Quantification of gliadin levels to the picogram level by flow cytometry.
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 alpha-, beta-, gamma-, and omega-gliadins.


METHODS
In the one-site assay, the antigen (gliadin standard or food extract) was adsorbed on 3-mum 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.


CONCLUSIONS
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.
Capparelli
Rosanna
R
School of Biotechnological Sciences, University of Naples Federico II, Via Università 133, 80055 Portici, Italy. capparel@unina.it
Ventimiglia
Ilaria
I
Longobardo
Luigi
L
Iannelli
Domenico
D
D000818:Animals; D000918:Antibody Specificity; D005434:Flow Cytometry / Q000379:methods* / Q000592:standards; D005903:Gliadin / Q000737:chemistry* / Q000276:immunology; D006041:Goats; D008832:Microchemistry / Q000379:methods* / Q000592:standards; D036103:Nanotechnology / Q000379:methods* / Q000592:standards; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D012015:Reference Standards
D010446:Peptide Fragments; D005903:Gliadin