Development of β-Lactoglobulin-Specific Chimeric Human IgEκ Monoclonal Antibodies for In Vitro Safety Assessment of Whey Hydrolysates

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.

Glycation of the Major Milk Allergen β‐Lactoglobulin Changes Its Allergenicity by Alterations in Cellular Uptake and Degradation

“…Furthermore, in our basophil activation experiments, glycation of BLG reduced its capacity to crosslink FcεRI receptors on RBL cells. In extensively glycated BLG, all residues containing free amino group (16 lysines, two arginines, and N‐terminal amino group of Leu 1) seem to be glycated, while all of the BLG‐specific chimeric human IgE monoclonal antibodies used in this study recognize peptides containing at least 1–2 residues with possible glycation sites . Therefore, it is highly likely that extensive BLG glycation reduced mAb IgE binding by changing epitope structure, and led to reduced basophil activation.…”

“…EXiLE assay was performed as described previously with slight modifications. RS‐ATL8 cells (5 × 10 4 cells per 50 μL per well) in 96‐wells flat bottom culture plate were sensitized overnight with a pool of six anti‐BLG chimeric IgE monoclonal antibodies (1 μg mL −1 each individual humanized IgE) . Cells were washed once with sterile PBS and then stimulated for 1 h at 37 °C in a 5% CO 2 incubator with 50 μL per well of native and glycated BLG diluted in MEM containing 10% FCS in four different concentrations (1, 10, 100, and 1000 ng mL −1 ) or with 1 μg mL −1 goat anti‐human IgE antibodies (DakoCytomation, Glostrup, Denmark) as control.…”

Peptide size profile and residual immunogenic milk protein or peptide content in extensively hydrolyzed infant formulas

“…This assay was developed with IgE directed against "allergenic" immunodominant regions/epitopes on bovine BLG, also recognized by serum IgE from CMPA infants. 7 A detailed description of the laboratory methods is provided in Table S2.…”

Hydrolyzed diets may stimulate food-reactive lymphocytes in dogs

“…As previously described [5,14], the antibodies used in the flow cytometry analysis included anti-dog CD4 rat antibody labeled with Alexa Fluor 647 (Clone YKIX302.9) (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and anti-human CD25 antibody labeled with phycoerythrin (Clone ACT-1) (DAKO Cytomation, Glostrup, Denmark), as well as isotype controls such as Alexa Fluor 647-labeled purified rat IgG2a (Bio-Rad Laboratories), and PE-labeled mouse IgG1 (Bio-Rad Laboratories). Stained dead cells were excluded by propidium iodide incorporation.…”

“…Flow cytometry analysis revealed that the type IV lymphocytes associated with hypersensitivity were CD4 + helper T-lymphocytes [5,21]. Theoretically, these types of lymphocytes are reactive to proteins or peptides that do not induce IgE-mediated reactions [10,14]. Therefore, it is clear that clinical use of hydrolyzed diets, as part of food elimination programs, may not be appropriate for all dogs with food hypersensitivity.…”