A cashew specific monoclonal antibody recognizing the small subunit of Ana o 3

Aryana

Clermont

et al. 2020

IJMS

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Nut-based milks and yogurts are gaining popularity, but may not offer the same benefits as dairy yogurts to consumers. Cashew nuts often cause severe allergic reactions, and cashew nut allergens are stable to several types of processing. To compare its characteristics to dairy yogurt and characterize the effects of fermentation on the Ana o 1–3 cashew nut allergens, a commercial yogurt made from cashew nuts (Cashewgurt) was evaluated for microbiological, physiochemical, and immunological properties. Average counts for lactobacilli and Streptococcus thermophilus were greater than 10 million colony forming units per milliliter, indicating the capacity to provide a health benefit. Cashewgurt pH and viscosity values were comparable to cow milk yogurts, and it was off white in color. SDS-PAGE analysis indicated a clear reduction in Ana o 1 and 2, and immuno-assay with polyclonal anti-cashew IgG antibody and cashew-allergic IgE indicated an overall reduction in allergen content. In contrast, SDS-PAGE, mass spectrometry, immunoblot, and ELISA all revealed that Ana o 3 was relatively unaffected by the fermentation process. In conclusion, Ana o 1 and Ana o 2 are sensitive to degradation, while Ana o 3 survives lactic acid bacterial fermentation during yogurt production. The analysis presented here indicates that cashew nut yogurt is not suitable for those with cashew nut allergy.

Section: Discussionmentioning

confidence: 64%

“…The rabbit polyclonal anti-cashew sera have been previously demonstrated to recognize Ana o 1, 2, and 3, as well as several other unidentified cashew nut proteins [16]. The 2H5 monoclonal anti-Ana o 3 peptide antibody has been previously characterized [24]. Blood samples from 12 cashew-allergic donors were obtained from PlasmaLab Int.…”

Section: Methodsmentioning

confidence: 99%

Microbiological, Physicochemical, and Immunological Analysis of a Commercial Cashew Nut-Based Yogurt

Aryana

Clermont

et al. 2020

IJMS

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“…Secondary infrared dye-labeled secondary antibodies (IRDye 680RD and IRDye 800CW) were purchased from LI−COR (Lincoln, NE, USA). Defatted nut extracts and purified native (from raw and roasted cashew nuts) and recombinant Ana o 3.0101 were generated as described in Mattison et al (2019) [ 21 ]. Monoclonal antibodies against whole cashew extract or against purified native or recombinant Ana o 3 immunogens were generated using Balb/c mice and standard immunization methods by Genscript (Piscataway, NJ, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Direct ELISA comparing monoclonal antibody binding to four micrograms (4 µg) of purified native Ana o 3, recombinant Ana o 3.0101, or defatted peanut, cashew, pistachio, pecan, walnut, or almond nut extract was carried out using either untreated samples or samples pretreated with DTT (5 mM). Recombinant Ana o 3.0101 was produced in E. coli as a fusion to thioredoxin, as described in Mattison et al (2019) [ 21 ]. The recombinant Ana o 3 protein was used in experiments only after the removal of the thioredoxin moiety.…”

Section: Methodsmentioning

confidence: 99%

“…Ana o 3 is a small protein (migrating at approximately 13 kDa under non-reducing conditions) whose solubility properties and resistance to digestion and processing make it a useful target for cashew allergen detection [ 20 ]. The 2H5 monoclonal antibody was generated previously using an Ana o 3.0101 peptide (H2N-CQRQFEEQQRFRNCQR−OH) immunogen and has been demonstrated to recognize properly folded and denatured native Ana o 3 purified from cashew extract, improperly folded recombinant Ana o 3.0101 produced in E. coli , and Ana o 3 in fermented cashew yogurt [ 21 , 22 ]. More reagents are needed to study immuno-dominant allergens, such as Ana o 3, so we used four distinct immunogens to develop new antibodies towards Ana o 3.0101 and other Ana o 3 isoforms.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Characterization of Anti-Ana o 3 Monoclonal Antibodies and Their Application in Comparing Brazilian Cashew Cultivars

Vant-Hull²,

Castro

et al. 2021

Antibodies

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Ana o 3 is an immuno-dominant cashew nut allergen. Four monoclonal antibodies to Ana o 3 (2H5, 6B9C1, 19C9A2, and 5B7F8) were characterized by ELISA and in silico modeling. The 2H5 antibody was the only antibody specific for cashew nut extract. In addition to cashew nut extract, the 6B9C1 and 19C9A2 antibodies recognized pistachio extract, and the 5B7F8 recognized pecan extract. All four antibodies recognized both recombinant Ana o 3.0101 and native Ana o 3. ELISA assays following treatment of purified Ana o 3 with a reducing agent indicated that the 6B9C1 and 19C9A2 antibodies likely recognize conformational epitopes, while the 2H5 and 5B7F8 antibodies likely recognize linear epitopes. In silico modeling predicted distinct epitopes for each of the anti-Ana o 3 antibodies. Screening extracts from 11 Brazilian cashew nut cultivars using all four antibodies showed slight differences in Ana o 3 bindings, demonstrating that these antibodies could identify cultivars with varying allergen content.

Cross-Serological Reaction of Glandless Cottonseed Proteins to Peanut and Tree Nut Allergic IgE

Zhang

et al. 2023

Molecules

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Food allergy is a potentially life-threatening health concern caused by immunoglobulin E (IgE) antibodies that mistakenly recognize normally harmless food proteins as threats. Peanuts and tree nuts contain several seed storage proteins that commonly act as allergens. Glandless cottonseed, lacking the toxic compound gossypol, is a new food source. However, the seed storage proteins in cottonseed may act as allergens. To assess this risk, glandless cottonseed protein extracts were evaluated for IgE binding by peanut and tree nut allergic volunteers. ELISA demonstrated that 25% of 32 samples had significant binding to cottonseed extracts. Immunoblot analysis with pooled sera indicated that IgE recognized a pair of bands migrating at approximately 50 kDa. Excision of these bands and subsequent mass-spectrometric analysis demonstrated peptide matches to cotton C72 and GC72 vicilin and legumin A and B proteins. Further, in silico analysis indicated similarity of the cotton vicilin and legumin proteins to peanut vicilin (Ara h 1) and cashew nut legumin (Ana o 2) IgE-binding epitopes among others. The observations suggest both the cotton vicilin and legumin proteins were recognized by the nut allergic IgE, and they should be considered for future allergen risk assessments evaluating glandless cottonseed protein products.