Limits...
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.

Show MeSH

Related in: MedlinePlus

The lack of hordeins in extracts of the hordein double- line ULG 2.0.Total hordeins (2 µg) from ULG 2.0 were subject to 1D SDS-PAGE, blotted and visualised as above and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). The location of known proteins was confirmed by protein sequencing of replicate gels [33]: 6, γ-3-hordein; 7, β-glucosidase; 8, LTP-1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g002: The lack of hordeins in extracts of the hordein double- line ULG 2.0.Total hordeins (2 µg) from ULG 2.0 were subject to 1D SDS-PAGE, blotted and visualised as above and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). The location of known proteins was confirmed by protein sequencing of replicate gels [33]: 6, γ-3-hordein; 7, β-glucosidase; 8, LTP-1.

Mentions: The composition of alcohol soluble extracts from the wild type, single and double hordein- grains were analysed by western blot with three anti-hordein antibodies (Fig. 1 & 2). The banding pattern of cv Sloop, Risø 56 and Risø 1508 showed the anti-gliadin-HRP conjugate (Sigma) detected all hordein families: D-hordein was observed as a faint band at ∼90 kDa in Sloop (not detected in ULG 2.0); C-hordeins were observed at 55 and 70 kDa in Risø 56; B-hordeins were observed at 43–45 kDa in Sloop and Risø 1508; and the three γ-hordeins, γ-1, γ-2 and γ-3, were observed at 45, 38 and 35 kDa respectively in Risø 56 (Fig. 1A, 1–6 respectively). The γ-hordeins are normally obscured by co-migrating B-hordeins which do not accumulate in Risø 56. These blots were stripped of antibodies, re-blocked and re-probed with alternate anti-hordein antibodies. The rabbit and mouse antibodies also reacted with all hordein families (Fig. 1B & C). However, the reaction with γ-hordein bands was less intense with both alternate antibodies. A faint western band corresponding to γ-hordein-3 was seen (Fig. 2B, 6). It was noted that both antibodies also reacted with a band corresponding to β-glucosidase at 65 kDa and LTP1 at approximately 10 kDa in extracts of ULG 2.0 (Fig. 2∶7 and 8 respectively). The lower level of hordein in ULG 2.0 results in a much lower level of signal from the respective western blot (Fig. 2) compared to hordein wild-type or single- (Fig. 1).


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

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

The lack of hordeins in extracts of the hordein double- line ULG 2.0.Total hordeins (2 µg) from ULG 2.0 were subject to 1D SDS-PAGE, blotted and visualised as above and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). The location of known proteins was confirmed by protein sequencing of replicate gels [33]: 6, γ-3-hordein; 7, β-glucosidase; 8, LTP-1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056456-g002: The lack of hordeins in extracts of the hordein double- line ULG 2.0.Total hordeins (2 µg) from ULG 2.0 were subject to 1D SDS-PAGE, blotted and visualised as above and compared to standard proteins (M; Benchmark Protein Ladder, Invitrogen). The location of known proteins was confirmed by protein sequencing of replicate gels [33]: 6, γ-3-hordein; 7, β-glucosidase; 8, LTP-1.
Mentions: The composition of alcohol soluble extracts from the wild type, single and double hordein- grains were analysed by western blot with three anti-hordein antibodies (Fig. 1 & 2). The banding pattern of cv Sloop, Risø 56 and Risø 1508 showed the anti-gliadin-HRP conjugate (Sigma) detected all hordein families: D-hordein was observed as a faint band at ∼90 kDa in Sloop (not detected in ULG 2.0); C-hordeins were observed at 55 and 70 kDa in Risø 56; B-hordeins were observed at 43–45 kDa in Sloop and Risø 1508; and the three γ-hordeins, γ-1, γ-2 and γ-3, were observed at 45, 38 and 35 kDa respectively in Risø 56 (Fig. 1A, 1–6 respectively). The γ-hordeins are normally obscured by co-migrating B-hordeins which do not accumulate in Risø 56. These blots were stripped of antibodies, re-blocked and re-probed with alternate anti-hordein antibodies. The rabbit and mouse antibodies also reacted with all hordein families (Fig. 1B & C). However, the reaction with γ-hordein bands was less intense with both alternate antibodies. A faint western band corresponding to γ-hordein-3 was seen (Fig. 2B, 6). It was noted that both antibodies also reacted with a band corresponding to β-glucosidase at 65 kDa and LTP1 at approximately 10 kDa in extracts of ULG 2.0 (Fig. 2∶7 and 8 respectively). The lower level of hordein in ULG 2.0 results in a much lower level of signal from the respective western blot (Fig. 2) compared to hordein wild-type or single- (Fig. 1).

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