Detection and Stability of the Major Almond Allergen in Foods

Abstract

Almond major protein (AMP or amandin), the primary storage protein in almonds, is the major allergen recognized by almond-allergic patients. A rabbit antibody-based inhibition ELISA assay for detecting and quantifying AMP in commercial foods has been developed, and this assay, in conjunction with Western blotting analyses, has been applied to the investigation of the antigenic stability of AMP to harsh food-processing conditions. The ELISA assay detects purified AMP at levels as low as 87 +/-16 ng/mL and can detect almond at between 5 and 37 ppm in the tested foods. The assay was used to quantify AMP in aqueous extracts of various foods that were defatted and spiked with known amounts of purified AMP or almond flour. In addition, AMP was quantified in commercially prepared and processed almond-containing foods. Neither blanching, roasting, nor autoclaving of almonds markedly decreased the detectability of AMP in subsequent aqueous extracts of almonds. Western blots using both rabbit antisera and sera from human almond-allergic patients confirm a general stability of the various peptides that comprise this complex molecule and show that the rabbit antibody-based assay recognizes substantially the same set of peptides as does the IgE in sera from almond-allergic patients.

Identification and Characterisation of the IgE-Binding Proteins 2S Albumin and Conglutin γ in Almond <i>(Prunus dulcis)</i> Seeds

“…These antibodies, specific to almonds, retained partial cross-reactivity with some tree nuts (black walnut, hazelnut, brazil nut). A similar approach was used by Roux et al [10]for the detection of the almond major protein allergen in foods. In a study by Eigenmann et al [25], the specificity of IgE binding in peanut was analysed using a preliminary absorption to soybean proteins of cross-reacting antibodies.…”

“…In one recent study, IgE-mediated allergy to almonds was found in 22% of symptomatic patients by means of positive skin prick tests and ImmunoCAP ® with IgE antibodies specific to almond [8]. Another recent report on the identification of almond allergens was based on a single case allergy [9], but only one major IgE-binding almond protein has been characterised as an allergen, namely almond major protein or amandin [10]. In this present work, two IgE-binding proteins isolated from almond seeds are reported and identified by sequence homology as 2S albumin and conglutin γ proteins.…”

Tree Nut Allergens

“…As a tree nut allergen, it ranks third (15% reactive) behind walnut and cashew nut in the FAAN self reporting survey of tree nut allergies [45]and can be found in many foods. Despite these statistics and despite the fact that the proteins in almonds have been biochemically characterized [60, 61, 62, 63, 64, 65, 66, 67], relatively little has been reported regarding the identity of the allergens involved in almond sensitivity.…”

“…Nineteen other polypeptides were also resolved, however, these were of lesser quantitative importance as judged by staining by Coomassie Brilliant Blue. Probing of blotted amandin with human almond-allergic sera shows that the 42- to 44-kD bands are strongly reactive [60, 67]. Preliminary sequence data indicate that these peptides are from a protein in the legumin family [unpubl.…”

Opinion of the Scientific Panel on Dietetic products, nutrition and allergies [NDA] on a request from the Commission relating to the evaluation of allergenic foods for labelling purposes

“…Several allergens have been described and some have been cloned. For most nuts ELISA methods have been described with high sensitivity and detection levels reaching as low as 0.2 mg/kg (Almond: Hlywka et al, 2000;Roux et al, 2001;Brazil nut: Clemente et al, 2004;Cashew nut: Wei et al, 2003;Hazelnut: Scheibe et al, 2001;Koppelman et al, 1999;Holzhauser and Vieths, 1999;Blais and Phillippe, 2001;Ben Rejeb et al, 2003;Stephan et al, 2002;Walnut: Niemann and Hefle, 2003). For specific nut allergen detection various ELISA test kits and DNA-based PCR and PCR-ELISA methods are commercially available .…”