Editor’s note: This is part 1 of a two-part series on allergen residue results. Part 1 focuses on how to confirm a positive result and Part 2, which will publish in our February/March 2021 issue, will focus on steps to take once a result has been affirmed.
Positive results in allergen testing of food products can sometimes be quite unexpected and disconcerting. This is particularly true when testing finished food products ready for distribution and sales, or ingredients procured for a manufacturing run in the near future. Positive allergen residue results are not so surprising when testing to assess the effectiveness of cleaning protocols on shared equipment, especially when qualifying a new cleaning procedure, but a positive result is disturbing if the cleaning protocol has been assessed on numerous previous occasions with no positive results.
Oftentimes, ingredient or finished product samples are sent to external commercial laboratories to confirm that detectable allergen residues are not present. Swabs and final clean-in-place rinse waters are also sent to commercial laboratories, typically as a third-party check to demonstrate consistency with in-plant analysis using qualitative methods such as lateral flow devices (LFDs). In some cases, the product, ingredient, and/or process have been checked on multiple occasions with no previous positive detection of allergen residues. Even in cases where allergen testing has not been performed previously or testing was sporadic, the positive result is still an unpleasant surprise. The situation becomes even more alarming if the testing was performed by a valued customer, an auditor, or a regulatory inspector.
What should you do when you get that unexpected positive result? First of all, take three deep, calming breaths, and step back from the ledge. Then, get into investigative mode. Sometimes, the recipients of an unexpected positive result will immediately wonder if the external laboratory made a testing error and/or claim that the result is a false positive. That possibility needs to be assessed but is not the only possible explanation. Other possibilities include a manufacturing error (cross contact), a contaminated ingredient, a packaging error, or a failure of the allergen cleaning protocol.
We know from experience that recipients of unexpected positive results from an external lab report may immediately reach out to their external laboratory or reach out to the Food Allergy Research and Resource Program (FARRP) for assistance. While we are okay with sharing your pain at this stage, know that our initial goal will be to put you into investigative mode to get enough information so that we might really be able to help you with a thorough root cause analysis and/or risk assessment.
Carefully Examine That Laboratory Report
The laboratory report should, but will not always, contain critical information. First, double check the sample identification information on the report to ensure the laboratory report contains the test result for the sample(s) that you sent for analysis. The laboratory report will contain the analytical result(s), but most importantly, it should also contain the units, usually ppm for allergen analysis. However, these laboratory reports may not always list the calibrant. For example, when testing for milk residues, 10 ppm β-lactoglobulin (BLG) is not the same as 10 ppm non-fat dry milk (NFDM). In fact, 10 ppm BLG is equivalent to 286 ppm NFDM because BLG is roughly 10% of total milk protein and NFDM contains about 35% protein. The laboratory report should, but will not always, contain the identity of the method used to acquire the result. Many external laboratories use commercial enzyme-linked immunosorbent assays (ELISAs) for detection of allergen residues. Citing milk allergen analysis again as our example, at least six different ELISA kit companies (even more on an international basis) produce milk ELISA kits, and several of them make multiple milk ELISA kits with different targets (total milk, casein, or BLG) and different calibrators (non-fat dry milk, soluble milk protein, casein, or BLG). The choice of the milk ELISA kit can make a difference for your results and will certainly affect the interpretation of the seriousness of the finding to the potential risk of the product.
If you have used this commercial laboratory on numerous occasions in the past, you may have confidence that they are excellent analysts; however, you need to find out which ELISA test was conducted and the units/calibrants associated with that method if this information is not clearly provided on the analytical report. If the laboratory report does not contain information on the method used or the calibrant, you will need to contact the laboratory to get that information. The choice of the external lab can also be important. ISO-accredited labs must undergo an audit and approval process for specific methods to be included on their scope of accredited procedures. Be aware that ISO-accredited labs may not have all test methods within their ISO scope. If you want to use an ISO-accredited lab, make sure that the allergen test methods for your samples are included within their ISO scope or that that the laboratory follows a similar degree of care with procedures that are not under their ISO scope.
In addition to commercial ELISA kits, allergen analysis may also be performed by polymerase chain reaction (PCR), which detects DNA from the allergenic source, or by mass spectrometry, which detects specific proteins from the allergenic source. PCR typically has high specificity, but quantification of results and correlation to how much protein came from the allergenic source of concern can be difficult. Mass spectrometry methods hold great promise for the future, but few well-validated methods currently exist that can be used in the wide variety of ingredient and food matrices that are commonly analyzed by the food industry.
