Trace Allergen Detection Methods for Food Manufacturing

Ask any QA director what keeps them up at night, and undeclared allergens are usually near the top of the list — right up there with the coffee that isn't strong enough to get them through it.

It's a fair concern. Undeclared allergens remain a leading cause of food recalls, and for manufacturers operating shared equipment or producing complex formulations, even small amounts of unintended allergen material can create significant food safety and compliance challenges. The FDA identifies allergen cross-contact as a hazard manufacturers must control through appropriate current good manufacturing practices and preventive controls.

For food safety directors, QA leaders, and plant managers, the challenge isn't simply knowing that an allergen could be present. It's being able to determine whether it is present, how much may be present, and whether the analytical method used can provide a result you can actually trust.

That's where allergen detection becomes more than a checkbox.

ELISA, PCR, and LC-MS/MS each answer different analytical questions. Food matrix, processing conditions, sample type, target allergen, and required sensitivity can all influence which method makes the most sense.

Here's how manufacturers can evaluate the options — and build a testing strategy that supports a more defensible Allergen Control Plan.

What Is Trace Allergen Detection?

Trace allergen detection is the analytical process used to identify or quantify unintended allergenic material in ingredients, finished products, or targeted environmental and food-contact samples.

Testing can support several manufacturing decisions, including:

• Screening higher-risk raw materials
• Verifying allergen removal after cleaning
• Testing finished products
• Investigating unexpected allergen results
• Evaluating difficult or highly processed food matrices
• Supporting allergen control and validation programs

The FDA identifies ELISA, DNA-based polymerase chain reaction (PCR), and mass spectrometry among the analytical approaches used for allergen testing.

The important distinction is that these technologies do not all detect the same thing. Choosing a method based solely on the allergen name isn't enough. The analytical question and the characteristics of the sample matter just as much.

Which Allergen Detection Method Is Best?

There isn't one universally "best" allergen detection method.

ELISA, PCR, and LC-MS/MS each have different analytical targets and strengths. Recent research continues to identify ELISA and PCR as established approaches while highlighting the growing role of mass spectrometry for protein identification and multiplex allergen analysis.

The takeaway is simple: choose the method to fit the question, not the other way around.

ELISA for Routine Allergen Screening

Enzyme-linked immunosorbent assay (ELISA) uses antibodies to detect specific allergenic proteins or protein fractions. It remains a widely used approach for routine allergen analysis because it can provide sensitive, practical testing for many common allergens.

Common applications include:

• Raw material screening
• Finished product testing
• Cleaning verification
• Food-contact surface swabbing
• Targeted environmental sampling

But ELISA isn't immune to the challenges created by food processing and formulation.

Heating, hydrolysis, fermentation, and other processing conditions can alter protein structure and affect antibody recognition. Food matrix components can also influence extraction and detection.

That's why method performance should be evaluated for the actual product or sample type being tested rather than assumed from performance in another matrix.

AOAC INTERNATIONAL's updated 2024 Appendix M guidance provides recommendations for validating immunoassay-based food allergen methods, including study design, testing materials, and statistical considerations.

PCR for Allergen-Related DNA Detection

Polymerase chain reaction (PCR) detects DNA associated with a particular species or ingredient.

That distinction matters.

PCR does not directly measure allergenic protein. Detecting DNA can provide evidence that a particular species or ingredient is present, but it does not establish the concentration of allergenic protein in the sample.

Likewise, the absence of detectable DNA does not necessarily prove the absence of allergenic protein.

This becomes particularly important in highly processed foods, where processing can affect DNA and proteins differently.

PCR can therefore be useful when the analytical question involves ingredient or species identification, but it should be selected with an understanding of what the method actually measures. Research comparing allergen detection technologies specifically notes this distinction between DNA detection by PCR and protein detection by other analytical approaches.

LC-MS/MS for Complex and Processed Food Matrices

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identifies characteristic peptide markers associated with allergenic proteins.

