title: Development of β-lactoglobulin-specific chimeric human IgEκ monoclonal antibodies for in vitro safety assessment of whey hydrolysates.
abstract: BACKGROUND
  Cow'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.
  
  
  OBJECTIVE
  An 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.
  
  
  METHODS
  Mice 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.
  
  
  RESULTS
  After 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.
  
  
  CONCLUSIONS
  Usage 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.
authors.affiliation: Nutricia Research B.V., Utrecht, The Netherlands; Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Karen
authors.initials: K
authors.lastname: Knipping
authors.affiliation: Bioceros Holding B.V., Utrecht, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Simons
authors.affiliation: Nutricia Research B.V., Utrecht, The Netherlands.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Buelens-Sleumer
authors.affiliation: Bioceros Holding B.V., Utrecht, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: Cox
authors.affiliation: Bioceros Holding B.V., Utrecht, The Netherlands.
authors.forename: Marcel
authors.initials: M
authors.lastname: den Hartog
authors.affiliation: Bioceros Holding B.V., Utrecht, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: de Jong
authors.affiliation: Division of Foods, National Institute of Health Sciences, Tokyo, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.affiliation: Nutricia Research B.V., Utrecht, The Netherlands; Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Garssen
authors.affiliation: Bioceros Holding B.V., Utrecht, The Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: Boon
authors.affiliation: Nutricia Research B.V., Utrecht, The Netherlands; Division of Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Léon M J
authors.initials: LM
authors.lastname: Knippels
mesh_terms: D000485:Allergens / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002364:Caseins / Q000276:immunology; D002417:Cattle; D002460:Cell Line; D002648:Child; D005260:Female; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007782:Lactoglobulins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D016269:Milk Hypersensitivity / Q000276:immunology; D011492:Protein Hydrolysates / Q000276:immunology; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology; D015203:Reproducibility of Results; D000067796:Whey / Q000276:immunology*; D000067816:Whey Proteins / Q000276:immunology
chemical_list: D000485:Allergens; D000911:Antibodies, Monoclonal; D002364:Caseins; C475664:FCER1A protein, human; D007782:Lactoglobulins; D011492:Protein Hydrolysates; D017455:Receptors, IgE; D000067816:Whey Proteins; D007073:Immunoglobulin E