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Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference

BACKGROUND: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be repr...

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Detalles Bibliográficos
Autores principales: Tanner, Gregory J., Blundell, Malcolm J., Colgrave, Michelle L., Howitt, Crispin A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585327/
https://www.ncbi.nlm.nih.gov/pubmed/23509607
http://dx.doi.org/10.1371/journal.pone.0056456
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author Tanner, Gregory J.
Blundell, Malcolm J.
Colgrave, Michelle L.
Howitt, Crispin A.
author_facet Tanner, Gregory J.
Blundell, Malcolm J.
Colgrave, Michelle L.
Howitt, Crispin A.
author_sort Tanner, Gregory J.
collection PubMed
description BACKGROUND: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. AIM: The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. RESULTS: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. CONCLUSIONS: Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.
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spelling pubmed-35853272013-03-18 Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference Tanner, Gregory J. Blundell, Malcolm J. Colgrave, Michelle L. Howitt, Crispin A. PLoS One Research Article BACKGROUND: Coeliacs require a life-long gluten-free diet supported by accurate measurement of gluten (hordein) in gluten-free food. The gluten-free food industry, with a value in excess of $6 billion in 2011, currently depends on two ELISA protocols calibrated against standards that may not be representative of the sample being assayed. AIM: The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined. RESULTS: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt. Primary hordein standards were purified by FPLC. ELISA detected different classes of purified hordeins with vastly different sensitivity. The dissociation constant (Kd) for a given ELISA reaction with different hordeins varied by three orders of magnitude. The Kd of the same hordein determined by ELISA using different antibodies varied by up to two orders of magnitude. The choice of either ELISA kit or hordein standard may bias the results and confound interpretation. CONCLUSIONS: Accurate determination of hordein requires that the hordein standard used to calibrate the ELISA reaction be identical in composition to the hordeins present in the test substance. In practice it is not feasible to isolate a representative hordein standard from each test food. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes which may vary between different hordeins and may not be related to the absolute hordein concentration. MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms. Public Library of Science 2013-02-28 /pmc/articles/PMC3585327/ /pubmed/23509607 http://dx.doi.org/10.1371/journal.pone.0056456 Text en © 2013 Tanner et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Tanner, Gregory J.
Blundell, Malcolm J.
Colgrave, Michelle L.
Howitt, Crispin A.
Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference
title Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference
title_full Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference
title_fullStr Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference
title_full_unstemmed Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference
title_short Quantification of Hordeins by ELISA: The Correct Standard Makes a Magnitude of Difference
title_sort quantification of hordeins by elisa: the correct standard makes a magnitude of difference
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3585327/
https://www.ncbi.nlm.nih.gov/pubmed/23509607
http://dx.doi.org/10.1371/journal.pone.0056456
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