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

Tanner GJ, Blundell MJ, Colgrave ML, Howitt CA - PLoS ONE (2013)

Bottom Line: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt.In practice it is not feasible to isolate a representative hordein standard from each test food.MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.

View Article: PubMed Central - PubMed

Affiliation: Commonwealth Scientific and Industrial Research Organisation Plant Industry, Canberra, Australian Capital Territory, Australia.

ABSTRACT

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.

Show MeSH

Related in: MedlinePlus

The hordein content of wild type flours varies with variety and environment.The response of ELISA Systems anti-gluten sandwich assays to triplicate 10 ng alcohol soluble protein from different wild type flours. Mean A450±SE is shown for triplicate extracts for cv Sloop grown at CSIRO Ginninderra Experiment Station, Canberra, 1 or Yanco Agricultural Institute (NSW Dept. of Agriculture, Yanco, NSW), 2; cv Himalaya, 3; cv Bomi, 4; cv Carlsberg II, 5; cv Hindmarsh, 6; and cv Commander, 7, all grown at CSIRO Ginninderra Experiment Station, Canberra. Total protein was dissolved in Urea/DTT, the protein content measured, diluted 1/500 with ESD, and duplicate aliquots containing 10 ng of protein in approximately 2 µL added to ELISA wells containing a final volume of 100 µL ESD and the sandwich assay carried out. The untransformed data were analysed by one-way ANOVA and the LSD is shown. Columns with the same letter were not significantly different (GenStat).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g010: The hordein content of wild type flours varies with variety and environment.The response of ELISA Systems anti-gluten sandwich assays to triplicate 10 ng alcohol soluble protein from different wild type flours. Mean A450±SE is shown for triplicate extracts for cv Sloop grown at CSIRO Ginninderra Experiment Station, Canberra, 1 or Yanco Agricultural Institute (NSW Dept. of Agriculture, Yanco, NSW), 2; cv Himalaya, 3; cv Bomi, 4; cv Carlsberg II, 5; cv Hindmarsh, 6; and cv Commander, 7, all grown at CSIRO Ginninderra Experiment Station, Canberra. Total protein was dissolved in Urea/DTT, the protein content measured, diluted 1/500 with ESD, and duplicate aliquots containing 10 ng of protein in approximately 2 µL added to ELISA wells containing a final volume of 100 µL ESD and the sandwich assay carried out. The untransformed data were analysed by one-way ANOVA and the LSD is shown. Columns with the same letter were not significantly different (GenStat).

Mentions: The hordeins in 10 ng of alcohol soluble protein from wild type flours varied significantly (Fig. 10). Extracts of cv Commander were the most reactive, followed by extracts of cv Sloop. Extracts of cv’s Himalaya, Bomi, Carlsberg II, and Hindmarsh were not significantly different from each other. Extracts of cv Sloop grown at two different locations also varied significantly. The hordein concentration in developing grains is sensitive to growth conditions especially to the level of sulfate [43], [44], nitrogen [45] or day length [46]. It is therefore not surprising that hordein content differed between cultivars and locations. This observation adds an additional difficulty in choosing a suitable flour to act as a universal calibration standard.


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

Tanner GJ, Blundell MJ, Colgrave ML, Howitt CA - PLoS ONE (2013)

The hordein content of wild type flours varies with variety and environment.The response of ELISA Systems anti-gluten sandwich assays to triplicate 10 ng alcohol soluble protein from different wild type flours. Mean A450±SE is shown for triplicate extracts for cv Sloop grown at CSIRO Ginninderra Experiment Station, Canberra, 1 or Yanco Agricultural Institute (NSW Dept. of Agriculture, Yanco, NSW), 2; cv Himalaya, 3; cv Bomi, 4; cv Carlsberg II, 5; cv Hindmarsh, 6; and cv Commander, 7, all grown at CSIRO Ginninderra Experiment Station, Canberra. Total protein was dissolved in Urea/DTT, the protein content measured, diluted 1/500 with ESD, and duplicate aliquots containing 10 ng of protein in approximately 2 µL added to ELISA wells containing a final volume of 100 µL ESD and the sandwich assay carried out. The untransformed data were analysed by one-way ANOVA and the LSD is shown. Columns with the same letter were not significantly different (GenStat).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g010: The hordein content of wild type flours varies with variety and environment.The response of ELISA Systems anti-gluten sandwich assays to triplicate 10 ng alcohol soluble protein from different wild type flours. Mean A450±SE is shown for triplicate extracts for cv Sloop grown at CSIRO Ginninderra Experiment Station, Canberra, 1 or Yanco Agricultural Institute (NSW Dept. of Agriculture, Yanco, NSW), 2; cv Himalaya, 3; cv Bomi, 4; cv Carlsberg II, 5; cv Hindmarsh, 6; and cv Commander, 7, all grown at CSIRO Ginninderra Experiment Station, Canberra. Total protein was dissolved in Urea/DTT, the protein content measured, diluted 1/500 with ESD, and duplicate aliquots containing 10 ng of protein in approximately 2 µL added to ELISA wells containing a final volume of 100 µL ESD and the sandwich assay carried out. The untransformed data were analysed by one-way ANOVA and the LSD is shown. Columns with the same letter were not significantly different (GenStat).
Mentions: The hordeins in 10 ng of alcohol soluble protein from wild type flours varied significantly (Fig. 10). Extracts of cv Commander were the most reactive, followed by extracts of cv Sloop. Extracts of cv’s Himalaya, Bomi, Carlsberg II, and Hindmarsh were not significantly different from each other. Extracts of cv Sloop grown at two different locations also varied significantly. The hordein concentration in developing grains is sensitive to growth conditions especially to the level of sulfate [43], [44], nitrogen [45] or day length [46]. It is therefore not surprising that hordein content differed between cultivars and locations. This observation adds an additional difficulty in choosing a suitable flour to act as a universal calibration standard.

Bottom Line: A simple alcohol-dithiothreitol extraction protocol successfully extracts the majority of hordeins from barley flour and malt.In practice it is not feasible to isolate a representative hordein standard from each test food.MS quantification is undertaken using peptides that are specific and unique enabling the quantification of individual hordein isoforms.

View Article: PubMed Central - PubMed

Affiliation: Commonwealth Scientific and Industrial Research Organisation Plant Industry, Canberra, Australian Capital Territory, Australia.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus