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 effect of DTT, hydrogen peroxide and urea on the ELISA response.The response of ELISA Systems sandwich assay containing total Sloop hordein (500 ppb) and either (A) DTT, or H2O2 or (B) urea, diluted in ED buffer and added to the ELISA wells at the concentration indicated above and processed as described.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g007: The effect of DTT, hydrogen peroxide and urea on the ELISA response.The response of ELISA Systems sandwich assay containing total Sloop hordein (500 ppb) and either (A) DTT, or H2O2 or (B) urea, diluted in ED buffer and added to the ELISA wells at the concentration indicated above and processed as described.

Mentions: Where large volumes of IPA/DTT extract were used, DTT carry-over disrupted antibody binding and reduced the final A450 (Fig. 7A) [42]. Excess DTT of 0.5, 1.0 or 2.0 mM reduced the A450 of 40 ng of Sloop-T hordein by 0, 30% or 69% respectively. Attempts to remove the DTT by precipitating the proteins (2D Clean Up Kit, GE Health Care) were unsatisfactory due to inconsistent re-solubilisation of the precipitate in the aqueous based ELISA Systems diluent. The use of excess H2O2 (2 mM) restored the A450 of total Sloop hordein, however, concentrations higher than 10 mM resulted in progressive reduction of the A450 signal. An excess of 40 mM H2O2 reduced the A450 of total Sloop hordein by 50% (Fig. 7A). Excess urea up to 100 mM had no significant effect on the A450 of total Sloop hordein (Fig. 7B).


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 effect of DTT, hydrogen peroxide and urea on the ELISA response.The response of ELISA Systems sandwich assay containing total Sloop hordein (500 ppb) and either (A) DTT, or H2O2 or (B) urea, diluted in ED buffer and added to the ELISA wells at the concentration indicated above and processed as described.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056456-g007: The effect of DTT, hydrogen peroxide and urea on the ELISA response.The response of ELISA Systems sandwich assay containing total Sloop hordein (500 ppb) and either (A) DTT, or H2O2 or (B) urea, diluted in ED buffer and added to the ELISA wells at the concentration indicated above and processed as described.
Mentions: Where large volumes of IPA/DTT extract were used, DTT carry-over disrupted antibody binding and reduced the final A450 (Fig. 7A) [42]. Excess DTT of 0.5, 1.0 or 2.0 mM reduced the A450 of 40 ng of Sloop-T hordein by 0, 30% or 69% respectively. Attempts to remove the DTT by precipitating the proteins (2D Clean Up Kit, GE Health Care) were unsatisfactory due to inconsistent re-solubilisation of the precipitate in the aqueous based ELISA Systems diluent. The use of excess H2O2 (2 mM) restored the A450 of total Sloop hordein, however, concentrations higher than 10 mM resulted in progressive reduction of the A450 signal. An excess of 40 mM H2O2 reduced the A450 of total Sloop hordein by 50% (Fig. 7A). Excess urea up to 100 mM had no significant effect on the A450 of total Sloop hordein (Fig. 7B).

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