PCR & ELISA Allergen Testing: How Manufacturers Detect Hidden Risks

1-Minute Summary

ELISA allergen testing and PCR allergen testing are the two most widely used methods for detecting allergens in food, supplements, and cosmetics.
Sandwich ELISA is ideal for protein-based allergens; PCR is preferred when DNA is more stable than protein.
Each method uses a different detection strategy: ELISA identifies proteins, while PCR finds DNA.
HPLC-MS is available for more complex matrices or confirmation of results.

Recall Risks: Why Allergen Testing Matters for Manufacturers

Undeclared allergens were the top reason for food product recalls in 2024, a trend that has been consistent for several years. Undeclared allergens are also a growing risk in dietary supplements, especially as nutraceuticals, botanical extracts, and complex matrices gain popularity.

The problem affects cosmetics, too. In fact, one analysis found 334 product recalls from 2011-2023, affecting ~77 million units. About 11% of those recalls were ascribed to “Incorrect or undisclosed ingredients” or “Labeling/packaging errors”, in which undeclared allergens is most likely to fall.

For manufacturers, allergen testing is no longer optional. It’s a critical element of safety and compliance, especially when managing diverse ingredient sources, shared equipment, or novel product formats.

But questions abound regarding how allergen testing labs carry out this work. What technologies are available? Which is right for your samples? We provide answers in this article.

How Allergen Testing Is Performed: ELISA, PCR, HPLC, & More

Several laboratory technologies are used today to detect allergens in food, supplements, and cosmetics. These include:

ELISA allergen testing (Enzyme-Linked Immunosorbent Assay) – detects allergenic proteins.
PCR allergen testing (Polymerase Chain Reaction) – detects DNA from allergenic ingredients.
HPLC-MS (High-Performance Liquid Chromatography–Mass Spectrometry) – detects allergenic peptides.
Lateral Flow Devices (LFDs) – rapid, portable screening tools.

Consider This if Using ELISA and PCR Allergen Test Kits

Most people use commercially available test kits to obtain and send samples to a lab. The technicians in the lab process those samples using one of these technologies. These kits are validated, easy to implement, and widely used for both routine testing and investigation of potential allergen cross-contact.

Each method has advantages depending on the product matrix, processing, and regulatory needs. However, ELISA and PCR remain the most widely used and validated platforms for routine allergen testing. They offer a strong balance of sensitivity, specificity, speed, and cost-effectiveness for real-world quality and safety programs.

For that reason, we shall explore how each one works using practical analogies to help make sense of the science. Let’s start with ELISA.

Why Accreditation and Validation Matter When Using ELISA & PCR Allergen Test Kits

It’s not enough to use ELISA or PCR test kits — the way these tests are performed makes a critical difference. Laboratories that are ISO 17025-accredited offer a higher level of confidence in their allergen testing results for these reasons:

Method Validation: Each test method should be validated in the product matrix to ensure it performs reliably (specificity, accuracy, precision, LOD/LOQ). Regulatory agencies such as the FDA and EU authorities (e.g., SANTE/11312/2021 and AOAC SMPRs) require this.
Matrix-Specific Reliability: Chocolate, cosmetics, supplements, and protein powders all pose different challenges (e.g., interfering compounds or extraction difficulties).
Repeatability and Reproducibility: ISO 17025 requires labs to demonstrate that results are consistent across time and technicians.

What to Ask Your Lab

Is your lab ISO 17025-accredited for allergen testing?
Have your ELISA or PCR methods been validated for my product matrix?
What’s your false positive/false negative mitigation protocol?

Understanding ELISA Allergen Testing: Why Sandwich Format Dominates

ELISA allergen testing is a versatile testing platform used to detect specific proteins within a complex sample. What makes ELISA so adaptable is that it can be performed in several different formats, depending on the target molecule and the analytical goal.

Here are the four main ELISA formats and how they differ:

Format	How It Works	Best For
Direct ELISA	One antibody binds directly to the target and is enzyme-labeled for detection.	Simple antigens; low interference matrices
Indirect ELISA	Unlabeled primary antibody binds the target, then a labeled secondary antibody detects it.	Boosting signal strength; research assays
Sandwich ELISA	One antibody captures the target, a second binds to a different site, and carries the detection enzyme.	Allergen proteins, biomarkers, regulatory testing
Competitive ELISA	The target in the sample competes with a labeled version for antibody binding. Signal decreases as concentration increases.	Small molecules with only one binding site (e.g., drugs)

Graphics that show the four formats of ELISA allergen testing.

