Sandwich ELISAs for Assessing Gluten Content of Foods
Sandwich ELISAs can be used to quantitatively assess ingredients and finished food products for gluten. Three sandwich ELISAs are discussed below. The R5 (Mendez) ELISA and the omega-gliadin (Skerritt) ELISA are two of the assays used most frequently in the United States. The Morinaga Wheat Protein ELISA is used less frequently but is presented here because it, along with the R5 ELISA are the only two commercially available ELISAs validated at the levels used for regulatory purposes and official governmental methods (see note below). The R5 and Morinaga ELISAs also are included in the FDA’s proposed gluten-free labeling rule as possible methods for rule enforcement.
Note: The omega-gliadin (Skerritt) ELISA is validated at a level considerably higher than levels required for regulatory purposes. Higher sensitivity versions are available but they have not been validated in a multi-lab collaborative trial.
One drawback of sandwich ELISAs is they can not adequately quantify gluten that has been highly hydrolyzed. Sandwich ELISAs require two epitopes or antibody binding sites. When a protein is hydrolyzed, the various fragments may not contain two epitopes. As a result these fragments will not be measured. A Competitive ELISA may be used to help assess gluten content of hydrolyzed foods. However, the gluten content still may be over or underestimated depending on the degree of hydrolysis. For more information, please see:
None of the three sandwich ELISAs discussed below is perfect; they all have limitations. New and better assays will continue to be developed and current limitations will be improved upon.
Assay: R5 (Mendez)
Validation: Validated in a collaborative trial conducted by the Prolamin Working Group of the Codex Alimentarius Commission.
What it measures: The R5 monoclonal antibody (an antibody that binds to a specific substance) to potentially celiac toxic epitope (amino acid sequence) QQPFP (glutamine-glutamine-proline-phenylalanine-proline) which is present in all fractions of wheat gliadin, barley hordein, and rye secalin.
Limit of quantification: 5 parts per million of gluten (2.5 parts per million of gliadin).
Limit of detection: 3 parts per million of gluten (1.5 parts per million of gliadin).
Intended use: To quantitatively asses the gluten content of heated and unheated foods.
Method of extraction: Cocktail (mixture of extraction compounds).
Strengths: The epitope QQPFP is resistant to heat; this assay can assess the gluten content of native and heated proteins; this assay recognizes all fractions of wheat gliadin, barley hordein, and rye secalin.
Weaknesses: This assay overestimates barley protein content by a factor of 2 in barley-contaminated foods when the Prolamin Working Group gliadin standard is used; this assay, like all sandwich ELISAs, can not adequately quantify gluten that has been highly hydrolyzed.
Additional notes: In 2006, the Codex Committee on Methods of Analysis and Sampling endorsed the R5 (Mendez) ELISA as a type l method for determination of gluten content in gluten-free foods. It is the method of analysis for gluten in the Codex standard for gluten-free foods. At present, this assay is widely viewed as the best commercially available validated sandwich ELISA for gluten determination.
Assay: Morinaga Wheat Gliadin
Validation: Validated in an interlaboratory study supported by The Japanese Ministry of Health, Labor and Welfare.
What it measures: Polyclonal antibody to wheat gliadin (detects multiple epitopes as compared to a monoclonal antibody that detects one epitope).This antibody also cross-reacts with barley hordein and rye secalin.
Limit of quantification: 0.3 parts per million wheat protein.
Limit of detection: 0.3 parts per million wheat protein.
Intended use: To quantitatively assess wheat protein content of processed and unprocessed food.
Method of extraction: Cocktail (mixture of extraction compounds).
Strengths: High recovery of gluten from processed foods.
Weaknesses: This assay underestimates barley protein content in barley contaminated foods (cross-reactivity approximately 42 percent). This assay also underestimates rye protein in rye contaminated foods (cross-reactivity approximately 61 percent).
Additional notes: This assay was developed in Japan where they have mandatory allergen labeling of egg, milk, wheat, peanut, and buckwheat protein. In general, polyclonal antibodies are more sensitive than monoclonal antibodies. But because they detect more than one epitope they are les specific than monoclonal antibodies. Thank you to Thomas Grace of Bia Diagnostics for this simplified explanation.
Assay: Omega-Gliadin (Skerritt)
Validation: Validated in a collaborative trial (official AOAC method).
What it measures: Monoclonal antibody to the omega-gliadin fraction of wheat gliadin.
Limit of quantification: 160 parts per million of gluten (official AOAC method—higher sensitivity versions available).
Intended use: To assess the gluten content of heated and unheated foods.
Method of extraction (official method): 40% ethanol.
Strengths: The omega-gliadin fraction of wheat protein is not denatured when heated; this assay can assess gluten content of heated and unheated proteins.
Weaknesses: This assay underestimates barley protein content in barley contaminated foods (cross-reactivity of 4 to 8 percent). This assay, like all sandwich ELISAs can not adequately quantify gluten that has been highly hydrolyzed. This assay is validated at a level considerably higher than levels required for regulatory purposes. Higher sensitivity versions are available but they have not been validated in a multi-lab collaborative trial.
Additional notes: When the omega-gliadin (Skerritt) ELISA was first developed in 1991 it was considered state of the art. Now that there are assays that have improved on some of its important limitations, this ELISA is no longer characterized in this fashion.
Sources of information:
Thompson T, Mendez E. Commercial Assays to Assess Gluten Content of Gluten-Free Foods: Why They Are Not Created Equal. J Am Diet Assoc. 2008;108:1682-1687. The Abstract for this article is available at: http://www.glutenfreedietitian.com/newsletter/commercial-assays-to-assess-gluten-content-of-gluten-free-foods-why-they-are-not-created-equal/
Matsuda et al. Interlaboratory Evaluation of Two Enzyme-Linked Immunosorbent Assay Kits for the Detection of Egg, Milk, Wheat, Buckwheat, and Peanut in Foods. Journal of AOAC International. 2006;89:1600-1608. The abstract for this article is available at: http://www.ncbi.nlm.nih.gov/pubmed/17225608
Wheat protein ELISA Kit available from the Morinaga Institute of Biological Science, Inc website http://www.miobs.com/english/product/food_allergen_elisa_kits/dl/gdrev1.pdf
Wheat protein ELISA Kit (Gliadin) from the Chrystal Chem, Inc website http://www.crystalchem.com/products/wheat-gliadin-elisa-kits.html
Copyright © February 28, 2011 by Tricia Thompson, MS, RD