Its ability to identify specific peptides and support multiplex analysis makes LC-MS/MS particularly valuable when manufacturers are dealing with complex formulations, highly processed foods, or situations where greater analytical selectivity is needed.

Potential applications include:

• Complex food formulations
• Highly processed products
• Multi-allergen analysis
• Confirmatory testing
• Investigations involving difficult matrices
• Method development and validation

But LC-MS/MS isn't a magic wand.

Like other analytical approaches, its performance depends on sample preparation, extraction, digestion, and the characteristics of the food matrix. Recent research shows that matrix composition and pretreatment can significantly influence allergen quantification, including LC-MS/MS-based analysis.

The technology is powerful. The method still has to be fit for purpose.

Why Does the Food Matrix Matter in Allergen Testing?

Here's where allergen testing gets especially interesting for manufacturers.

The same allergen can behave differently depending on what surrounds it.

Proteins, fats, carbohydrates, polyphenols, salts, and other components of a food can affect extraction and detection. Processing can also alter allergenic proteins or the molecular targets analytical methods rely on.

Recent research identifies factors such as reduced allergen solubility, epitope masking, nonspecific interactions, and sample preparation as important considerations when quantifying allergens in food.

High-Fat Foods

Chocolate, nut butters, fillings, and other high-fat products can make extraction more challenging because target proteins may interact with other components of the food matrix.

Polyphenol-Rich Foods

Cocoa, spices, tea, and other polyphenol-rich ingredients can introduce compounds that interfere with analytical recovery or signal.

Hydrolyzed and Fermented Products

Hydrolysis and fermentation can break proteins into smaller fragments and alter the molecular targets available for detection.

That means a method that performs well on one product may not automatically perform the same way on another.

Matrix-specific evaluation matters.

The result isn't simply a number on a laboratory report. It's a measurement whose reliability depends on how well the analytical method fits the product being tested.

How Should Manufacturers Choose an Allergen Detection Method?

Start with the question you're trying to answer.

Are you screening an incoming ingredient? Verifying cleaning? Testing a finished product? Investigating an unexpected positive? Evaluating a highly processed formulation?

The answer can help determine which analytical approach is appropriate.

This doesn't mean every manufacturer needs every technology.

It means the testing strategy should be built around the risk, matrix, target, and decision the result will support.

How Can Allergen Testing Support an Allergen Control Plan?

Analytical testing works best when it is connected to the rest of the allergen control program.

FDA guidance emphasizes controls such as appropriate sanitation, segregation, production scheduling, and procedures designed to prevent or significantly minimize allergen cross-contact.

Testing can provide another layer of evidence that those controls are working.

Verify Higher-Risk Raw Materials

Supplier documentation can help establish expectations for incoming materials, but targeted independent testing can provide additional evidence for higher-risk ingredients or lots when appropriate.

Verify Cleaning Procedures

Testing can help determine whether a sanitation process is effectively controlling allergen residues under actual operating conditions.

Depending on the risk assessment, sampling may include:

• Food-contact surface swabs
• Equipment rinse samples
• Final CIP rinse samples
• Finished product
• Targeted difficult-to-clean locations

Investigate Unexpected Results

A positive result should prompt a thoughtful investigation rather than simply a pass/fail reaction.

Potential contributing factors can include:

• Inadequate cleaning
• Equipment design
• Ingredient variability
• Supplier issues
• Rework practices
• Production sequencing
• Employee or material movement
• Airborne particulate transfer
• Sampling or analytical variability

When results are trended over time, they can help identify whether an isolated result is truly isolated — or part of a larger pattern.

What Should Food Manufacturers Look for in an Allergen Testing Laboratory?

Choosing an allergen testing laboratory based on price alone can create a bigger problem later: data that doesn't adequately answer the question you need to make a manufacturing decision.