Why Sandwich ELISA Is Most Common for Allergen Testing

Most allergenic targets — such as casein (milk), ovomucoid (egg), or Ara h1 (peanut) — are large enough to allow two antibodies to bind simultaneously. This makes them ideal for sandwich ELISA, which is:

Highly specific (dual antibody recognition).
Quantitative (color intensity correlates with concentration).
Validated in commercial test kits for food, supplements, and cosmetics.
Resistant to false positives, especially in complex or processed matrices.

Because of these advantages, nearly all commercial ELISA allergen testing kits use the sandwich format. Let’s look at how it works.

ELISA Allergen Testing Explained: A Two-Key Security System

Let’s explain with an analogy. Picture your product sample as a massive building filled with thousands of locked rooms. Each room contains one specific ingredient, compound, or trace molecule — but you don’t know which room contains what. Behind one of those doors, an allergenic protein like milk, soy, or peanut may be hiding. You need a way to find it quickly and confirm it accurately.

Enter ELISA.

It works like a precision security system using two specially made keys. These keys are actually antibodies, each designed to bind only to a very specific protein — in this case, a known allergen.

Step 1: The First Key Fits the Lock

A custom key (the capture antibody) is sent out to try every lock. Only one door — the one containing the allergenic protein — will accept this key. But this key alone can’t open the door.

Step 2: The Second Key Unlocks the Door and Triggers an Alarm

Next, a second key (the detection antibody) is introduced. This key is also custom-built for the same allergen and only works if the first key is already in place. If both keys are present and matched to the same protein, the door finally opens.

And when it does? It sets off an alarm that changes the door’s color.

In real-world terms, this is an enzyme-based color change that occurs inside the test well. The signal is visible and measurable. The stronger the color, the more of that allergen is present in the sample.

Why This Double-Key System Works So Well

Accuracy: Both keys must fit to trigger the alarm. This dual step helps eliminate false positives and ensures you’re detecting the right protein.
Quantification: The intensity of the alarm signal (color) gives you a reliable estimate of how much allergen is in the sample, often down to parts per million (ppm).
Speed and Scalability: ELISA tests can be run in batches across multiple samples, making them ideal for routine QC, cleaning validation, or verifying “free-from” claims.

PCR Allergen Testing: Finding DNA with a “Molecular Photocopier”

Sometimes, the allergenic protein is degraded — but the DNA remains. This is where PCR allergen testing comes in.

PCR doesn’t look for the allergen’s protein, it looks for its genetic blueprint.

Step 1: You Know What the Blueprint Looks Like

Imagine you’re looking for traces of peanut in a supplement ingredient. The peanut’s DNA sequence is like a unique architectural blueprint. You know exactly what sections to look for.

You prepare short DNA sequences called primers. These act like search commands that tell your copier: “Only copy this blueprint if you find it.”

Step 2: The Copier Starts Scanning the Building

Your sample (the product) is scanned by the PCR machine — like sending a technician through every room of the building, looking for even a scrap of the peanut blueprint.

If a match is found, the copier kicks into gear.

Step 3: Copying Scales Quickly

Once the target sequence is found, PCR makes millions of copies of it. It’s like a copier that duplicates the same blueprint over and over, incredibly fast. This is what scientists mean by “amplification”.

Even if the original piece of DNA was nearly invisible, once it’s been copied millions of times, you can detect it easily. The more copies are made (faster and earlier in the process), the more of that allergen DNA was originally present.

Why This Matters for Quality and Safety

Sensitivity: PCR can detect allergen DNA even when the protein is destroyed, degraded, or diluted.
Specificity: The copier only turns on if it finds an exact DNA match, so it avoids copying the wrong blueprint.
Utility in Complex Matrices: PCR is ideal for products with multiple botanicals, processed ingredients, or emulsifiers, which are areas where ELISA might struggle.

