title: Intra- and inter-laboratory validation of an innovative huFcεRIα-RBL-2H3 degranulation assay for in vitro allergenicity assessment of whey hydrolysates.
abstract: Cow's milk-derived whey hydrolysates are milk substitutes for cow's milk allergic infants. Safety assessment of these hydrolysates is crucial. Currently, huFcεRIα-RBL-2H3 cells, sensitized with serum IgE from cow's milk allergic patients, are used to assess in vitro residual allergenicity. However, limited availability and high inter-lot variation of sera impede the standardization of safety testing. Recently, we generated an oligoclonal pool of chimeric human (chu)IgE antibodies against bovine β-lactoglobulin (BLG) as an alternative for human serum. These antibodies demonstrated increased sensitivity, specificity and reproducibility. An inter-laboratory ring trial using our new degranulation assay with different whey-based hydrolysates was performed at four independent laboratories to investigate the robustness and reproducibility. RBL-2H3 cells expressing huFcεRIα were sensitized with our oligoclonal pool of anti-BLG chuIgE antibodies. The cells were subsequently incubated with an amino-acid based formula (AAF), two extensively hydrolyzed formulas (eHF) and three partially hydrolyzed formulas (pHF) to assess the degranulation upon challenge. Results demonstrated a very strong inter-laboratory correlation and the intra- and inter-laboratory variations were acceptable. The AAF and both eHFs showed no degranulation, whereas all pHFs demonstrated degranulation. The study showed that this degranulation assay is robust and reproducible within and between laboratories. This new in vitro degranulation assay seems predictive for allergenicity outcome and might therefore be considered as a relevant substitute for animal models.
authors.affiliation: Nutricia Research, Utrecht, The Netherlands; Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands. Electronic address: karen.knipping@danone.com.
authors.forename: Karen
authors.initials: K
authors.lastname: Knipping
authors.affiliation: Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Manon
authors.initials: M
authors.lastname: van Roest
authors.affiliation: Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands.
authors.forename: Laura
authors.initials: L
authors.lastname: Kruijssen
authors.affiliation: Utrecht University of Applied Sciences, Utrecht, The Netherlands.
authors.forename: Mieke
authors.initials: M
authors.lastname: Smits
authors.affiliation: Utrecht University of Applied Sciences, Utrecht, The Netherlands.
authors.forename: Marc
authors.initials: M
authors.lastname: Teunis
authors.affiliation: Bioceros Holding BV, The Netherlands.
authors.forename: Linda
authors.initials: L
authors.lastname: Cox
authors.affiliation: Bioceros Holding BV, The Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: de Jong
authors.affiliation: Bioceros Holding BV, The Netherlands.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Simons
authors.affiliation: Bioceros Holding BV, The Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: Boon
authors.affiliation: National Institute of Health Sciences, Division of Foods, Tokyo, Japan.
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.affiliation: FrieslandCampina Domo, Wageningen, The Netherlands.
authors.forename: Marjan
authors.initials: M
authors.lastname: Gros
authors.affiliation: Nutricia Research, Utrecht, The Netherlands.
authors.forename: Diane
authors.initials: D
authors.lastname: Kegler
authors.affiliation: Nutricia Research, Utrecht, The Netherlands; Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Johan
authors.initials: J
authors.lastname: Garssen
authors.affiliation: Nutricia Research, Utrecht, The Netherlands; Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: Léon M J
authors.initials: LM
authors.lastname: Knippels
authors.affiliation: Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands; Utrecht University of Applied Sciences, Utrecht, The Netherlands.
authors.forename: Raymond
authors.initials: R
authors.lastname: Pieters
mesh_terms: D000485:Allergens / Q000276:immunology*; D000818:Animals; D001681:Biological Assay; D015550:Cell Degranulation; D002460:Cell Line; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D007753:Laboratories; D007782:Lactoglobulins / Q000276:immunology*; D008407:Mast Cells / Q000502:physiology*; D008892:Milk / Q000276:immunology; D017455:Receptors, IgE / Q000276:immunology*; D015203:Reproducibility of Results
chemical_list: D000485:Allergens; D007782:Lactoglobulins; D017455:Receptors, IgE; D007073:Immunoglobulin E
keywords: Chimeric; IgE; Monoclonal antibodies; RBL; Whey hydrolysates; β-Lactoglobulin