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Lacorn & Weiss.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o
. 5, 2015
1349
for the analysis of fermented and hydrolyzed food (e.g., beer,
starch syrup, starch, malt extract, sourdough, and soy sauce)
that are declared as “gluten-free.” The kit is not applicable for
measurement of intact gluten.]
Caution
: Stop solution contains 0.5 M sulfuric acid; avoid
skin and eye contact (
see
Material Safety Data
Sheet).
A. Principle
The method is based on an enzyme immunoassay format
using a monoclonal antibody that can determine hydrolyzed
gluten derived from wheat, rye and barley. The antibody
binds to the short amino acid sequence QQPFP and to
related sequences, which exist as motifs on all the prolamin
subunits (9). Some of these sequences are potentially celiac
immuno-stimulatory (10, 11). Since the assay is calibrated to
a prolamin hydrolysate mixture form wheat, rye, and barley, a
conversion to “gluten” content is achieved by the conversion
factor of 2 set by the Codex Alimentarius. No cross-reactivity
has been observed to oats, maize, rice, millet, teff, buckwheat,
quinoa, or amaranth. Protein fragments for gluten measurement
from food are extracted by using ethanol. After centrifugation,
the supernatant is used in a competitive method.
The basis of the test is the antigen-antibody reaction. The
microtiter wells are coated with a constant amount of gliadin.
Standards (mixture of hydrolysates from wheat, rye, and barley
prolamins) or sample solutions are pipetted, and peroxidase
labeled antigliadin antibody (conjugate with monoclonal
R5 antibodies) is added and incubated for 30 min. During
incubation, free and immobilized analyte competes for the
antibody binding sites (competitive enzyme immunoassay).
Any unbound enzyme conjugate is then removed by a washing
step. Substrate/chromogen is added to the wells and incubated
for 10 min. Bound enzyme conjugate converts the chromogen
into a blue product. Addition of the stop solution causes a color
change from blue to yellow. The measurement is performed
photometrically at 450 nm. The absorption is inversely
proportional to the gluten concentration. The response of sample
extracts is compared with response observed with calibrators.
B. Apparatus
Apparatus specified here has been tested in the laboratory;
equivalent apparatus may be used.
(
a
)
Laboratory mincer/grinder, mortar and pestle, or Ultra-
Turrax.
—e.g., Mr. Magic, ds-produkte GmbH, Gallin, Germany.
(
b
)
Rotator or shaker.
—e.g., Roto Shaker Genie (Scientific
Industries Inc., Bohemia, NY).
(
c
)
Centrifuge
.—e.g., Minifuge RF, Kendro, Hanau,
Germany.
(
d
)
Microtiter plate reader
.—e.g., Tecan Sunrise Remote
(Tecan Group, Maennedorf, Switzerland).
(
e
)
Micropipets
.—Variable 20–200 µL and 200–1000 µL.
(
f
)
Graduated pipets
.
(
g
)
Graduated cylinders
.—Up to 1000 mL, plastic or glass.
(
h
)
Centrifugal glass vials with screw tops
.
C. Reagents
Items (
a
)–(
g
) are available as a test kit (RIDASCREEN
®
Gliadin competitive, R-Biopharm AG). All reagents are stable
at least over a period of 15 months at 2–8°C (36–46°F) from
the date of manufacture. Please refer to the kit label for current
expiration.
(
a
)
Microtiter plate
.—Coated with gliadin (96 wells).
(
b
)
Five standard solutions
.—Labeled 0, 20, 60, 180, and
540 ng/mLgluten, 1.3 mL each; ready to use, transparent-capped
bottles.
(
c
)
Conjugate.—
Horseradish peroxidase labeled R5 antibody;
0.7 mL, as an 11-fold concentrate, red-capped bottle.
(
d
)
Red Chromogen Pro.—
Substrate/chromogen; 10 mL,
ready to use, brown-capped bottle.
(
e
)
Stop solution
.—14 mL, ready to use, yellow-capped
bottle.
(
f
)
Sample diluent.—
60 mL, as a 5-fold concentrate,
white-capped bottle.
(
g
)
Washing buffer
.—100 mL, as a 10-fold concentrate,
brown-capped bottle.
Necessary or recommended but not provided with the test kit:
(
h
)
Distilled water
.
(
i
)
Ethanol
.—99% reagent grade.
(
j
)
Fish gelatin.—
Sigma, St. Louis, MO; Part No. G-7765 or
Serva, Heidelberg, Germany; Part No. 22156.
Table 2015.05. Performance statistics for overall competitive R5 ELISA results without outlier (gluten concentrations are
shown)
Sample ID
a
Symbol
1
2
3
4
5
6
7
Total No. of labs
p
13
12
11
13
13
13
13
Total No. of replicates
Sum(n(L))
26
24
22
26
26
26
26
Overall mean of all data (grand mean), mg/kg
XBARBAR 2.36
26.2
119.5
1.29
10.6
48.4
145.6
Repeatability SD, mg/kg
s
r
2.31
7.92
37.2
2.03
1.73
11.2
28.4
Reproducibility SD, mg/kg
s
R
2.98
9.67
37.2
3.05
3.65
12.5
40.0
Repeatability RSD, %
RSD
r
98.0
30.2
31.2
157.3
16.3
23.1
19.5
Reproducibility RSD, %
RSD
R
126.1
36.8
31.2
236.1
34.4
25.9
27.5
Recovery, %
—
b
87
119
— — 69
97
a
See
Table 1.
b
— = Not applicable.