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Measuring hordein (gluten) in beer--a comparison of ELISA and mass spectrometry.

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

Bottom Line: Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten.Several barley beers also contained undeclared wheat proteins.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: Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.

Methods: We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).

Results: Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60-140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.

Conclusions: ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this 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. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.

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Related in: MedlinePlus

Coomassie stained protein gels (16.7 µg total protein loaded per lane) of flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); or ULG 2.0 (D); were calibrated against Benchmark 10 kDa protein standards (Lane M, Invitrogen).The positions of serpin Z4 (*1) and LTP (*2) are indicated.
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pone-0056452-g001: Coomassie stained protein gels (16.7 µg total protein loaded per lane) of flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); or ULG 2.0 (D); were calibrated against Benchmark 10 kDa protein standards (Lane M, Invitrogen).The positions of serpin Z4 (*1) and LTP (*2) are indicated.

Mentions: Staining of total protein extracts from flour samples following SDS-PAGE visualised a large number of proteins ranging from 10 kDa to 100 kDa (Fig. 1: flour). Similar analysis of malt samples identified fewer protein species with an increased intensity observed for smaller peptides (less than 10 kDa), indicating that significant proteolysis had occurred during germination and malting (Fig. 1: malt). In contrast SDS-PAGE of wort and beer, together with in-gel protein digestion and analysis, indicated that the protein composition had been dramatically enriched for two protein families, serpin Z4 at 43 kDa and LTP1 at approximately 9 kDa, accounting for the bulk of the protein in these fractions (Fig. 1; 1 & 2 respectively, Supplementary Results in Information S1, and Fig. S1 & S2 in Information S1). There were differences in the serpin Z4 proportion of the beer produced by different lines, with the most Z4 produced by beer made from cv Sloop, intermediate levels produced by Risø 56, and relatively lower proportions of serpin Z4 produced by Risø 1508 and ULG 2.0.


Measuring hordein (gluten) in beer--a comparison of ELISA and mass spectrometry.

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

Coomassie stained protein gels (16.7 µg total protein loaded per lane) of flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); or ULG 2.0 (D); were calibrated against Benchmark 10 kDa protein standards (Lane M, Invitrogen).The positions of serpin Z4 (*1) and LTP (*2) are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585340&req=5

pone-0056452-g001: Coomassie stained protein gels (16.7 µg total protein loaded per lane) of flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); or ULG 2.0 (D); were calibrated against Benchmark 10 kDa protein standards (Lane M, Invitrogen).The positions of serpin Z4 (*1) and LTP (*2) are indicated.
Mentions: Staining of total protein extracts from flour samples following SDS-PAGE visualised a large number of proteins ranging from 10 kDa to 100 kDa (Fig. 1: flour). Similar analysis of malt samples identified fewer protein species with an increased intensity observed for smaller peptides (less than 10 kDa), indicating that significant proteolysis had occurred during germination and malting (Fig. 1: malt). In contrast SDS-PAGE of wort and beer, together with in-gel protein digestion and analysis, indicated that the protein composition had been dramatically enriched for two protein families, serpin Z4 at 43 kDa and LTP1 at approximately 9 kDa, accounting for the bulk of the protein in these fractions (Fig. 1; 1 & 2 respectively, Supplementary Results in Information S1, and Fig. S1 & S2 in Information S1). There were differences in the serpin Z4 proportion of the beer produced by different lines, with the most Z4 produced by beer made from cv Sloop, intermediate levels produced by Risø 56, and relatively lower proportions of serpin Z4 produced by Risø 1508 and ULG 2.0.

Bottom Line: Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten.Several barley beers also contained undeclared wheat proteins.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: Subjects suffering from coeliac disease, gluten allergy/intolerance must adopt a lifelong avoidance of gluten. Beer contains trace levels of hordeins (gluten) which are too high to be safely consumed by most coeliacs. Accurate measurement of trace hordeins by ELISA is problematic.

Methods: We have compared hordein levels in sixty beers, by sandwich ELISA, with the level determined using multiple reaction monitoring mass spectrometry (MRM-MS).

Results: Hordein levels measured by ELISA varied by four orders of magnitude, from zero (for known gluten-free beers) to 47,000 µg/mL (ppm; for a wheat-based beer). Half the commercial gluten-free beers were free of hordein by MS and ELISA. Two gluten-free and two low-gluten beers had zero ELISA readings, but contained significant hordein levels (p<0.05), or near average (60-140%) hordein levels, by MS, respectively. Six beers gave false negatives, with zero ELISA readings but near average hordein content by MS. Approximately 20% of commercial beers had ELISA readings less than 1 ppm, but a near average hordein content by MS. Several barley beers also contained undeclared wheat proteins.

Conclusions: ELISA results did not correlate with the relative content of hordein peptides determined by MS, with all barley based beers containing hordein. We suggest that mass spectrometry is more reliable than ELISA, as ELISA enumerates only the concentration of particular amino-acid epitopes; this 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. This outlines the problem of relying solely on ELISA determination of gluten in beverages such as beer and highlights the need for the development of new sensitive and selective quantitative assay such as MS.

Show MeSH
Related in: MedlinePlus