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© Romer Labs, 2016
AOAC Official Method 2014.03
Gluten in Rice Flour and Rice-Based
Food Products G12 Sandwich ELISA
First Action 2014
1. Test principle
The method is based on an enzyme immunoassay format using a monoclonal G12 antibody
that can determine gluten derived from wheat, rye, barley, and cross-bred varieties. The
G12 antibody binds to the celiac toxic amino acid sequence QPQLPY and related sequences
in rye and barley. The antibody detects prolamins in non-heated and heated food by using
a specific proprietary extraction solution.
Gluten is extracted from samples using proprietary extraction solution containing reducing
agents followed by ethanol extraction. After centrifugation the supernatant is used in a
sandwich enzyme-linked immunoassay. When incubated on monoclonal antibody-coated
microwells, the analyte is forming an antibody-antigen complex. After a washing step, an
enzyme-conjugated monoclonal antibody is applied to the well and incubated. After a
second washing step, an enzyme substrate is added and blue color develops. The intensity
of the color is directly proportional to the concentration of gluten in the sample or standard.
A stop solution is then added which changes the color from blue to yellow. The microwells
are measured optically using a microwell reader with a primary absorbance filter of 450
nm (OD450). The optical densities of the samples are compared to the standards and an
interpolated result is determined.
2. Method Applicability
The test method is applicable for determination of gluten in rice flour and rice-based
unprocessed and processed foods. No limitations of the intended use have been discovered
over the past two years.
None of the reagents or required apparatuses have changed since the first approval of the
method. The same general instructions, precautions and instructions for solution and
sample preparation, extraction, assay and reading and calculation of the results still apply
as stated in the method publication.
The method performs as desired in the above mentioned matrices and furthermore shows
satisfying results routine samples composed of other matrices as well.
3. Safety Concerns
No additional safety concerns have been identified.
The following general instructions and precautions still apply:
It is recommended to perform the assay in a room separate from that used for sample
preparation and extraction in order to maintain a gluten-free environment. The lab and
equipment has to be kept clean, ideally by using a 70% alcoholic solution. Good laboratory
practice applies when performing the assay.
Instructions in the package insert need to be followed accurately to ensure reliable test
results.
4. Reference materials
No certified reference material for gluten is available at the time of submission.