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

Response of ELISA Systems sandwich assay to log [hordein] concentration.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


getmorefigures.php?uid=PMC3585327&req=5

pone-0056456-g011: Response of ELISA Systems sandwich assay to log [hordein] concentration.The ELISA response to different hordein fractions, added in the concentrations indicated, are shown above in order of decreasing sensitivity.

Mentions: Purified hordein standards were used to calibrate the response of ELISA kits. The response of the ELISA systems antibody to increasing hordein concentration may be illustrated for wide substrate concentrations by plotting on a log axes (Fig. 11). The curves of best fit approximate sigmoidal curves. However, when plotted on a linear scale, the curves of best fit accurately approximate standard Michaelis-Menton curves with individual R2 values >0.97. The sensitivity of the assay is described by the dissociation constant, Kd, analogous to the Km of a Michaelis-Menton curve. The Kd is a measure of the amount of hordein required to produce a half-maximal response in the ELISA assay (Table 1). The lower the Kd, the more sensitive was the detection, e.g. 57 ppb of ULG 2.0 total hordein, produced a half-maximal response in this assay, compared to 670 ppb Sloop total hordein required to produce the same colour (Fig. 11, Table 1). Thus ULG 2.0 hordein was detected approximately 10-fold more sensitively than total hordeins from Sloop. Thus the ELISA systems sandwich assay was very sensitive to ULG 2.0 and Risø 1508 total hordeins (Fig. 11, Table 1). Preparations enriched for B-hordeins (e.g. Table 1: Sloop_T, Sloop_B) were detected poorly. Likewise, the γ-hordein fraction from Risø 56 was also poorly detected, suggesting that the response to ULG 2.0 was due mainly to the D-hordein content as ULG 2.0 accumulates only D- and γ-3-hordein [33]. Preparations enriched for C-hordeins (e.g. Sloop_C, Risø 56_C, and Risø 56_T) were detected with medium sensitivity indicated by Kd values intermediate between those for total hordein from ULG 2.0 total and B-hordein (Table 1). One way analysis of variance (ANOVA) of log10 transformed Kd values confirmed each of these groups differed significantly (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 ELISA Systems sandwich assay to log [hordein] concentration.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-g011: Response of ELISA Systems sandwich assay to log [hordein] concentration.The ELISA response to different hordein fractions, added in the concentrations indicated, are shown above in order of decreasing sensitivity.
Mentions: Purified hordein standards were used to calibrate the response of ELISA kits. The response of the ELISA systems antibody to increasing hordein concentration may be illustrated for wide substrate concentrations by plotting on a log axes (Fig. 11). The curves of best fit approximate sigmoidal curves. However, when plotted on a linear scale, the curves of best fit accurately approximate standard Michaelis-Menton curves with individual R2 values >0.97. The sensitivity of the assay is described by the dissociation constant, Kd, analogous to the Km of a Michaelis-Menton curve. The Kd is a measure of the amount of hordein required to produce a half-maximal response in the ELISA assay (Table 1). The lower the Kd, the more sensitive was the detection, e.g. 57 ppb of ULG 2.0 total hordein, produced a half-maximal response in this assay, compared to 670 ppb Sloop total hordein required to produce the same colour (Fig. 11, Table 1). Thus ULG 2.0 hordein was detected approximately 10-fold more sensitively than total hordeins from Sloop. Thus the ELISA systems sandwich assay was very sensitive to ULG 2.0 and Risø 1508 total hordeins (Fig. 11, Table 1). Preparations enriched for B-hordeins (e.g. Table 1: Sloop_T, Sloop_B) were detected poorly. Likewise, the γ-hordein fraction from Risø 56 was also poorly detected, suggesting that the response to ULG 2.0 was due mainly to the D-hordein content as ULG 2.0 accumulates only D- and γ-3-hordein [33]. Preparations enriched for C-hordeins (e.g. Sloop_C, Risø 56_C, and Risø 56_T) were detected with medium sensitivity indicated by Kd values intermediate between those for total hordein from ULG 2.0 total and B-hordein (Table 1). One way analysis of variance (ANOVA) of log10 transformed Kd values confirmed each of these groups differed significantly (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