Instead, evaluate the laboratory's ability to support your specific products, matrices, allergens, sample types, and testing objectives.

Look for:

• Method capability — ELISA, PCR, LC-MS/MS, and other relevant technologies
• Matrix expertise — experience with products similar to yours
• Method validation — evidence that methods are appropriate for their intended use
• Sensitivity and selectivity — aligned with the analytical question
• Multi-allergen capability — when multiple targets are relevant
• Technical support — assistance interpreting results and selecting methods
• Sampling support — appropriate collection supplies and guidance
• Turnaround time — aligned with production and release decisions
• Data management — accessible results and documentation

AOAC INTERNATIONAL's current food allergen validation guidance reinforces the importance of appropriate study design, testing materials, and statistical evaluation when validating immunoassay-based allergen methods.

The principle is straightforward: the value of an allergen testing program depends on how well the analytical method fits the question you're asking.

Building a More Defensible Allergen Testing Strategy

Allergen management is increasingly data-driven.

Visual inspections, sanitation procedures, segregation, production scheduling, and preventive controls remain essential. Analytical testing adds another layer of evidence by helping manufacturers determine whether those controls are achieving the intended result.

A strong testing strategy asks:

• Where is allergen cross-contact most likely to occur?
• Which ingredients or products present the greatest risk?
• What exactly does the selected analytical method detect?
• Does the method fit the food matrix?
• Has the method been appropriately validated?
• Is the result an isolated finding or part of a trend?
• Can the data support a sanitation, release, investigation, or corrective-action decision?

The goal isn't simply to generate more test results.

It's to generate useful evidence.

Partner With Mérieux NutriSciences for Allergen Testing

Defending a brand against allergen cross-contact takes more than ordering a test. It requires an analytical strategy built around the products, equipment, matrices, and risks that matter to your operation.

Mérieux NutriSciences supports food manufacturers across North America with allergen testing and chemistry capabilities designed to help answer complex analytical questions.

Capabilities include:

• ELISA, PCR, and LC-MS/MS
• Testing for FDA-recognized major food allergens
• Canadian and EU allergen testing support
• Gluten-free testing, including fermented and hydrolyzed matrices
• Allergen swab kits
• Raw material, finished product, and environmental sample testing
• Matrix-specific testing support
• Method development and validation
• Sampling and shipping support
• MyMXNS online results access

A strong Allergen Control Plan is only as defensible as the data behind it.

Put analytical testing to work turning your allergen controls into measurable, verifiable safeguards for your products — and your brand.

Explore Mérieux NutriSciences Chemistry & Allergen Testing Services or contact our technical team at (877) 777-6375 to discuss your allergen testing needs.

References

AOAC INTERNATIONAL. (2024). *Guidance on food allergen immunoassay validation*. In G. W. Latimer Jr. (Ed.), *Official Methods of Analysis of AOAC INTERNATIONAL* (22nd ed., Appendix M). Oxford University Press. https://doi.org/10.1093/9780197610145.005.013 

Birse, N., et al. (2023). Food allergen analysis: A review of current gaps and the potential to fill them by matrix-assisted laser desorption/ionization. *Comprehensive Reviews in Food Science and Food Safety, 22*(5), 3984–4003. https://doi.org/10.1111/1541-4337.13216 

Hu, R., et al. (2024). Quantifying allergenic proteins using antibody-based methods or liquid chromatography–mass spectrometry/mass spectrometry: A review about the influence of food matrix, extraction, and sample preparation. *Comprehensive Reviews in Food Science and Food Safety*. https://doi.org/10.1111/1541-4337.70029 

U.S. Food and Drug Administration. (2023, September 26). *The current food allergen landscape*. https://www.fda.gov/food/conversations-experts-food-topics/current-food-allergen-landscape 

Wang, Y., et al. (2024). Research progress on detection methods for food allergens. *Journal of Food Composition and Analysis*. https://doi.org/10.1016/j.jfca.2024.106479