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

Abstract

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.

Prevention of Allergy to a Major Cow's Milk Allergen by Breastfeeding in Mice Depends on Maternal Immune Status and Oral Exposure During Lactation

“…As BLG detection relied on the use of an immunometric assay, BLG might not be detectable in the former milks due to a masking effect of specific antibodies present in the breastmilk and/or the presence of BLG mainly as immune complexes. The fact that not all mothers were found to be excretors in various studies might result from the same masking effect (7)(8)(9)(10)(11)(12)(14)(15)(16)(17). This might then imply that all mothers are actually excretors of allergens in their BM, but as a free and/or complexed form depending on the levels of exposure.…”

“…Notably, breastmilk might influence immune system development via the transfer of various immunomodulatory molecules directly acting on the epithelial and immune system, or acting via the microbiota, such as regulatory/proinflammatory cytokines, miRNA, immunoglobulins, nutrients, but also metabolic products from the microbiota (2)(3)(4)(5). Human breastmilk also contains food antigens, which have been ingested by the mother (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). While the factors controlling food antigen shedding in breastmilk are poorly identified, the excretion of food antigens, at low doses and over a long period of time after ingestion (>24 h), appears as a natural process.…”

Circulating Ara h 6 as a marker of peanut protein absorption in tolerant and allergic humans following ingestion of peanut‐containing foods

“…Immunoassays generally remain the method of choice in detecting food allergens in complex food matrices due to their high sensitivity, specificity and simplicity, and have also been applied to the detection of peanut allergens such as Ara h 6 in human biological samples. 10,11,32,33 However, endogenous proteins, particularly in the blood, With this strategy, we were successful in detecting Ara h 6 in serum samples collected from both healthy and allergic volunteers following ingestion of much lower amounts of peanut (<1 g) than that previously reported in the literature. Actually, we were able to detect Ara h 6 following consumption of 300 mg peanut protein (equivalent to ~1½ peanuts) by all healthy volunteers, with Ara h 6 detection increasing in a dose-dependent manner.…”

“…3 Food proteins have also been detected at low levels in placental tissue [4][5][6] and in breastmilk. [7][8][9][10][11] The transfer of food antigens may be enhanced in diseases where intestinal barrier integrity is impaired, such as coeliac disease 12 and in subjects with underlying food allergy, [13][14][15][16] although data are limited. Intestinal permeability is not predictive of food allergy, 17 but may play a role in specific conditions such as wheat-dependent exercise-induced anaphylaxis.…”

Recent Advances in Experimental Allergy

“…Moutsoglou and Dreskin [63] investigated the role of long-lived antibody-secreting cells in peanut allergy and the potential of the proteasomal inhibitor bortezomib as a therapy in this context. While they found that bortezomib decreased long-lived anti-peanut IgE in mice, a correlative decrease in allergic symptoms was not observed.…”