How to Manage a Positive Allergen Residue Result when Testing Your Food Product

Beyond cross-reactivity, commercial ELISA kits can occasionally experience matrix interference. This is more common with foods or ingredients containing chemicals that might react with proteins in the sample or the antibodies used in the ELISA kits. For example, spices containing high amounts of phenolic compounds can generate false positives, often due to non-specific binding to the proteins and antibodies contained in the ELISA wells. We have also observed false positives in several commercial peanut ELISAs when testing caramel color, especially the darkest class of caramel color. These matrix-associated false positives typically yield weak positive results—usually <10 ppm. If, after dilution, your sample gives a positive result of 50 to >5000 ppm, the result is rather unlikely to be a false positive. With a true positive sample, the absorbance values that are measured from the sample wells will decrease rather linearly with each additional dilution until the absorbance reaches the absorbance associated with the buffer control. When a matrix interference is observed, the absorbance values will remain stable with each subsequent dilution. When the dilution is factored into the calculation of the final concentration for each dilution, an increase in the ppm value will be observed with each additional dilution. This is counterintuitive and provides a good indication of a matrix interference.

If you suspect a false positive result, what steps can be taken? The FARRP Laboratory has an investigational approach used for such situations, so it is prudent to contact us at this stage. First, we might repeat the analysis done by the original laboratory, because the result is more reliable if confirmed in two laboratories. This approach also examines the possibility that the original result was caused by laboratory error. Of course, this approach requires a duplicate sample and is dependent on the homogenous (non-particulate) distribution of the analyte in the sample. The original laboratory should be asked to do a repeat analysis if they have sample remaining that could be used for this purpose. If the original result is confirmed by the FARRP Laboratory, then we will recommend testing samples of this product or ingredient with multiple (3 to 4 if available) commercial ELISA kits for that analyte. This recommendation is generally predicated upon the original result falling below 10 ppm. In our experience, it is unlikely that higher concentrations are a result of a matrix interference but are rather true positive results. Since each commercial ELISA kit uses their own proprietary antibodies, extraction methods, and so on, they are not likely to experience the same matrix issues. If we find positive results in multiple ELISA kits, the likelihood of a true positive is enhanced. Conversely, if only the original ELISA kit gives a positive result, the possibility of a false positive is increased. We might also arrange for a PCR and/or a mass spectrometry analysis for confirmation of the positive result if such methods are available.

If the unexpected positive result was obtained by testing of an ingredient, the supplier may try to claim that the result was a false positive. Testing can readily demonstrate if that explanation is plausible. If testing is arranged on multiple lots (perhaps from multiple suppliers) of the same ingredient and only the suspicious lot tests positive, then matrix interference cannot be the answer. Matrix interference will be quite consistent if the test samples have a similar composition.

Sub-sampling can also contribute to analytical uncertainty. If the analyte is present in particulate form—e.g., chopped peanuts or whole sesame seeds—the analyte may not be present in every sample taken for analysis. In this case, the unexpected positive result may not be confirmed in every subsequent test. The only solution to this possibility is to test multiple samples. If you suspect that potential cross-contact allergens may be particulate in nature, taking multiple samples at the outset is an excellent strategy.