Detection of wheat gliadin contamination of gluten-free foods by a monoclonal antibody dot immunobinding assay

Background—Future European Community regulations will require a sensitive and specific assay for measurement of coeliac toxic gluten proteins in foods marketed as gluten-free. To avoid spurious cross reactions with non-toxic proteins, specific antibodies and target antigens are required. A synthetic 19 amino acid peptide of A gliadin has been shown to cause deterioration in the morphology of small intestinal biopsy specimens of coeliac patients in remission.Aims—To develop an assay for detection of gluten in foods, based on measurement of a known toxic peptide.Methods—A monoclonal antibody raised against the toxic A gliadin peptide, with a polyclonal anti-unfractionated gliadin capture antibody, was used to develop a double sandwich enzyme linked immunosorbent assay (ELISA) for the measurement of gluten in foods.Results—Standard curves for gliadin and for rye, barley, and oat prolamins were produced. The sensitivity of the assay was 4 ng/ml of gliadin, 500 ng/ml for rye prolamins, and 1000 ng/ml for oat and barley prolamins. The assay could detect gluten in cooked foods, although at reduced sensitivity. Prolamins from coeliac non-toxic rice, maize, millet, and sorghum did not cross react in the assay. A variety of commercially available gluten- free foods were analysed; small quantities of gluten were detected in some products.Conclusion—The assay may form the basis of a sensitive method for measurement of gluten in foods for consumption by patients with coeliac disease.

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Monoclonal Antibodies to Measure Wheat Gliadin in Gluten-Free Foods

Clinical Science ◽

10.1042/cs071069p ◽

1986 ◽

Vol 71 (s15) ◽

pp. 69P-69P

Author(s):

A.R. Freedman ◽

G. Galfre ◽

P.J. Ciclitira

Keyword(s):

Monoclonal Antibodies ◽

Gluten Free ◽

Wheat Gliadin

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Production of monoclonal antibodies to immunogenic peptides of ω-glaidin/c-hordein and rye-secalin for gluten free food screening

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v15i1.1360 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Widya Abd Wahab ◽

H Julia Ellis ◽

Paul J Ciclitira

Keyword(s):

Monoclonal Antibody ◽

Monoclonal Antibodies ◽

Free Food ◽

Enzyme Linked Immunosorbent Assay ◽

Gluten Free ◽

Immunization Schedule ◽

Booster Injection ◽

Immunogenic Peptides ◽

T Cell Lines ◽

Class Antibody

Introduction: The ω-glaidin/c-hordein (QPFPQPEQPFPW) and rye-secalin (QPFPQPQQPIPQ) peptides have previously demonstrated immunogenicity in sensitised coeliac T cell lines. The development of monoclonal antibody to those immunogenic peptides is presented with a view of developing an improved enzyme linked immunosorbent assay (ELISA) as a reliable tool to screen the safety of foods specialised for coeliac disease (CD) patients. Methods: Balb/C mice were fed with gluten free food. The immunogens were conjugated to purified tuberculin protein derivative (PPD) with glutaraldehyde and emulsified in Freund’s adjuvant. The employed immunization schedule included 3-5 weeks intervals, followed by a final intravenous injection without adjuvant, 3-4 days prior to fusion. Results: The antibody produced was IgM rather than IgG, although the hybridoma was successfully generated. The IgM class antibody is known to be relatively unstable, so could not be used in kits designed for food screening. Conclusions: The immunogenic peptides could possibly be used to raise monoclonal antibodies for gluten screening. However, a single booster injection might be insufficient to stimulate the spleen directly or the mice should be immunized with higher concentration of immunogen. This could be improved by including multiple booster administrations and increasing the dose.

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Assessment of the gluten content in gluten-free labeled foods: comparison of two gluten detection methods

Segurança Alimentar e Nutricional ◽

10.20396/san.v17i2.8634794 ◽

2015 ◽

Vol 17 (2) ◽

pp. 70

Author(s):

Álvaro Macedo Laureano ◽

Themis Reverbel da Silveira

Keyword(s):

Monoclonal Antibody ◽

Free Product ◽

Screening Test ◽

Reliable Method ◽

Kappa Coefficient ◽

Detection Methods ◽

Immunochromatographic Test ◽

Gluten Free ◽

Elisa Kit ◽

Significant Difference

This study was designed to compare the effectiveness of the R5-ELISA and immunochromatographic assays in detecting gluten in foods labeled gluten-free and to determine if the immunochromatographic method is a sensitive and reliable method for detecting gluten at safe levels for celiac patients. We analyzed seventy different commercially foods available in Brazil, labeled “gluten-free”. Gluten was extracted by ethanol precipitation and, subsequently, analyzed using a commercial immunochromatographic test and ELISA kit, both based in a monoclonal antibody. The analysis of sensitivity and specificity was made using the kappa coefficient. More than a quarter of the samples (28.6%) analyzed by ELISA contained levels of gluten greater than 5 mg/kg. Almost half of these (12.9%) exhibited levels that exceeded 20 mg/kg, the maximum gluten level recommended by the Codex Alimentarius for a naturally gluten-free product. We found 27.1% of the samples tested positive in the immunochromatographic test. There was no statistically significant difference between the results of the ELISA (detection value ≥ 5 mg/kg) and the immunochromatographic test. Comparing the ELISA (≥ 5 mg/kg) and immunochromatographic test, we obtained 90% sensitivity and 98% specificity (Kappa of 0.89). We found gluten in a high proportion of the samples tested using both methods. In this study we also demonstrate that the immunochromatographic method is nearly as sensitive as the ELISA in detecting gluten levels and thus may serve as an inexpensive and rapid alternative to the R5-ELISA screening test.

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Anti-IL-15 monoclonal antibody for the treatment of coeliac disease: the light at the end of the gluten free diet tunnel?

Digestive Medicine Research ◽

10.21037/dmr-20-28 ◽

2020 ◽

Vol 3 ◽

pp. 107-107

Author(s):

Stefan L. Popa ◽

Giuseppe Chiarioni

Keyword(s):

Monoclonal Antibody ◽

Coeliac Disease ◽

Gluten Free Diet ◽

Gluten Free

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A web-like network of type vi collagen in human skin is revealed by immuno-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103978 ◽

1988 ◽

Vol 46 ◽

pp. 382-383

Author(s):

Douglas R. Keene ◽

Robert W. Glanville ◽

Eva Engvall

Keyword(s):

Electron Microscopy ◽

Monoclonal Antibody ◽

Human Skin ◽

Basement Membranes ◽

Primary Antibody ◽

Fat Cells ◽

Secondary Antibody ◽

Type Vi Collagen ◽

Dermal Matrix ◽

Immuno Electron Microscopy

A mouse monoclonal antibody (5C6) prepared against human type VI collagen (1) has been used in this study to immunolocalize type VI collagen in human skin. The enbloc method used involves exposing whole tissue pieces to primary antibody and 5 nm gold conjugated secondary antibody before fixation, and has been described in detail elsewhere (2).Biopsies were taken from individuals ranging in age from neonate to 65 years old. By immuno-electron microscopy, type VI collagen is found to be distributed as a fine branching network closely associated with (but not attached to) banded collagen fibrils containing types I and III collagen (Fig. 1). It appears to enwrap fibers, to weave between individual fibrils within a fiber, and to span the distance separating fibers, creating a “web-like network” which entraps fibers within deep papillary and reticular dermal layers (Fig. 2). Relative to that in the dermal matrix, the concentration of type VI collagen is higher around endothelial basement membranes limiting the outer boundaries of nerves, capillaries, and fat cells (Fig. 3).

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