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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.

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Related in: MedlinePlus

Validation of hordein extraction methods.Half seed extracts were sequentially extracted from cv Sloop (A), Risø 56 (B), Risø 1508 (C), ULG 2.0 (D) in water (1), then 0.5 M NaCl (2), then IPA/DTT (3), then Urea/DTT (4), and 20 µg of total protein from each extract was subject to SDS-PAGE, blotted, interrogated with commercial anti-gliadin-HRP (Sigma) and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). Note lanes 2 and 3 are reversed in panels B and C. This blot was deliberately overloaded with protein, to maximise detection of hordeins in the aqueous and salt fractions, causing over exposure of lane 3. When loaded at a lower protein loading of 2 µg per lane a well resolved banding pattern was seen as in Fig. 1A.
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pone-0056456-g003: Validation of hordein extraction methods.Half seed extracts were sequentially extracted from cv Sloop (A), Risø 56 (B), Risø 1508 (C), ULG 2.0 (D) in water (1), then 0.5 M NaCl (2), then IPA/DTT (3), then Urea/DTT (4), and 20 µg of total protein from each extract was subject to SDS-PAGE, blotted, interrogated with commercial anti-gliadin-HRP (Sigma) and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). Note lanes 2 and 3 are reversed in panels B and C. This blot was deliberately overloaded with protein, to maximise detection of hordeins in the aqueous and salt fractions, causing over exposure of lane 3. When loaded at a lower protein loading of 2 µg per lane a well resolved banding pattern was seen as in Fig. 1A.

Mentions: We verified the efficacy of a simple IPA/DTT solvent extraction by western blots of total protein from half seed extracts of cv Sloop, Risø 56, Risø 1508 and ULG 2.0 interrogated with the Sigma commercial anti-gluten polyclonal antibody (Fig. 3). Very little antibody reactive protein was extracted by water or 0.5 M NaCl. In some cases, traces of the dominant bands seen in the IPA/DTT lane were also present in the water, NaCl and urea extracts. However, in all grains, the majority of the gluten antibody reactive proteins were extracted by IPA/DTT. Alternatively, lyophilised preparations may be dissolved with Urea/DTT or Urea/SDS solutions; the latter is particularly useful as a solubilisation step prior to SDS-PAGE.


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

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

Validation of hordein extraction methods.Half seed extracts were sequentially extracted from cv Sloop (A), Risø 56 (B), Risø 1508 (C), ULG 2.0 (D) in water (1), then 0.5 M NaCl (2), then IPA/DTT (3), then Urea/DTT (4), and 20 µg of total protein from each extract was subject to SDS-PAGE, blotted, interrogated with commercial anti-gliadin-HRP (Sigma) and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). Note lanes 2 and 3 are reversed in panels B and C. This blot was deliberately overloaded with protein, to maximise detection of hordeins in the aqueous and salt fractions, causing over exposure of lane 3. When loaded at a lower protein loading of 2 µg per lane a well resolved banding pattern was seen as in Fig. 1A.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056456-g003: Validation of hordein extraction methods.Half seed extracts were sequentially extracted from cv Sloop (A), Risø 56 (B), Risø 1508 (C), ULG 2.0 (D) in water (1), then 0.5 M NaCl (2), then IPA/DTT (3), then Urea/DTT (4), and 20 µg of total protein from each extract was subject to SDS-PAGE, blotted, interrogated with commercial anti-gliadin-HRP (Sigma) and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). Note lanes 2 and 3 are reversed in panels B and C. This blot was deliberately overloaded with protein, to maximise detection of hordeins in the aqueous and salt fractions, causing over exposure of lane 3. When loaded at a lower protein loading of 2 µg per lane a well resolved banding pattern was seen as in Fig. 1A.
Mentions: We verified the efficacy of a simple IPA/DTT solvent extraction by western blots of total protein from half seed extracts of cv Sloop, Risø 56, Risø 1508 and ULG 2.0 interrogated with the Sigma commercial anti-gluten polyclonal antibody (Fig. 3). Very little antibody reactive protein was extracted by water or 0.5 M NaCl. In some cases, traces of the dominant bands seen in the IPA/DTT lane were also present in the water, NaCl and urea extracts. However, in all grains, the majority of the gluten antibody reactive proteins were extracted by IPA/DTT. Alternatively, lyophilised preparations may be dissolved with Urea/DTT or Urea/SDS solutions; the latter is particularly useful as a solubilisation step prior to SDS-PAGE.

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