Most allergen analysis is performed using commercial ELISA kits. The level of sensitivity of those kits is typically in the low ppm range but can vary between kits. It is important to remember that the choice of calibrant affects the result (10 ppm BLG = 286 ppm NFDM) as well as the choice of protein target(s) that the kit detect. Allergen ELISA kits also have a dynamic range associated with the quantitative standards supplied with the kits. For example, the dynamic range might be 2.5–25 ppm. Many commercial laboratories will not dilute the sample if the result exceeds the upper limit of the dynamic range. Thus, you might get a result of, for example, >25 ppm. Your first question will be: How much greater than 25 ppm? Some commercial laboratories will not answer or even be able to answer this question. Some external laboratories will dilute samples, but some of these laboratories charge extra for taking that step. The FARRP Laboratory immediately dilutes samples that fall outside the dynamic range of the kit up to a maximum of 200-fold dilution (5000 ppm, or 0.5%). From a risk assessment perspective, knowing the specific, quantitative result is imperative for a thorough exposure assessment.
When you receive an unexpected result, you may want the laboratory to repeat the analysis. Some commercial laboratories will not be able to do that because they do not save any of the sample to use for a repeat analysis. Other laboratories will conduct a repeat analysis upon request, but some of them will charge again to do that test. For these reasons, we suggest that a best practice is to take duplicate or even triplicate samples, saving the extra ones at your place of business in case a repeat analysis is desired.
Could the Result Be a False Positive?
Commercial ELISA kits are typically quite specific. The manufacturers of these commercial kits assess the likelihood of cross-reactivity across a range of foods and food ingredients. Significant cross-reactivity is not often encountered except for very closely related foods such as walnut and pecan (these two nuts are botanical cousins). ELISA kit manufacturers publish written inserts that are distributed with the kits and typically include information on cross-reactivity. Commercial laboratories should be aware of cross-reactivity issues and decline to test samples that might generate false positives due to cross-reactivity in that specific test. Alternative methods should be considered if available; however, the laboratories do not always know the compositional nature of the submitted sample. Often laboratories receive unknown, food-grade powders for analysis. It is impossible for the laboratory to know what the composition of a powder may be, so it is critical to develop a good line of communication with your external laboratory. For example, we are aware of a commercial peanut ELISA kit that yielded weak positive results on samples containing high amounts of pea protein, another legume, that were reported by another commercial laboratory. This situation prompted a recall before we had the chance to inform the company that the analytical result was likely a false positive. Another best practice is to inform the external laboratory about the general composition of the test sample and ask them to verify that there are no known cross-reactivities in the ELISA kit being selected for the analysis that may affect the reliability of the results.
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.
Sample preparation can further contribute to analytical uncertainty, especially with certain types of particulates. For example, whole sesame seeds are difficult to break apart. If laboratories are using blenders to homogenize the sample before sub-sampling and extraction, they may find that the sesame seed remains intact. In that case, the sesame proteins will not be well distributed within the sample.
Every commercial ELISA kit has a stated dynamic range, such as 2.5 to 25 ppm in the earlier example. The kits should come supplied with a standard curve, and one of those standards should be the lowest concentration from the dynamic range—2.5 ppm in our example. In that situation, 2.5 ppm would be the lower limit of quantitation (LLOQ) of the ELISA method. But ELISAs also have lower limits of detection (LOD) that are below the LLOQ. The LOD is typically determined in a very passive matrix such as buffer. Some commercial laboratories will report positive results between the LOD and the LLOQ. This practice is questionable in our opinion because the matrix interference from the food being tested can generate weak low positive results between the LOD and LLOQ. Thus, it is important to find out the LLOQ of the ELISA method being used on your samples by the external laboratory and be mistrustful of any positive result reported below the LLOQ. These results could be false positives that will be very difficult to confirm.
Dr. Taylor is professor emerita of food science and technology and co-founder and co-director of the Food Allergy Research and Resource Program (FARRP) at the University of Nebraska-Lincoln. Reach him at email@example.com. Dr. Jayasena is a post-doctoral and senior researcher at FARRP. Niemann, Lambrecht and Kraft are lab managers at FARRP. Reach them at firstname.lastname@example.org, email@example.com, and firstname.lastname@example.org. Dr. Baumert is associate professor in the department of food science and technology at the University of Nebraska-Lincoln and co-director of FARRP. Reach him at email@example.com.