Part 3: Wheat vs Gluten – Testing Perspective
FACTS receives many inquiries about the ‘significant’ cereal group, also called ‘gluten-containing’ cereals elsewhere in the world. We sympathetically refer to this as ‘Wheat vs Gluten confusion’. The goal of this four-part series is to help you go from confusion to clarity.
In part 3, we look at the different testing methods available to test for the presence of wheat and gluten.
Why do we need accurate wheat and gluten testing methods?
Why do we need accurate wheat and gluten testing methods?There are various reasons for samples to be tested for the presence of wheat or gluten, including but not limited to:
- Supporting wheat- and gluten-free claims
- Checking for accidental contamination
- Validating and verifying gluten and wheat cross-contact management controls
The popularity of (especially) gluten-free products has risen sharply in the last decade. Multiple countries, including South Africa, have established prerequisites for wheat- and gluten-free food labelling that include tolerance standards for gluten-related claims, making accurate and sensitive analytical methods imperative.
The evolution of wheat and gluten testing methods
In the past, enzyme-linked immunosorbent assay (ELISA) and Polymerase chain reaction (PCR) methods were widely used to detect and quantify gluten, while PCR was generally utilised to detect wheat. Gluten ELISAs can be used to detect wheat indirectly. But recent years have seen a dramatic change in the allergen- and gluten-testing landscape. The most prominent shift has been due to the recognition and refining of targeted proteomics (TP) as an alternative and reference method. Recent studies have confirmed and emphasised the benefits of TP, highlighting this important shift.
It is important to note that no analytical method is bulletproof, and that each method serves a purpose.
Although the most widely used method for quantitative allergen testing, in general ELISA suffers from several well-documented drawbacks. False positives (due to cross-reactivity of ELISA antibodies) lead to overestimation of risk; while false negatives (due to modification of the target epitope) lead to underestimation of risk, and expose consumers to potential reactions. Some common food-processing methods cause partial or complete denaturation or hydrolysis of gluten. This leads to a loss of immunoreactive epitopes, resulting in the testing process underestimating how much gluten is present, or not detecting it at all.
Great strides have been made to optimise the accuracy of gluten ELISA kits. Although they may still suffer some of the drawbacks mentioned above, gluten ELISAs are the exception to the rule, because they include the use of specialised monoclonal antibodies, and also due to the resources that have been committed to their development.
However, care should still be taken. Some studies have pointed out that the accuracy of the results generated by the kits may be problematic. Depending on the source of the gluten and the specific antibody employed, kits may still over- or underestimate the actual gluten content in a sample. The different commercially available ELISA kits have been shown to respond differently to gluten from different cereals (wheat, barley and rye). This is because the target protein fraction is present in different ratios in each cereal. Several multi-laboratory studies have been conducted in an attempt to emphasise and ensure the accuracy of ELISA results. The data suggest that although ELISAs are precise, they may not be accurate. This may be particularly true of the R5 Mendez ELISA.
Targeted Proteomic (TP) methods
Due to its specificity, TP by Liquid chromatography tandem mass spectrometry (LC-MS/MS) can easily detect wheat protein and differentiate between gluten species, by directly detecting multiple peptides from the proteins of interest. This high degree of specificity provides confidence in the results, and reduces the risk of false positives and false negatives.
Previous challenges with the implementation of LC-MS/MS for routine gluten and allergen detection included the need for specialised knowledge to operate the equipment, and a lack of information on target proteins. These challenges have been overcome through widespread experimentation and the publication of information on a variety of methods and targets. As the primary goal is the detection and quantification of trace amounts of gluten and allergens in food matrices, TP by LC-MS/MS is the best tool for the job, given the current technology. TP presents a great opportunity for improved food-protein analysis, on account of its sensitivity, precision, accuracy and robust quantitative ability; these characteristics provide greater assurance to consumers and to food businesses.
WANT TO KNOW MORE?
Mass spectrometry (MS) is familiar to many in the food industry as a widely used analytical tool. Hyphenated methods such as LC-MS/MS, coupling a separation technique with MS, allow the direct and absolute identification and quantification of analytes. Mass spectrometric methods for protein quantification are routinely performed at the peptide scale, making detection independent of the tertiary structure of the allergen, extremely specific, and still achievable after food processing.
LC-MS has been used in proteomics research and clinical diagnostics for over 20 years. However, until recently it has only been used routinely in commercial food testing for small-molecule contaminant detection, such as veterinary drug residue and pesticide detection.
