Abstract
Despite a high level of sequence identity between cow's, goat's, and sheep's milk (CM, GM, and SM, respectively) proteins, some patients tolerant to CM are allergic to GM and SM. In most cases, this specificity is due to the presence of IgE antibodies that bind only to caprine and ovine caseins. The patients may then develop severe allergic reactions after ingestion of CM products contaminated with low amounts of GM or SM. We thus aimed to develop an assay able to detect traces of caprine/ovine β-caseins in different food matrices, irrespective of the presence of the bovine homolog. We produced monoclonal antibodies (mAbs) specific to caprine caseins in mice tolerized to the bovine whole casein then sensitized to the caprine whole casein. In order to develop a two-site immunometric assay, we selected mAbs that could discriminate the caprine β-casein from its bovine homolog. Characteristics and performances of two tests were determined with various dairy products. Results were analyzed in relation with the IgE-immunoreactivity of the food matrices, thanks to sera from CM, GM/SM allergic patients. Our two-site immunometric assays demonstrated a high sensitivity with a detection limit of 1.6–3.2 ng/mL of caprine and ovine β-caseins. The tests were able to detect contaminations of GM in CM at the ppm level. Heat-treatment, ripening and coagulation processes, usually applied to dairy products that exhibit a very high IgE-immunoreactivity, did not impair the test sensitivity. These quantitative assays could then be useful for the risk assessment of food products potentially contaminated with GM and SM in order to prevent adverse reactions in patients specifically allergic to these milks.
Highlights
Milk is one of the foods causing the most frequently an adverse reaction [1, 2]
Among the substitutions occurring between the caprine and bovine β-CN sequences, we further identified the critical role of the substitution T63P in the major non-cross reactive IgE-binding epitope of caprine β-CN [25]
The monoclonal antibodies (mAb) SCB1 and SCB4 recognized the N-terminal fragment f(29-107) of β-CN. These mAbs were able to bind to a synthetic peptide covering the domain 49–79 but not to the caprine β-CN carrying the sole substitution of Thr residue at position 63 by the corresponding bovine Pro residue
Summary
Milk is one of the foods causing the most frequently an adverse reaction [1, 2]. Cross-allergenicity between milks from different ruminants are generally observed, rendering the use of goat’s and sheep’s milks (GSM) as substitute of cow’s milk (CM) unsafe for patients allergic to CM [3,4,5]. Immunoassays Specific to Caprine β-casein similar protein compositions and from a high sequence homology between protein components of ruminant milks [6,7,8] Despite these structural similarities, allergic reactions to GM or SM can occur in patients tolerant to CM. Since the first report by Wuthrich, this allergy has been regularly observed, mainly in European countries [8,9,10,11,12] The development of this specific allergy to GSM does not always rely on earlier sensitization to CM proteins as only 19% of the GSM-allergic/CM-tolerant patients had suffered from an outgrown CM allergy [12]. This assessment requires the development of sensitive methods able to detect GSM protein contaminations along the entire food chain process
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