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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

Response of Ridascreen sandwich assay to purified hordein standards.The ELISA response to different hordein fractions, added in the concentrations indicated, are shown above in order of decreasing sensitivity.
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pone-0056456-g012: Response of Ridascreen sandwich assay to purified hordein standards.The ELISA response to different hordein fractions, added in the concentrations indicated, are shown above in order of decreasing sensitivity.

Mentions: The Ridascreen antibody kit also bound to hordein proteins with varying affinity (Fig. 12, Table 1). The response of the Ridascreen kit to purified hordein fractions also accurately fitted sigmoidal curves when plotted on a log axis (Fig. 12). Kd values can be similarly extracted from linear plots. Not unexpectedly, the sensitivity of these antibodies to individual hordeins differs from that of the ELISA Systems antibodies. Thus the Ridascreen kit was relatively more sensitive to fractions enriched for C-hordeins, such as total hordeins from RisØ 56 and C-hordeins fractions purified from RisØ 56 or Sloop, having Kd values 15, 30 and 26 times lower (more sensitive) than the respective Kd values for the ELISA Systems kit (Table 1, Fig. 12). Preparations enriched for B-hordeins (e.g. Sloop_B) were detected with low sensitivity (Table 1). The γ-hordein fraction from Risø 56 was detected more sensitively by the Ridascreen kit, with a Kd value 6.5 times lower than that obtained with the ELISA Systems kit. However, hordein preparations enriched for D-hordeins, such as total hordein from ULG 2.0 and Risø 1508, were not detected at these dilutions by the Ridascreen kit (Fig. 12). Thus the Ridascreen kit was more sensitive to C-hordeins, but relatively insensitive to B-, D- and γ-hordeins. One way ANOVA analysis of log10 transformed Kd values for the Ridascreen kit showed there were two groups of hordeins, that containing the C-hordeins, and total hordeins from RisØ 56 and cv Sloop, which differed significantly from that containing gliadin, and B- and γ-hordeins (p<0.001; Table 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)

Response of Ridascreen sandwich assay to purified hordein standards.The ELISA response to different hordein fractions, added in the concentrations indicated, are shown above in order of decreasing sensitivity.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056456-g012: Response of Ridascreen sandwich assay to purified hordein standards.The ELISA response to different hordein fractions, added in the concentrations indicated, are shown above in order of decreasing sensitivity.
Mentions: The Ridascreen antibody kit also bound to hordein proteins with varying affinity (Fig. 12, Table 1). The response of the Ridascreen kit to purified hordein fractions also accurately fitted sigmoidal curves when plotted on a log axis (Fig. 12). Kd values can be similarly extracted from linear plots. Not unexpectedly, the sensitivity of these antibodies to individual hordeins differs from that of the ELISA Systems antibodies. Thus the Ridascreen kit was relatively more sensitive to fractions enriched for C-hordeins, such as total hordeins from RisØ 56 and C-hordeins fractions purified from RisØ 56 or Sloop, having Kd values 15, 30 and 26 times lower (more sensitive) than the respective Kd values for the ELISA Systems kit (Table 1, Fig. 12). Preparations enriched for B-hordeins (e.g. Sloop_B) were detected with low sensitivity (Table 1). The γ-hordein fraction from Risø 56 was detected more sensitively by the Ridascreen kit, with a Kd value 6.5 times lower than that obtained with the ELISA Systems kit. However, hordein preparations enriched for D-hordeins, such as total hordein from ULG 2.0 and Risø 1508, were not detected at these dilutions by the Ridascreen kit (Fig. 12). Thus the Ridascreen kit was more sensitive to C-hordeins, but relatively insensitive to B-, D- and γ-hordeins. One way ANOVA analysis of log10 transformed Kd values for the Ridascreen kit showed there were two groups of hordeins, that containing the C-hordeins, and total hordeins from RisØ 56 and cv Sloop, which differed significantly from that containing gliadin, and B- and γ-hordeins (p<0.001; Table 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