Allergen testing – is a positive actually a positive?

Most allergen tests look for proteins relating to the species that contain that allergen (ELISA testing) or a section of DNA associated with that allergenic species (qPCR testing). Sometimes the protein or section of DNA used within the test is indistinguishable between closely related species.

Essentially, most of the tests look to identify the species and not necessarily the specific allergenic component within the species. This means that for some tests it is possible to get a positive result for an allergen that is actually a false positive. So it has detected another, closely related species, that does not contain the allergenic component. Allergens we know to be challenging to test for include peanut and mustard. Many peanut tests cross-react with pulses, for example.

Appropriate tests are usually selected based on the declared ingredients list. Different tests and different kits have different cross-reactivity matrices. In most instances, the laboratories will (if asked) provide a list of species where cross-reactivity does not occur and where known cross-reactivity has been documented. However, this does not mean that a positive result is not a false positive.

Peanut allergen testing

Where we have seen positive results for peanuts, further testing using next generation sequencing (NGS) has often identified an undeclared ingredient/species that is known to cross-react with that specific test or a legume that is not declared on the cross-reactivity information.

This information is helpful and could avert a product recall. It does still, however, identify a significant issue with the presence of a non-declared ingredient. Is this there by design (fraud), incidental (i.e., field contamination of raw materials) or human error? Bear in mind that testing cannot identify intent, it can only report on the outcome seen.

Mustard allergen testing

As mentioned, mustard allergen is also challenging to test for. This has been a hot topic for some time, particularly after Italy moved to apply precautionary (alibi) labelling on all flour and pasta products indicating they ‘may contain mustard’.

There are three mustard species where allergenic proteins are found (Brassica juncea - mustard greens, Brassica nigra - black mustard, and Sinapis alba - white mustard); however, there is often cross-reactivity in the test with other, similar, non-allergenic Brassica species, such as rapeseed. As a result, we recommend should you receive an unexpected, positive allergen test for mustard, further DNA testing is undertaken to specifically target these three target species, either using NGS or targeted PCR testing.

There are documented primer sequences that target sections of the DNA that are different between closely related species, which, when used in combination, can allow the discrimination of these different species.

Given these allergens are often detected at very low levels this can still prove problematic. Before undertaking DNA testing looking for mustard allergen, ask some targeted questions about what level of discrimination the laboratory can achieve. At Food Forensics we are using a combination of primers (targeting different parts of the genome) to enable discrimination between the different species within the Brassica family. Even with this level of sophistication, where the percentage inclusion of the allergen is very low, it can still be tricky to detect sufficient sequences of the allergen to conclusively determine the species.

Takeaway messages

• A positive allergen result does not necessarily mean the allergen is present – ask questions on cross-reactivity and false positives regarding the specific test you have undertaken.
• If you have a positive detection, consider a cross-validation test, but, make sure you investigate the test limitations before you test.
• Make sure you have a robust plan on how you will react to a positive allergen result before you find yourself in that situation.