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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

Western blot of 8.3 µg of total protein per lane visualised with 1/2000 diluted anti-gliadin-HRP from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D).The location of known proteins was confirmed by protein sequencing of replicate gels [5]: 1, Serpin Z4; 2, LTP1; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ-2-hordein; 7, γ-3-hordein; and 8, D-hordein.
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pone-0056452-g002: Western blot of 8.3 µg of total protein per lane visualised with 1/2000 diluted anti-gliadin-HRP from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D).The location of known proteins was confirmed by protein sequencing of replicate gels [5]: 1, Serpin Z4; 2, LTP1; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ-2-hordein; 7, γ-3-hordein; and 8, D-hordein.

Mentions: Analysis of a replicate anti-gliadin western blot identified the main hordein families (Fig. 2: flour). These westerns were deliberately loaded with a high protein load to maximise the detection of hordeins in wort and beer. In all cases hordein bands in wort and beer were almost absent, indicating very low hordein levels in all wort and beer fractions (Fig. 2; wort and beer). The relative lack of hordeins in flour extracts from the hordein double- line ULG 2.0 can be seen when anti-hordein western blots are compared with those from parental lines Risø 56, Risø 1508 and the wild type cv Sloop. Only two significant bands corresponding to known hordeins, D- and γ-3-hordein (Fig. 2D, 7 & 8), were seen in blots of total protein from ULG 2.0. At this high protein loading other proteins, in addition to hordeins, were detected presumably due to a weak homology to the epitopes detected by the antibody. This is particularly clear in the extracts of ULG 2.0 flour, where faint western bands are seen at approximately 15, 20, 32, 48, 50, and 60 kDa. Previous protein sequencing of ULG 2.0 has shown that ULG 2.0 only accumulates γ-3 hordein and D-hordein [5]. Additional bands due to serpin Z4 and LTP (Fig. 2; 1 and 2 respectively) were also seen in all wort and beer fractions, however, no hordein bands were seen in these fractions. The location of known proteins was confirmed by protein sequencing of replicate gels (Fig. 2: 1, serpin Z4; 2, LTP 1 and 2; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ -2-hordein; 7, γ-3-hordein and 8, D-hordein [5].


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)

Western blot of 8.3 µg of total protein per lane visualised with 1/2000 diluted anti-gliadin-HRP from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D).The location of known proteins was confirmed by protein sequencing of replicate gels [5]: 1, Serpin Z4; 2, LTP1; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ-2-hordein; 7, γ-3-hordein; and 8, D-hordein.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056452-g002: Western blot of 8.3 µg of total protein per lane visualised with 1/2000 diluted anti-gliadin-HRP from flour, malt, wort, and beer produced from: cv Sloop (A); Risø 56 (B); Risø 1508 (C); and ULG 2.0 (D).The location of known proteins was confirmed by protein sequencing of replicate gels [5]: 1, Serpin Z4; 2, LTP1; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ-2-hordein; 7, γ-3-hordein; and 8, D-hordein.
Mentions: Analysis of a replicate anti-gliadin western blot identified the main hordein families (Fig. 2: flour). These westerns were deliberately loaded with a high protein load to maximise the detection of hordeins in wort and beer. In all cases hordein bands in wort and beer were almost absent, indicating very low hordein levels in all wort and beer fractions (Fig. 2; wort and beer). The relative lack of hordeins in flour extracts from the hordein double- line ULG 2.0 can be seen when anti-hordein western blots are compared with those from parental lines Risø 56, Risø 1508 and the wild type cv Sloop. Only two significant bands corresponding to known hordeins, D- and γ-3-hordein (Fig. 2D, 7 & 8), were seen in blots of total protein from ULG 2.0. At this high protein loading other proteins, in addition to hordeins, were detected presumably due to a weak homology to the epitopes detected by the antibody. This is particularly clear in the extracts of ULG 2.0 flour, where faint western bands are seen at approximately 15, 20, 32, 48, 50, and 60 kDa. Previous protein sequencing of ULG 2.0 has shown that ULG 2.0 only accumulates γ-3 hordein and D-hordein [5]. Additional bands due to serpin Z4 and LTP (Fig. 2; 1 and 2 respectively) were also seen in all wort and beer fractions, however, no hordein bands were seen in these fractions. The location of known proteins was confirmed by protein sequencing of replicate gels (Fig. 2: 1, serpin Z4; 2, LTP 1 and 2; 3, B-hordeins; 4, C-hordeins; 5, γ-1-hordein; 6, γ -2-hordein; 7, γ-3-hordein and 8, D-hordein [5].

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