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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

Repeated freeze thaw cycles do not affect the ELISA response.One mL aliquots of either total Sloop hordein (1.0 mg/mL; in a solution containing 8 M urea, 1% (w/v) DTT, 20 mM triethanolamine-HCl adjusted to pH 6 (A) or Sloop beer (B) were repeatedly frozen in liquid nitrogen and thawed in a water bath at RT. Aliquots were retained at 4°C after the indicated freeze-thaw cycle and diluted with ED buffer as required and duplicates analysed for hordeins by an ELISA Systems kit. The mean A450±SE is shown. Error bars are not shown where the S.E. is less than the line thickness. In each experiment the means were not significantly different (P<0.05) by one-way ANOVA (GenStat) and the least significant difference (LSD) is shown. Final concentration of hordein was 1000 ppb. Final dilution of Sloop beer was 1/500.
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pone-0056456-g009: Repeated freeze thaw cycles do not affect the ELISA response.One mL aliquots of either total Sloop hordein (1.0 mg/mL; in a solution containing 8 M urea, 1% (w/v) DTT, 20 mM triethanolamine-HCl adjusted to pH 6 (A) or Sloop beer (B) were repeatedly frozen in liquid nitrogen and thawed in a water bath at RT. Aliquots were retained at 4°C after the indicated freeze-thaw cycle and diluted with ED buffer as required and duplicates analysed for hordeins by an ELISA Systems kit. The mean A450±SE is shown. Error bars are not shown where the S.E. is less than the line thickness. In each experiment the means were not significantly different (P<0.05) by one-way ANOVA (GenStat) and the least significant difference (LSD) is shown. Final concentration of hordein was 1000 ppb. Final dilution of Sloop beer was 1/500.

Mentions: A: The eluant A280 from a 1 mL injection of Urea/DTT extract from cv Sloop, Risø 1508, Risø 56 or ULG 2.0 is shown with individual curves offset for clarity. The identity of peaks containing oxidised DTT (DTTox), B- (B-Hor), C- (C-Hor), and γ (γ-Hor)-hordeins of cv Sloop are shown. The eluate from 20 mL to 80 mL was pooled and lyophilised for total hordeins from each line. B & C: Fractions enriched for C-hordeins from cv Sloop (B, peak 3) and Risø 56 (C, peak 8); and γ-hordeins from Risø 56 (C, peaks at 9) were isolated by pooling the indicated eluate (–). Peak 2 did not contain any protein. Peaks 4, 5, 6 were not analysed.


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

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

Repeated freeze thaw cycles do not affect the ELISA response.One mL aliquots of either total Sloop hordein (1.0 mg/mL; in a solution containing 8 M urea, 1% (w/v) DTT, 20 mM triethanolamine-HCl adjusted to pH 6 (A) or Sloop beer (B) were repeatedly frozen in liquid nitrogen and thawed in a water bath at RT. Aliquots were retained at 4°C after the indicated freeze-thaw cycle and diluted with ED buffer as required and duplicates analysed for hordeins by an ELISA Systems kit. The mean A450±SE is shown. Error bars are not shown where the S.E. is less than the line thickness. In each experiment the means were not significantly different (P<0.05) by one-way ANOVA (GenStat) and the least significant difference (LSD) is shown. Final concentration of hordein was 1000 ppb. Final dilution of Sloop beer was 1/500.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g009: Repeated freeze thaw cycles do not affect the ELISA response.One mL aliquots of either total Sloop hordein (1.0 mg/mL; in a solution containing 8 M urea, 1% (w/v) DTT, 20 mM triethanolamine-HCl adjusted to pH 6 (A) or Sloop beer (B) were repeatedly frozen in liquid nitrogen and thawed in a water bath at RT. Aliquots were retained at 4°C after the indicated freeze-thaw cycle and diluted with ED buffer as required and duplicates analysed for hordeins by an ELISA Systems kit. The mean A450±SE is shown. Error bars are not shown where the S.E. is less than the line thickness. In each experiment the means were not significantly different (P<0.05) by one-way ANOVA (GenStat) and the least significant difference (LSD) is shown. Final concentration of hordein was 1000 ppb. Final dilution of Sloop beer was 1/500.
Mentions: A: The eluant A280 from a 1 mL injection of Urea/DTT extract from cv Sloop, Risø 1508, Risø 56 or ULG 2.0 is shown with individual curves offset for clarity. The identity of peaks containing oxidised DTT (DTTox), B- (B-Hor), C- (C-Hor), and γ (γ-Hor)-hordeins of cv Sloop are shown. The eluate from 20 mL to 80 mL was pooled and lyophilised for total hordeins from each line. B & C: Fractions enriched for C-hordeins from cv Sloop (B, peak 3) and Risø 56 (C, peak 8); and γ-hordeins from Risø 56 (C, peaks at 9) were isolated by pooling the indicated eluate (–). Peak 2 did not contain any protein. Peaks 4, 5, 6 were not analysed.

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