title: Improved ELISA method for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods.
abstract: For monitoring the occurrence of IgE antibody specific for novel proteins in genetically modified (GM) foods, ELISA is the most convenient method. The levels of IgE specific for recombinant proteins, phosphinothricin-N-acetyltransferase (PAT), CP4-EPSPS, and Cry9C were determined by ELISA using the sera from patients allergic to known allergens. Ovalbumin (OVA) and OVA-positive patient sera were used as positive control. In the ELISA, 20-fold-diluted sera tested were mostly negative for the specific IgE. However, the PAT-specific, but not CP4-EPSPS- or Cry9C-specific IgE in some patients was apparently higher than that of the healthy volunteers. To clarify the binding specificity of the antibody, we pre-incubated the sera with soluble PAT, but the inhibition was marginal, suggesting that the binding was non-specific. Therefore, we used 1M NaCl as a washing buffer to remove IgE non-specifically bound to the coated PAT. This washing step efficiently decreased non-specific binding. In contrast, OVA-specific IgE binding to OVA-coated plate was not affected by the washing. Finally, in this pilot study significant levels of IgE antibodies specific for the three proteins were not detected in the sera of Japanese food-allergy patients.
authors.affiliation: Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
authors.forename: Kayoko
authors.initials: K
authors.lastname: Takagi
authors.forename: Reiko
authors.initials: R
authors.lastname: Teshima
authors.forename: Osamu
authors.initials: O
authors.lastname: Nakajima
authors.forename: Haruyo
authors.initials: H
authors.lastname: Okunuki
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Sawada
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000276:immunology*; D000123:Acetyltransferases / Q000276:immunology*; D000485:Allergens / Q000276:immunology; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D004731:Endotoxins / Q000276:immunology*; D004781:Environmental Exposure; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005512:Food Hypersensitivity / Q000276:immunology; D006460:Hemolysin Proteins; D006801:Humans; D007073:Immunoglobulin E / Q000097:blood* / Q000276:immunology; D010047:Ovalbumin / Q000276:immunology; D010572:Pest Control, Biological; D030821:Plants, Genetically Modified / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology; D013025:Glycine max / Q000009:adverse effects* / Q000235:genetics / Q000276:immunology
chemical_list: D000485:Allergens; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D007073:Immunoglobulin E; D010047:Ovalbumin; D000123:Acetyltransferases; C056243:phosphinothricin N-acetyltransferase; D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase