title: Application of human Fc epsilon RI alpha-chain-transfected RBL-2H3 cells for estimation of active serum IgE.
abstract: We transfected the alpha-chain of human FcepsilonRI into rat basophilic leukemia cell line RBL-2H3, established several stable transfected cells, and screened them by beta-hexosaminidase release induced by sensitization with human IgE and stimulation with anti-human IgE antibody. A cloned cell line RBL-hEIa-2B12 was the strongest responder among the transfected cell clones. The concentrations of cytosolic free Ca2+ concentration in the human IgE-sensitized cells increased after stimulation with anti-human IgE antibody. Thus, it is suggested that the alpha-chain of human FcepsilonRI is associated with the beta-chain and/or gamma-chain of rat FcepsilonRI, and that they form functional high affinity IgE receptor complexes. The total IgE concentrations of the sera from allergic patients were determined by using the beta-hexosaminidase release assay, where the transfected cells were sensitized with diluted and heat-inactivated (at 56 degrees C for 30 min) serum and stimulated with anti-human IgE antibody. The IgE concentration obtained correlated with those measured by an enzyme immunoassay method. beta-Hexosaminidase release induced by stimulation with 5 times diluted serum was sometimes less than the release induced by the same serum; diluted 25 times or 125 times, suggesting that these serum contained factors that blocked IgE binding to FcepsilonRI or cross-linking by anti-human IgE antibody. The results suggested that our system will be useful for detecting FcepsilonRIalpha-bindable IgE in human serum.
authors.affiliation: National Institute of Health Sciences, Division of Biochemistry and Immunochemistry, Tokyo, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Takagi
authors.forename: Ryosuke
authors.initials: R
authors.lastname: Nakamura
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada
mesh_terms: D000818:Animals; D015550:Cell Degranulation; D006801:Humans; D007124:Immunoenzyme Techniques; D007073:Immunoglobulin E / Q000032:analysis / Q000097:blood* / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D017455:Receptors, IgE; D007073:Immunoglobulin E