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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

Hordeins do not bleed off in aqueous washes.Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).
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pone-0056456-g004: Hordeins do not bleed off in aqueous washes.Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).

Mentions: Hordeins did not “bleed-off” with extended aqueous washes of barley flour (Fig. 4). Western blots of successive duplicate aqueous washed pellets which were then extracted in IPA-DTT give a similar pattern on Coommassie stained gels with no evidence of the dominant proteins decreasing in subsequent pellets (Fig. 4A) and increasing in subsequent supernatants (Fig. 4C). Similarly, hordeins revealed by western blotting also did not decrease in the IPA-DTT soluble pellets (Fig. 4B) and did not increase in the aqueous supernatants (Fig. 4D). The signal from the supernatant (Fig. 4D) was much less intense than from the pellet (Fig. 4B), confirming that hordeins were not soluble in the aqueous supernatant. However, a small amount of low molecular weight protein between 10 and 40 kDa was extracted by aqueous washes and was visualised by additional protein bands in supernatant S1, S2, S3 (Fig. 4C, arrowed) and a depletion of similar bands in pellets P and P1 (Fig. 4A, arrowed). This material did not contain hordeins since it did not react with western blots (Fig. 4B & D). Two aqueous washes were sufficient to remove this water soluble protein that was partially soluble in IPA-DTT.


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

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

Hordeins do not bleed off in aqueous washes.Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g004: Hordeins do not bleed off in aqueous washes.Duplicate 50 mg aliquots of Sloop flour were extracted in 1 mL of 50% IPA containing 1% (w/v) DTT (IPA/DTT) and an aliquot containing 20 µg protein dried in a SpeedyVac, dissolved in Urea/SDS and resolved on duplicate SDS-PAGE gels (lanes P) which were either stained in colloidal Coomassie Blue (A and C) or blotted to nitrocellulose (iBLOT Promega), blocked in PBST containing 5% skim milk powder, 1% (w/v) Tween overnight at 4°C. The blot was exposed to anti-gliadin-HRP (Sigma) diluted at 1/2000 for 30 min, then washed in PBST, and the signal produced by ECL reagent (Amersham) exposed to Hyperfilm (Amersham) (B and D). Similarly, 50 mg of duplicate flour samples were first washed in 1 mL of water, and centrifuged to give a supernatant and a pellet, which was dissolved in IPA/DTT. Aliquots containing 20 µg protein from both pellet (P1) and supernatant (S1) were dried, redissolved and subject to SDS-PAGE as above. The process was repeated with additional aqueous washing steps to produce supernatants S2–4 and pellets P2–4. Low molecular weight proteins were removed from pellets (A: arrowed) and extracted into supernatants (C: arrowed) after 2 washes. Proteins were calibrated with Benchmark protein ladder (St; Invitrogen).
Mentions: Hordeins did not “bleed-off” with extended aqueous washes of barley flour (Fig. 4). Western blots of successive duplicate aqueous washed pellets which were then extracted in IPA-DTT give a similar pattern on Coommassie stained gels with no evidence of the dominant proteins decreasing in subsequent pellets (Fig. 4A) and increasing in subsequent supernatants (Fig. 4C). Similarly, hordeins revealed by western blotting also did not decrease in the IPA-DTT soluble pellets (Fig. 4B) and did not increase in the aqueous supernatants (Fig. 4D). The signal from the supernatant (Fig. 4D) was much less intense than from the pellet (Fig. 4B), confirming that hordeins were not soluble in the aqueous supernatant. However, a small amount of low molecular weight protein between 10 and 40 kDa was extracted by aqueous washes and was visualised by additional protein bands in supernatant S1, S2, S3 (Fig. 4C, arrowed) and a depletion of similar bands in pellets P and P1 (Fig. 4A, arrowed). This material did not contain hordeins since it did not react with western blots (Fig. 4B & D). Two aqueous washes were sufficient to remove this water soluble protein that was partially soluble in IPA-DTT.

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