume, seed, and tree nut extracts (<0.01%, except for Ara h 1 in the Ara h 2 ELISA <0.1%). Ara h 2 was detectable in breast milk samples from 14/40 (35%) of the participants in concentrations from 2.3 to 184 ng/mL, Ara h 6 appeared in 9/40 (22.5%) of the lactating mothers between 1.1 and 9.7 ng/mL, and 1 highly positive sample with 79 ng/mL. Both allergens appeared at the same time points, but Ara h 6 in lower concentrations than Ara h 2. Conclusions: Sensitive and specific diagnostic tools for the determination of Ara h 2 and Ara h 6 in human breast milk were established. The kinetics of secreted Ara h 2 and Ara h 6 seem to be similar but with a difference in concentration. Follow-up investigations on their tolerogenic or sensitizing properties in breast milk become now accessible.

“…While Ara h 2 and Ara h 6 exhibit an intermediate level of overall sequence identity (53–57%), cases of isolated sensitization to Ara h 6 are rare as the two allergens share several, even if not all important IgE binding epitopes, and have been found to cross-react extensively [14]. …”

Section: Discussionmentioning

confidence: 99%

“…A variant of peanut 2S albumin, designated Ara h 6, has recently been described and found to be a relevant allergen in peanut allergy [3,11,12,13,14,15]. Since Ara h 2 and Ara h 6 belong to the same protein family, are structurally similar and extensively cross-reactive, it is uncertain to what extent Ara h 6 plays a role in allergy to peanut, distinct from that of Ara h 2, and if evaluation of IgE to Ara h 6 would offer diagnostic value additional to that of Ara h 2.…”

Section: Introductionmentioning

confidence: 99%

Anaphylaxis to Peanut in a Patient Predominantly Sensitized to Ara h 6

Asarnoj

Glaumann²,

Elfström³

et al. 2012

Diagnosis of peanut allergy has improved thanks to component-resolved diagnostics. Peanut allergen component Ara h 2 is considered to indicate true peanut allergy. The component Ara h 6 is structurally similar to Ara h 2, but the diagnostic value of analyzing IgE antibodies to Ara h 6 is unclear. A boy sensitized (≥0.35 kU_A/l) to Ara h 8 but not to Ara h 1, Ara h 2 and Ara h 3 was challenged with peanut and developed grade II anaphylaxis. In serum collected at the time of challenge a doubling of IgE to the peanut allergen extract was observed compared to allergy testing 9 months earlier. In contrast, IgE levels to Ara h 1, Ara h 2, Ara h 3 and to Ara h 8 were rather unchanged. After another 2 months, Ara h 6 was analyzed and revealed a level of 24 kU_A/l whilst Ara h 2 was 0.12 kU_A/l. We suggest that IgE sensitization to Ara h 6 caused the reaction and conclude that analyses of IgE levels to peanut and peanut components should be performed in connection with a challenge. Furthermore, levels to Ara h 2 below 0.35 kU_A/l may still indicate a risk of severe reaction at the time of challenge since in rare cases, Ara h 6 IgE antibodies may be present without occurrence of IgE antibodies to Ara h 2.