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Quantification of gliadin levels to the picogram level by flow cytometry
Author(s) -
Capparelli Rosanna,
Ventimiglia Ilaria,
Longobardo Luigi,
Iannelli Domenico
Publication year - 2005
Publication title -
cytometry part a
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.316
H-Index - 90
eISSN - 1552-4930
pISSN - 1552-4922
DOI - 10.1002/cyto.a.20109
Subject(s) - gliadin , fluorescein isothiocyanate , antibody , flow cytometry , chemistry , gluten , antigen , gluten free , detection limit , microbiology and biotechnology , chromatography , biochemistry , immunology , biology , fluorescence , physics , quantum mechanics
Background Celiac disease is a widely prevalent enteropathy caused by intolerance to gliadin, one of the gluten proteins. We developed two methods for the analysis of gliadin levels. Both methods use flow cytometry and rat antibodies against a 16‐residue peptide of gliadin. The peptide is common to the α‐, β‐, γ‐, and ω‐gliadins. Methods In the one‐site assay, the antigen (gliadin standard or food extract) was adsorbed on 3‐μm latex particles. Sensitized particles were then incubated, in this order, with rat anti‐gliadin peptide antibodies and anti‐rat immunoglobulin G antibodies labeled with fluorescein isothiocyanate. In the two‐site assay, the antigen was trapped on the latex particles by rat anti‐gliadin antibodies and then measured by the same antibodies labeled with fluorescein. Results Detection limits were 1 ng/ml for the one‐site assay and 10 pg/ml for the two‐site assay. The two‐site assay displayed gliadin at concentrations above the limit proposed by the Codex Alimentarius in 2 of 40 gluten‐free products. Conclusion There is a growing concern that gliadin, even when present in gluten‐free foods within the limit fixed by the Codex Alimentarius, over the long term may become toxic to patients with celiac disease. The techniques described in this study provide an opportunity to further decrease the acceptable limit of gliadin in gluten‐free foods. © 2005 Wiley‐Liss, Inc.