Beyond Immuno-Based Allergen Testing

Most commercially available kits for food allergen testing rely on the application of immuno-based methods such as ELISA or lateral flow devices (strip tests). To carry out ELISA, trained personnel are required but numerous samples can be analyzed in parallel by using 48-well or 96-well microtiter plates. In general, the analysis can take between 30 minutes and a few hours.
At present, ELISA is the most widely applied method for the detection and quantification of food allergens. However, although many samples can be analyzed at the same time, these samples can only be tested for one analyte.

Limitations to consider

Due to the high specificity of antibodies towards only one particular allergenic protein and technology related limitations, a separate kit has to be used for each allergen. Furthermore, the high degree of specificity to one allergen might lead to false negative results.
Food processing steps like heat treatment, the addition of acidic compounds or fermentation can modify the target protein structure. These modified allergens can lose their immunological properties and the antibody – target protein complex cannot be formed anymore. This leads to false negative results or reduced quantifications. Strip tests are inexpensive, very easy to use, do not require laboratory equipment, and give results usually in a few minutes. However, most strip tests are only qualitative and rely on antibodies as recognition elements. Therefore, they suffer from the same problems as ELISA tests with highly processed food.
In recent years, alternative analysis methods have been established to overcome at least some of the restrictions of immuno-based tests systems.

Detecting allergens with DNA

PCR (polymerase chain reaction) is a relatively fast and inexpensive method for identifying DNA. This technology, developed in the 1980s, has improved continuously since then. PCR has been used for many years in the fields of medical diagnostics, forensics, environmental monitoring, and the quantification of genetically modified organisms in food and feed. In the early 2000s PCR was applied for the first time to identify the DNA of common food allergens like hazelnut and peanut. Until now, PCR assays for most of the US “big eight” and the 14 EU food allergens have been published.
PCR amplifies small fragments of a target DNA until a sufficient number of copies are obtained for visualization or quantification. By multiplying the analytical target by a factor of 107 to 109, the few molecules of allergen DNA obtained might just be sufficient for the successful detection of allergenic ingredients. Initially developed as a qualitative method, PCR was later modified to become a tool for quantitative analysis by the application of different fluorescent generating dyes or probes. The fact that PCR detects the extremely stable DNA molecule might be an advantage when analyzing highly processed food. DNA tends to be unaffected even by extreme conditions and can therefore still be detected even when most of the proteins have already been degraded or modified in some way. Furthermore, PCR can be used for allergens like celery which cannot be detected by antibodies. Celery has to be labeled in the EU but until now, all attempts to produce reliable antibodies have failed due to the close relationship between celery and other plants like parsley, carrot, coriander or fennel.
Over the last decade, newer DNA detection techniques have been developed. All these so-called isothermal amplification methods are in some way related to the conventional PCR but can be performed almost without any instrumentation.
A simple heating block is used to amplify the target DNA and the subsequent visual detection is realized via fluorescent dyes. Isothermal amplification is usually faster than PCR and less prone to any co-isolated impurities and in many cases even more sensitive.