Food Allergen Analysis

Quantitative assessment of cross contact allergens using analytical testing should consider the sporadic nature of cross contact allergens by using a comprehensive testing regime.

When determining an appropriate sample number for analysis, consider how the cross-contact allergens may be distributed. Even an extensive sample programming may not detect all cross-contact allergens. The sample number should consider the type of potential cross contact, and involve different time points within a batch, multiple batches, or possibly different production runs. To have confidence in the value of the results, it is important to have a robust sampling plan which considers the nature of the allergen (e.g. finely ground powder, paste, meal, whole or segmented nuts); and the nature of the process. If the process is a single continuous flow, or uses multiple depositor heads, run through heat exchange columns or single batch produced, the impact on the distribution of any cross-contact allergen will vary.

It is also important to consider any claims regarding the allergen status of a product . A stringent, risk based and ongoing sampling plan should be applied where any pack claim is made.

N.B When sampling, avoid composite samples for allergen analysis. Compositing samples occurs when several samples are combined into a single unit. Testing a composite sample may dilute the true allergen concentration and report a value that does not represent the level in each individual sample. If composite samples are used (e.g. for commodity testing) adjust the sensitivity of the test accordingly.

1. What are you intending to achieve, such as:

1. cleaning validation
2. cleaning verification
3. introduction of a new recipe
4. introduction of new equipment
5. investigation of the allergen profile of an ingredient
6. trouble shooting an out of specification ingredient or product
7. developing a baseline of allergen cross contact
8. mapping of potential allergen cross contact patterns
9. confirmation of a VITAL risk review. For a VITAL risk review confirmation, it may be helpful to advise the laboratory of the Reference Amount to ensure the method is suitably sensitive.

2. Information regarding the sample, such as:

1. the sensitivity you require
2. is quantitation required
3. is the sample a cleaning flush solution, a push through product, research and development investigation, known positive sample, customer complaint sample, etc.
4. does the sample have very high or very low ph
5. are there high levels of substances which may interfere in the analysis. e.g. phosphates, sugars salt, tannins, colouring agents
6. has the sample been exposed to extremes of temperature or pressure, fermentation or hydrolysis?

1. does the laboratory have expertise in allergen analysis?
2. does the laboratory have accreditor in the field of analysis?
3. is the method used accredited or validated for the ingredient or product that requires analysis?
4. will the allergen be detected by the method used (as an example, standard egg kits may not detect lysozyme and a specific kit to detect lysozyme may be required)?
5. will the matrix or process interfere in the analysis?
6. will the assay be the right sensitivity for my needs?
7. what units does the laboratory issue the result in? (i.e. mg /kg or ppm of the food or mg/kg or ppm of the protein. For example, peanut analysis results may be reported as 10 ppm total peanut, or, 2.6 ppm peanut protein).
8. does the laboratory confirm positive results and how?

There are a range of methods available which have their place in allergen analysis. The most common are listed below

There are a range of methods available which have their place in manufacturing site allergen analysis.

• ATP test kits
• Protein swabs
• Lateral Flow Device (LFD)

ATP Test Kits

ATP (Adenosine Triphosphate) test kits are less sensitive than specific allergen tests. They detect ATP from all sources (animal, vegetable, microorganisms, food protein residues etc) and are a general indicator of sanitation.

ATP test kits and protein swabs indicate general hygiene, as they are not specific to allergen residue, nor representative of allergen load. All swabbing may be impacted by sanitisers or residues and swabbing in general is qualitative and can only be used in easily accessed areas, so results may not accurately reflect the risk associated with hang up or closed systems.

Protein Swabs

Protein swabs use a simple colour indicator to identify the presence of protein on surfaces. The more protein present, the more pronounced the colour but the assays are not specific to allergens.

Lateral Flow Device (LFD)

Lateral Flow Devices may also be known as strip tests. These are an immunologically based analytical test similar to ELISA (Enzyme-Linked Immunosorbent Assay). They are used to screen for the presence of low level allergens in cleaning rinses and on environmental surfaces. They are suitable for surfaces and rinses but should only be used for raw materials or finished products where they have been validated for the matrix and allergen of interest. LFDs are a qualitative test (indicating present/absent), rapid, portable and relatively easy to use, however there are some technical limitations (e.g. where a high allergen load may be present) so always check from kit to kit. LFDs are not equivalent to laboratory-based ELISA testing.

There are a range of methods available which have their place in laboratory allergen analysis.

ELISA (Enzyme Linked Immunosorbent Assay) laboratory kits

ELISA (Enzyme Linked Immunosorbent Assay) test kits are the current analytical method of choice for food allergen detection and the most commonly used for routine food analysis. These test kits are available for detecting many of the common food allergen proteins. There is an increasing range of assays available as kit manufactures respond to industry needs.

ELISA test kits generally focus on specific ‘marker’ proteins. They should be specific (minimal false positives), quantitative (provide an allergen concentration) and sensitive (able to detect very low (ppm) levels of the protein. Although cost and time effective, and relatively easy to use, result interpretation requires a degree of technical expertise. There is no single ELISA kit available that will detect all the relevant allergens under the Food Code in a single assay.

LC-MS (Liquid Chromatography Mass Spectrometry)

Mass Spectrometry (MS) in the context of allergen detection is a process that identifies peptide markers from the allergic protein. The analysis can selectively identify very closely related proteins that may not able to be distinguished by other techniques. Allergen proteins that have been altered or broken down by food processing and which may not be detectable by antibody-based techniques may still be detectable by MS and the technique has the ability to directly analyze multiple allergens in a single analysis.

