57 / 363
© 2015 AOAC INTERNATIONAL
AOAC Official Method 2015.05
Partially Hydrolyzed Gluten
in Fermented Cereal-Based Products
R5 Competitive ELISA
First Action 2015
[RIDASCREEN
®
Gliadin competitive ELISA kit is used 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).
See
Table
2015.05
for performance statistics (without outliers)
supporting acceptance of the method.
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 (1). Some of these sequences
are potentially celiac immuno-stimulatory (2, 3). 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, USA).
(
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, Darmstadt, Germany). 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/mL gluten, 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, USA; 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
[ J. AOAC Int . 98 , 1346(2015)].
b
— = Not applicable.
Candidates for 2016 Method of the Year
56