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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

Optimisation of dilution and extraction protocol for ELISA Systems assay.The response of the ELISA Systems sandwich assays to a commercial lager, (Tanner this journal, this issue, Beer 7) extracted with different buffers was measured by adding triplicate samples of commercial beer (100 µL) to 0.9 mL of either: (A & B) dilute ELISA Systems extraction buffer; (C, D, and E) Ridascreen extraction cocktail; (F) 60% (v/v) ethanol; or (G) Urea/DTT. The solutions were mixed at either room temperature (A, C, D, F, and G) or 60°C (B, E) for 1 h and an aliquot diluted 1/100 fold with ED buffer (A, B, D, F, G). Solutions C and E were diluted with ten-fold with 80% (v/v) ethanol, and then diluted a further ten-fold with dilute RD buffer and assayed for hordein. Hordeins were measured by adding duplicate 50 µL aliquots to 50 µL of ED buffer in ELISA Systems wells and assayed as described. The mean A450±SE is shown. The maximum concentrations of urea and DTT in assays of solution G were 40 mM and 0.3 mM respectively. The untransformed data were analysed by one-way ANOVA and the LSD is shown. Columns with the same letter were not significantly different (GenStat).
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pone-0056456-g008: Optimisation of dilution and extraction protocol for ELISA Systems assay.The response of the ELISA Systems sandwich assays to a commercial lager, (Tanner this journal, this issue, Beer 7) extracted with different buffers was measured by adding triplicate samples of commercial beer (100 µL) to 0.9 mL of either: (A & B) dilute ELISA Systems extraction buffer; (C, D, and E) Ridascreen extraction cocktail; (F) 60% (v/v) ethanol; or (G) Urea/DTT. The solutions were mixed at either room temperature (A, C, D, F, and G) or 60°C (B, E) for 1 h and an aliquot diluted 1/100 fold with ED buffer (A, B, D, F, G). Solutions C and E were diluted with ten-fold with 80% (v/v) ethanol, and then diluted a further ten-fold with dilute RD buffer and assayed for hordein. Hordeins were measured by adding duplicate 50 µL aliquots to 50 µL of ED buffer in ELISA Systems wells and assayed as described. The mean A450±SE is shown. The maximum concentrations of urea and DTT in assays of solution G were 40 mM and 0.3 mM respectively. 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 composition of the initial dilution buffer had a significant effect on the final signal produced by beer in the ELISA Systems assay. An initial 10-fold dilution with Urea/DTT was most successful (Fig. 8; G), followed by initial dilutions with ED buffer (Fig. 8; A, B). Initial dilution with Ridascreen extraction cocktail or 60% (v/v) ethanol produced a lower final absorbance (Fig. 8; C, D, E, F). Altering the temperature at which this dilution was performed (at either room temperature or 60°C) did not significantly affect the final absorbance (Fig. 8; A vs B; C vs E). Further dilution of the Ridascreen extractions with either RD buffer (Fig. 8; C) or ED buffer (Fig. 8; D) did not increase the final signal. The signal produced following an initial dilution in 60% (v/v) ED buffer or EtOH, followed by dilution with ED buffer were not significantly different (Fig. 8; A vs F). Based on these results, the optimum dilution schedule for beer with the ELISA Systems kit was an initial 1/10 dilution with Urea/DTT, followed by a second dilution of at least 1/100 with ED buffer (Fig. 8; G).


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

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

Optimisation of dilution and extraction protocol for ELISA Systems assay.The response of the ELISA Systems sandwich assays to a commercial lager, (Tanner this journal, this issue, Beer 7) extracted with different buffers was measured by adding triplicate samples of commercial beer (100 µL) to 0.9 mL of either: (A & B) dilute ELISA Systems extraction buffer; (C, D, and E) Ridascreen extraction cocktail; (F) 60% (v/v) ethanol; or (G) Urea/DTT. The solutions were mixed at either room temperature (A, C, D, F, and G) or 60°C (B, E) for 1 h and an aliquot diluted 1/100 fold with ED buffer (A, B, D, F, G). Solutions C and E were diluted with ten-fold with 80% (v/v) ethanol, and then diluted a further ten-fold with dilute RD buffer and assayed for hordein. Hordeins were measured by adding duplicate 50 µL aliquots to 50 µL of ED buffer in ELISA Systems wells and assayed as described. The mean A450±SE is shown. The maximum concentrations of urea and DTT in assays of solution G were 40 mM and 0.3 mM respectively. 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

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getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g008: Optimisation of dilution and extraction protocol for ELISA Systems assay.The response of the ELISA Systems sandwich assays to a commercial lager, (Tanner this journal, this issue, Beer 7) extracted with different buffers was measured by adding triplicate samples of commercial beer (100 µL) to 0.9 mL of either: (A & B) dilute ELISA Systems extraction buffer; (C, D, and E) Ridascreen extraction cocktail; (F) 60% (v/v) ethanol; or (G) Urea/DTT. The solutions were mixed at either room temperature (A, C, D, F, and G) or 60°C (B, E) for 1 h and an aliquot diluted 1/100 fold with ED buffer (A, B, D, F, G). Solutions C and E were diluted with ten-fold with 80% (v/v) ethanol, and then diluted a further ten-fold with dilute RD buffer and assayed for hordein. Hordeins were measured by adding duplicate 50 µL aliquots to 50 µL of ED buffer in ELISA Systems wells and assayed as described. The mean A450±SE is shown. The maximum concentrations of urea and DTT in assays of solution G were 40 mM and 0.3 mM respectively. 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 composition of the initial dilution buffer had a significant effect on the final signal produced by beer in the ELISA Systems assay. An initial 10-fold dilution with Urea/DTT was most successful (Fig. 8; G), followed by initial dilutions with ED buffer (Fig. 8; A, B). Initial dilution with Ridascreen extraction cocktail or 60% (v/v) ethanol produced a lower final absorbance (Fig. 8; C, D, E, F). Altering the temperature at which this dilution was performed (at either room temperature or 60°C) did not significantly affect the final absorbance (Fig. 8; A vs B; C vs E). Further dilution of the Ridascreen extractions with either RD buffer (Fig. 8; C) or ED buffer (Fig. 8; D) did not increase the final signal. The signal produced following an initial dilution in 60% (v/v) ED buffer or EtOH, followed by dilution with ED buffer were not significantly different (Fig. 8; A vs F). Based on these results, the optimum dilution schedule for beer with the ELISA Systems kit was an initial 1/10 dilution with Urea/DTT, followed by a second dilution of at least 1/100 with ED buffer (Fig. 8; G).

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