Immunological techniques rely on the recognition and interaction of a target analyte and one or more antibodies. Antibodies recognise and bind to ‘epitopes’ – small portions of the whole protein molecule. The target epitopes may be linear or conformational. Linear epitopes are continuous strings of amino acids, and recognition is specific to the amino acid sequence (the primary structure of the protein). A conformational epitope may be a continuous or a discontinuous string of amino acids, and recognition depends on the three-dimensional shape of the protein (protein tertiary structure). Because antibodies only recognise epitopes, rather than the whole molecule, the specificity of an antibody depends on the uniqueness of the epitope. A lack of specificity may lead to false positives or false negatives. Most gluten ELISA methods target a defined section of the gluten protein. For example, the R5 assay targets epitopes with the peptide sequence glutamine-glutamine-prolinephenylalanine- proline (QQPFP), which can result in an over- or underestimation of the total gluten content.
Regulatory and Codex Requirements
The Regulations Relating to the Labelling and Advertising of Foodstuffs (R. 146/2010) recommend that R5 Mendez ELISA or other methods recommended by Codex Alimentarius are used to support ‘gluten-free’ claims. For more information, see Part 2 of this series.
Codex standard 118/1981 (‘Foods for special dietary use for persons intolerant to gluten’) refers to immunologic methods (ELISA), but makes provision for the use of other methods, provided they have at least equal sensitivity and specificity.
Additionally, it sets out performance characteristics for gluten detection methods:
- They must show sensitivity and specificity equal to immunologic methods.
- They must react with or target the cereal-protein fractions that are toxic for persons intolerant to gluten.
- They must not cross-react with other cereal proteins, constituents of foods, or ingredients.
- They must be validated and calibrated against a certified reference material, if available.
- Their detection limit must be appropriate, according to the state of the art and the technical standard.
- The detection limit should be 10mg gluten/kg or below.
- The qualitative analysis indicating the presence of gluten must be based on relevant methods.
Does the gluten LC-MS/MS method comply with Codex’s performance characteristics?
Yes it does. See the table below for more information:
| Sensitivity & specificity | Superior sensitivity and specificity, as described in the above sections. |
| Protein target | The LC-MS/MS method employed by FACTS targets a series of peptides from the following proteins: B1-hordein, B3-hordein, avenin-3, 75k gamma secalin, glutenin subunit DY 10 and PW212, Beta-amylase, alpha-amylase inhibitor 0.19. |
| Cross-reactivity | Non-specificity is less of a challenge with the proteomic methods. These methods target peptides from the above proteins; therefore, they are not cross-reactive with other cereal proteins or constituents of food or ingredients. |
| Validation | The method has been comprehensively validated to accurately and precisely detect and quantify gluten in a wide range of food matrices. Most Immunoassays are not validated against incurred materials. |
| Calibration | Limited reliable certified reference materials are available. FACTS uses wheat, barley and rye flour as calibrants. |
| Limit of Quantification (LOQ) | The LOQ is currently set at 1ppm, lower than the 10ppm performance criterion. |
| Method relevance | The relevance of LC-MS/MS as a gluten detection and quantification method is well documented in the literature. This is discussed in the sections above. |
In closing, accurate and reliable wheat and gluten testing is imperative to ensuring the integrity and safety of food products. The use of TP by LC-MS/MS allows direct and unequivocal identification and quantification of gluten. Because of its sensitivity, precision, accuracy and robust quantitative ability, LC-MS/MS provides greater assurance and protection (to both consumers and manufacturers) than other commercially available methods. Currently, the Regulations Relating to the Labelling and Advertising of Foodstuffs (R. 146/2010) state that R5 Mendez ELISA or other methods recommended by Codex must be used to support gluten-free claims. LC-MS/MS meets – and exceeds – the performance characteristics required for gluten-detection methods set out in Codex. Therefore, LC-MS/MS complies with the regulatory requirements as a suitable method for gluten analysis.
It is important to remember that no analytical method is bulletproof, and that each method serves a purpose.
If you have any questions, please contact us.
To view the full reference list of this article, please contact us.
Other articles and resources you may be interested in:
Part 1: Wheat vs Gluten – Medical Perspective
Part 2: Wheat vs Gluten – Regulatory Perspective
Part 4: Wheat vs Gluten – Hydrolysed gluten: health risk?
South Africa’s regulated common allergens
To claim or not to claim – the ‘Allergen-free’ controversy
Allergen labelling: Significant impact on product sales and market
‘Gluten-free’ under Scrutiny
VIDEO | Wheat vs Gluten
Allergen Testing Catalogue