Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

<h4>Background</h4>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 t...

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Autores principales:	Gregory J Tanner, Malcolm J Blundell, Michelle L Colgrave, Crispin A Howitt
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7e63977cae83488bb68c6441fa753ec3
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spelling	oai:doaj.org-article:7e63977cae83488bb68c6441fa753ec32021-11-18T07:55:16ZQuantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.1932-620310.1371/journal.pone.0056456https://doaj.org/article/7e63977cae83488bb68c6441fa753ec32013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23509607/?tool=EBIhttps://doaj.org/toc/1932-6203<h4>Background</h4>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.<h4>Aim</h4>The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.<h4>Results</h4>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.<h4>Conclusions</h4>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.Gregory J TannerMalcolm J BlundellMichelle L ColgraveCrispin A HowittPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 2, p e56456 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Gregory J Tanner Malcolm J Blundell Michelle L Colgrave Crispin A Howitt Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.
description	<h4>Background</h4>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.<h4>Aim</h4>The factors affecting the accuracy of ELISA analysis of hordeins in beer were examined.<h4>Results</h4>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.<h4>Conclusions</h4>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.
format	article
author	Gregory J Tanner Malcolm J Blundell Michelle L Colgrave Crispin A Howitt
author_facet	Gregory J Tanner Malcolm J Blundell Michelle L Colgrave Crispin A Howitt
author_sort	Gregory J Tanner
title	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_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_sort	quantification of hordeins by elisa: the correct standard makes a magnitude of difference.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/7e63977cae83488bb68c6441fa753ec3
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Quantification of Hordeins by ELISA: the correct standard makes a magnitude of difference.

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