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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

Isolation of hordein fractions by FPLC.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.
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pone-0056456-g005: Isolation of hordein fractions by FPLC.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.

Mentions: Primary hordein standards were purified by solvent extraction, precipitation, FPLC (Fig. 5) and lyophilisation. Identity of the FPLC peaks in Fig. 5 was established by SDS-PAGE of the lyophilised fractions (Fig. 6) and in-gel protein sequencing [33]. The large FPLC peak eluting at 15 mL corresponded to oxidised DTT (Fig. 5). The A280 elution profile was calibrated with extracts of the hordein wild-type (Fig. 5A: Sloop), hordein single-s (Fig. 5A: RisØ 1508 and RisØ 56) and hordein double- line (Fig. 5A: ULG 2.0). C-hordein eluted first at 45 mL, followed by B-hordein (55 mL) and the γ-hordeins as partially resolved peaks from 55–60 ml (Fig. 5C). Unfortunately, we have not succeeded in purifying D-hordein fractions by FPLC. The small A280 peak at 35 mL (Fig. 5B, peak 2) in extracts of Risø 1508 was not due to protein (Fig. 6, lane 2). Fractions enriched for C-hordeins were isolated from cv Sloop (Fig. 1B, peak 3) and Risø 56 (Fig. 1C, peak 8). Fractions enriched for γ-hordeins were isolated from Risø 56 (Fig. 1C, peaks at 9) by pooling the indicated eluate. C-hordeins (Fig. 6, lanes 3 and 8) were observed at 55, 65 and 80 kDa. The γ-1, -2 and -3 hordeins (Fig. 6, lane 9) were observed at 35, 40 and 45 kDa respectively. These bands are normally masked by overlapping B-hordein bands (Fig. 6, lane 4) which are observed at 45–50 kDa, but which do not accumulate in the B-hordein deletion mutant Risø 56 (Fig. 6, lane 7).


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

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

Isolation of hordein fractions by FPLC.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.
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Related In: Results  -  Collection

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pone-0056456-g005: Isolation of hordein fractions by FPLC.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.
Mentions: Primary hordein standards were purified by solvent extraction, precipitation, FPLC (Fig. 5) and lyophilisation. Identity of the FPLC peaks in Fig. 5 was established by SDS-PAGE of the lyophilised fractions (Fig. 6) and in-gel protein sequencing [33]. The large FPLC peak eluting at 15 mL corresponded to oxidised DTT (Fig. 5). The A280 elution profile was calibrated with extracts of the hordein wild-type (Fig. 5A: Sloop), hordein single-s (Fig. 5A: RisØ 1508 and RisØ 56) and hordein double- line (Fig. 5A: ULG 2.0). C-hordein eluted first at 45 mL, followed by B-hordein (55 mL) and the γ-hordeins as partially resolved peaks from 55–60 ml (Fig. 5C). Unfortunately, we have not succeeded in purifying D-hordein fractions by FPLC. The small A280 peak at 35 mL (Fig. 5B, peak 2) in extracts of Risø 1508 was not due to protein (Fig. 6, lane 2). Fractions enriched for C-hordeins were isolated from cv Sloop (Fig. 1B, peak 3) and Risø 56 (Fig. 1C, peak 8). Fractions enriched for γ-hordeins were isolated from Risø 56 (Fig. 1C, peaks at 9) by pooling the indicated eluate. C-hordeins (Fig. 6, lanes 3 and 8) were observed at 55, 65 and 80 kDa. The γ-1, -2 and -3 hordeins (Fig. 6, lane 9) were observed at 35, 40 and 45 kDa respectively. These bands are normally masked by overlapping B-hordein bands (Fig. 6, lane 4) which are observed at 45–50 kDa, but which do not accumulate in the B-hordein deletion mutant Risø 56 (Fig. 6, lane 7).

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