ELISA Allergen Testing vs. PCR Allergen Testing

To help compare the two methods side-by-side, here’s a reference table based on common performance attributes:

	PCR Allergen Testing (Polymerase Chain Reaction)	ELISA Allergen Tetsing (Enzyme-Linked Immunosorbent Assay)
Detects	DNA from allergenic ingredients	Allergenic proteins (actual allergens)
Target	Species-level DNA (peanut, soy, shellfish)	Specific allergenic proteins (Ara h 1 from peanuts)
Sensitivity	Very high (detects trace DNA)	High (sensitive to low protein levels)
Specificity	High, depends on primer design	High, depends on antibody quality
Quantification	Generally qualitative or semi-quantitative	Quantitative (can measure allergen concentration)
Processing Effects	DNA often survives heat, pressure, pH changes	Proteins may degrade with heat or processing
False Positives	Can detect DNA from non-functional allergens	Cross reactivity concerns
Time to Result	~4-5 days	~2-3 days
Cost per Test	Generally higher due to equipment and kit costs	Lower for routine testing

What About HPLC-MS?

High Performance Liquid Chromatography–Mass Spectrometry (HPLC-MS) is used when allergen detection requires greater sensitivity and specificity than ELISA or PCR can offer, especially in complex or processed products.

How It Works

The process begins by extracting proteins from the sample using appropriate solvents. These proteins are then enzymatically digested (commonly with trypsin) into smaller peptide fragments. After protein clean-up, the sample is analyzed using HPLC-MS, which separates and identifies peptides with high precision. The final step involves data analysis to confirm the presence of allergenic material.

Advantages

Highly sensitive and accurate.
Comprehensive profiling of protein fragments.
Low detection limits.
Ability to detect multiple allergens in one run (multiplexing).

Disadvantages

Higher cost and technical requirements.

Requires trained analysts and extensive sample preparation.

Time-consuming and maintenance-intensive.

While not typically used for routine screening, HPLC-MS is invaluable for confirmatory testing, method development, and detecting hydrolyzed or modified allergens that ELISA or PCR may miss.

Using PCR & ELISA Allergen Testing for Environmental Swabs & Cleaning Validation

ELISA and PCR can also be applied to environmental swabs, helping manufacturers validate their sanitation and allergen control programs.

Swab-Based ELISA & PCR Allergen Testing: Detects allergen residues on surfaces, equipment, or utensils after cleaning.
Routine Cleaning Validation: Confirms that your cleaning procedures are consistently removing allergens to below threshold levels.
FDA FSMA & GFSI Alignment: Allergen cleaning validation is required under FSMA’s Preventive Controls rule and by all major GFSI standards (SQF, BRCGS, etc.).

Top Challenges in Allergen Testing for Food, Supplements, & Cosmetics

Testing samples for the presence of allergens presents a host of challenges, which we summarize below:

Food Matrix Effects
Each product matrix has unique chemical and physical characteristics that can interfere with detection. For example, tannins in chocolate can bind to proteins and reduce assay sensitivity.
Physical Properties of the Sample
Some allergens are tightly bound within the sample, making extraction difficult. Poor recovery during sample prep can result in underestimation or false negatives.
Homogeneity and Allergen Distribution
If the allergen is unevenly dispersed in the product, test results can vary depending on the portion sampled. This is especially important in bulk materials or dry blends.
Processing Effects
Heat, pressure, pH, or enzymatic treatment can denature or degrade proteins, making them undetectable by ELISA, even if allergenic potential remains.
False Negatives and False Positives
Both are possible. Denatured allergens may escape detection (false negatives), while non-functional fragments or cross-reactive compounds may be mistakenly identified (false positives).
Emerging Novel Foods
Plant-based meats, insect proteins, and other novel ingredients may contain new or unknown allergens. They can also trigger cross-reactivity with existing allergens.
Cross-Reactivity
Some allergenic proteins share similar structures across different sources. For instance, proteins in canola seeds may cross-react with those in mustard seeds. This can lead to false positives if the test isn’t sufficiently specific.

Before using any commercial test method, it’s essential to:

Review the manufacturer’s validation data for known cross-reactivities.
Verify performance in your specific product matrix, especially for novel foods.

Get Support with ELISA or PCR Allergen Testing

Certified Group companies offer ISO 17025-accredited allergen testing at multiple labs across North American. In addition to competitive TAT and pricing, we provide:

ELISA allergen testing.
PCR allergen testing (only in Aurora, Ill. Note that our PCR validated method is not available for over-the-counter drug products).
Capability to test all major allergens.
Automated testing capability – Chemwell and Thunderbolt.
Ability to test raw materials, products, and swabs.
Chemical testing capabilities

Contact our team now to discuss your allergen testing needs.