More Efficient Extraction Procedure for
Gluten Using the R5 ELISA Method
By:
Thomas Grace
Luke Emerson-Mason, Teri Massey, Diana Butler
www.biadiagnostics.com
More Efficient Extraction Procedure for Gluten Using the R5 ELISA Method
By: Thomas Grace, Luke Emerson-Mason, Teri Massey, Diana Butler
Abstract:
To date the “Cocktail” Extraction Procedure (Mendez et al) has been recommended for
extraction of gluten in baked or processed foods. This utilizes 2-mercaptoethanol and
guanidine hydrochloride to reduce the disulfide prolamin cross linking and more efficiently
solubilizing the proteins. Here we aim to demonstrate a simpler, less toxic procedure that
has shown to be much more effective at extracting gluten from a large variety of baked
and processed foods.
It has been well documented that the thiol rich regions of gliadin (α/β, γ) undergo some
cross linking during high heat baking and processing and it has been shown that utilizing cold EtOH (60%) extraction of gluten in these products is less effective in recovering
the actual gluten content than Mendez’s Cocktail solution (250mM 2-mercaptoethanol, 2M
guanidine hydrochloride). Here we show that 60% EtOH with 5% Fish Gel and 2% PVP
(GEB = Gluten Extraction Buffer) heated to 60C in a shaking incubator for 45min produces
higher recovery on most naturally incurred matrices than the Cocktail solution.
Analysis Showing G.E.B. Higher Recovery
Samples
Processed Oat Fiber
Base D Crème
Carmel Apple Bar
FAPAS Gluten Positive Control
Cherry Choc Cookie Mix
Spice
DEHYDRATED SOUP
DEHYDRATED SOUP
DEHYDRATED SOUP
Buckwheat Hulled
Buckwheat Pancakes
Buckwheat Flour
Buckwheat Flour
WHOLE O'S CEREAL
HOT CEREAL
HOT CEREAL
HOT CEREAL
G.E.B.
Values in ppm
12.6
16.7
7.2
41.5
7.5
45
4.5
5.5
3.2
12
38.7
36
25.3
11.6
19.3
5.4
80.5
COCKTAIL Values
in ppm
3.1
12.7
4
25.5
2.5
36
BLD
BLD
BLD
7.5
28.4
17.8
8.6
4.1
6.3
1.4
62.9
Table 1 Analysis Showing G.E.B. with Higher Recovery than Cocktail Solution
page 2
www.biadiagnostics.com
BLD = Below Limit of
Detection
Procedure:
Over 150 random samples were analyzed using both the Cocktail extraction method and
GEB method in duplicate and with separate extractions . All samples were well homogenized and any sample that was shown to have greater than 20% variation in duplicate
results was eliminated from the study. Some samples were re-homogenized and retested
and were included in the study. A total of 30 samples were shown to have gluten by either method at or above the LOD for the R5 ELISA (3ppm).
17 samples had higher recovery using the GEB than the Cocktail, as much as 50% higher
(see Table 1). 8 samples had relatively the same recovery (see Table 4) and 5 had
lower recovery than the Cocktail extraction (see Table 5).
Also analyzed were lab incurred samples: sugar cookies baked at 350F for 20min spiked
with 0, 5, 10 and 20ppm wheat gluten (Sigma G5004) (see Table 3) and three different
nutritional bars spiked with 25ppm barley gluten (see Table 2).
Both GEB and Cocktail had virtually identical recoveries (100%) for all of the lab incurred
sugar cookies spiked with Sigma wheat gluten. While lab incurred nutritional bars spiked
with barley gluten had 100% recovery with GEB and only 50% with the Cocktail extraction procedure.
Analysis of Manually Incurred Samples with Barley
Gluten @ 25ppm
G.E.B.
Values in ppm
27.1
25.3
26.4
COCKTAIL Values
in ppm
14.3
16.1
13.8
Samples
Nutritional Bar (A)
Nutritional Bar (B)
Nutritional Bar (C)
Table 2 Analysis of Manually Incurred
Samples with Barley Gluten at 25ppm
Analysis of Manually Incurred Samples with Wheat
Samples
Cookie @ 0ppm
Cookie @ 5ppm
Cookie @ 10ppm
Cookie @ 20ppm
G.E.B.
COCKTAIL Values
Values in ppm
in ppm
BLD
BLD
6
5.2
8.6
8.1
19.2
21.5
Table 3 Analysis of Manually Incurred
Samples with Wheat Gluten
page 3
www.biadiagnostics.com
BLD = Below Limit of
Detection
Conclusion:
The GEB extraction method has been shown to be as robust, reproducible and reliable as the Cocktail extraction method at recovering gluten in complex food matrices.
In most cases the GEB reported the same or higher recovery then the Cocktail (57% of
the time higher, 21% the same and 13.5% lower). The only matrices that were shown
to have lower recovery were some thin wafer like products (heated to very high temperatures) and one rice/millet bread sample (with very low moisture content), all others
showed equivalent or better recovery with this method. If disulfide linkage alone was
responsible for lower recovery in these 5 samples it would seem also true that other
matrices heated to even higher temperatures would likewise have lower recovery (sugar
cookies, dehydrated soup, rosemary crackers, biscotti), but we do not see this, so it appears there must be other reasons for this lower recovery in these few samples. Overall
we see consistently higher recovery in almost all positive matrices we have tested using
the GEB as opposed the Cocktail procedure.
Analysis Showing the Same Recovery
Samples
Cajun Spice Blend
Vegi Pizza Mix Snacks
Biscotti
Oat Groats
Rosemary Crackers
Hot Cereal
Hot Cereal
Tortilla
G.E.B.
Values in ppm
25.6
84.0
11.6
4.2
14.4
2.7
3.8
84.0
COCKTAIL Values
in ppm
24.2
70.0
13.1
2.5
16.7
6.3
2.6
84.0
Table 4 Analysis Showing
G.E.B. and Cocktail Solution
with Similar Recovery
Analysis Showing the Lower Recovery
Table 5 Analysis Showing Cocktail
Solution with Higher Recovery than
G.E.B.
Samples
Tortilla
Tortilla
Millet Bread
Lental Chips
Snack Mix (crackers, pretzels)
page 4
www.biadiagnostics.com
G.E.B.
COCKTAIL Values
Values in ppm
in ppm
20.5
42.8
4.8
8.3
39.1
68.8
17.2
41.0
2.8
9.2