Abstract
BackgroundCow’s milk-derived whey hydrolysates are nutritional substitutes for allergic infants. Safety or residual allergenicity assessment of these whey hydrolysates is crucial. Currently, rat basophilic leukemia RBL-2H3 cells expressing the human IgE receptor α-chain (huFcεRIα-RBL-2H3), sensitized with serum IgE from cow’s milk allergic children, are being employed to assess in vitro residual allergenicity of these whey hydrolysates. However, limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays.ObjectiveAn oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (a major allergen in whey) was generated to increase sensitivity, specificity, and reproducibility of existing degranulation assays.MethodsMice were immunized with bovine β-lactoglobulin, and subsequently the variable domains of dissimilar anti-β-lactoglobulin mouse IgG antibodies were cloned and sequenced. Six chimeric antibodies were generated comprising mouse variable domains and human constant IgE/κ domains.ResultsAfter sensitization with this pool of anti-β-lactoglobulin chuIgEs, huFcεRIα-expressing RBL-2H3 cells demonstrated degranulation upon cross-linking with whey, native 18 kDa β-lactoglobulin, and 5–10 kDa whey hydrolysates, whereas a 3 kDa whey hydrolysate and cow’s milk powder (mainly casein) showed no degranulation. In parallel, allergic serum IgEs were less sensitive. In addition, our pool anti-β-lactoglobulin chuIgEs recognized multiple allergenic immunodominant regions on β-lactoglobulin, which were also recognized by serum IgEs from cow’s milk allergic children.ConclusionUsage of our ‘unlimited’ source and well-defined pool of β-lactoglobulin-specific recombinant chuIgEs to sensitize huFcεRIα on RBL-2H3 cells showed to be a relevant and sensitive alternative for serum IgEs from cow’s milk allergic patients to assess safety of whey-based non-allergic hydrolyzed formula.
Highlights
Food allergens, aeroallergens, medications and insect venoms are the most common allergens which are responsible for inducing Type I or immunoglobulin E (IgE)-mediated hypersensitivity reactions [1]
While breastfeeding is considered the golden standard for infant nutrition, hypoallergenic (HA) formulas are a good alternative for infants at risk of developing allergy or for infants diagnosed with cow’s milk allergy (CMA)
Assessment of residual allergenicity of hydrolyzed formulas by peptide size distribution analysis, residual allergen detection by ELISA and SDS-PAGE/western blotting followed by immune incubation with specific antibodies in combination with in vitro cellular degranulation assays is proposed as a strategy for the screening of new HA formulas aimed at preventing sensitization in atopic children and avoiding clinical symptoms in infants suffering from CMA [12]
Summary
Aeroallergens, medications and insect venoms are the most common allergens which are responsible for inducing Type I or immunoglobulin E (IgE)-mediated hypersensitivity reactions [1]. Assessment of residual allergenicity of hydrolyzed formulas by peptide size distribution analysis, residual allergen detection by ELISA and SDS-PAGE/western blotting followed by immune incubation with specific antibodies in combination with in vitro cellular degranulation assays is proposed as a strategy for the screening of new HA formulas aimed at preventing sensitization in atopic children and avoiding clinical symptoms in infants suffering from CMA [12]. One of these proposed in vitro cellular degranulation assays uses the rat basophilic leukaemia cell line RBL-2H3 stably transfected with the a-chain of the human FceRI (RBL-huFceRI) as target cells. Limited availability and inter-lot variation of these allergic sera impede standardization of whey hydrolysate safety testing in degranulation assays
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