This technique may provide a valuable reference method in the future.

PCR (Polymerase Chain Reaction)

Polymerase Chain Reaction (PCR) methods detect DNA sequences (of the allergenic species) not allergenic protein. PCR methods are used to amplify and detect the DNA of an allergen and can be used to detect more than two allergens at once. They are specific, sensitive, and qualitative, can verify or clarify an ELISA result and can detect potentially allergenic products for which no ELISA test is currently available. PCR is a complex, laboratory-based method that requires a skilled analyst and can be useful for food products containing hydrolysed proteins. However, like all detection methods there are limitations associated with the impact of food processing. Some processing methods can destroy detectable DNA, causing false negative results and food matrices may interfere in the assays. In addition, DNA methods are not suitable for the detection of certain allergens, where there are low levels of DNA e.g. egg and milk.

When choosing a method of allergen analysis, it is critical that there is a clear understanding of the analytical outcomes and the appropriate application of laboratory results for each allergen detection scenario. There are many factors that can affect the accuracy of analytical testing.

An inappropriate allergen technique can produce a false negative result (the allergen is present, but the test is not capable of detecting the allergen) or a false positive result (the allergen is not present, but the test incorrectly identifies it as being present). An incorrect choice of analysis test may result in an incorrect result.

Each technique is different and may not necessarily produce comparable results. When choosing a test method, ensure that the limitations of the test and the required outcome for the test are considered.

Consider that the technique must be:

• Appropriately sensitive
• Qualitative or quantitative
• Specific for the target substance
• Able to detect the target substance in the specific matrix (some matrices, like chocolate and meat, do not readily liberate proteins and produce false negatives)
• Able to be performed in an appropriate time frame (minutes to days)
• Able to be performed by the person doing the test (simple to requiring an experienced laboratory-based analysis)
• Economical ($ to $$$)
• Validated (independently for its intended use).

Identify required outcome

An understanding of the required outcome will help to choose the correct method of analysis. Some methods provide quick results and may be useful for production scenarios such as to check if a production line has been sufficiently cleaned. However, often tests which can be performed rapidly do not provide quantitative results. Scenarios where quantitative results may be required, such as finished product testing to verify a VITAL risk assessment, may be required to be performed under laboratory conditions and be more time-consuming.

Specificity of test

Care should be taken to choose a test kit which is looking for the correct target. Allergen testing may be highly specific for a particular target and it is important to establish which marker the test is looking for and whether it will be in the product being tested. For example, kits which detect Beta Lactoglobulin are specific to the beta lactoglobulin fraction of milk. If the cross-contact allergen comes from the casein fraction of milk (which does not contain beta lactoglobulin) then a Beta Lactoglobulin kit would provide a false negative result for milk.

Affect of Matrices

Different matrices will affect the validity of allergen testing. Some matrices inconsistently and reluctantly liberate target proteins for identification by test kits. Also, some matrices, such as cleaning or flush solutions, may destroy target proteins and give a negative result which does not accurately reflect the allergen status of the product or flush. Extreme ph levels, high levels of sugars and salts and polyphenol rich foods (e.g. blueberries, blackberries, vegetables including onion, cabbage, and legumes) may all present challenges for analysis and require specialised extraction protocols to provide correct results.

Affect of Processing

The target protein can be adversely affected by processing, with high temperatures and/or hydrolysis possibly altering the structure of the proteins and making it more difficult to detect. Whilst these changes generally reduce the detectability of the allergen, they may in fact enhance allergenicity.

• For example, soy trypsin inhibitor is a target used by some soy allergen test kits. If the product that you are testing contains hydrolysed soy, or has been exposed to hydrolysis, then the presence of soy will not be detected in this kit and could produce a false negative result.
• Cleaning products may hydrolyse allergenic proteins so it is particularly important that this is considered when testing after a clean.

Read more in the tabs below on choosing a method of analysis below:

When choosing an external contract laboratory chose one with:

• Expertise in the field of allergen analysis
• NATA or equivalent accreditation for the performance of allergen analysis
• Validated methods
• A verification program for kit performance in the sample type submitted or has evidence that the sample is suitable for analysis
• Participation in appropriate proficiency programs
• Robust quality control processes
• Reports with clear details around the method and the reporting units provided in the results

Whether using an external laboratory or performing in house analysis it is important to choose:

• Methods or kits from a company with an established history and record of performance.
• Methods where the performance has been peer reviewed, published or approved by an international body (eg. AOAC, AFNOR) or has external validation data to support performance.
• Methods which perform well in proficiency programs
• Methods which are appropriately sensitive, specific and robust and able to detect the target allergen
• Methods which are designed to detect the correct form of the allergen (eg. casein or whey or both dependent on which milk component has been used by the facility).

The preferred method to identify and quantify cross contact allergens in a VITAL risk assessment is to sum the cross contact which is identified by the supplier in each raw material with cross contact allergens identified during a physical audit of the production environment. However, there is a significant role for allergen analysis in:

1. validation of the VITAL risk assessment;
2. verifying ingredient allergen statements and potential raw material cross contact;
3. targeted analysis of problem pieces of processing equipment;
4. confirming assumptions made during the implementation of VITAL (such as validation of cleaning);
5. testing allergen status of the final product to compare with calculated results from VITAL assessment (this may be especially relevant to high risk environments); and,
6. monitoring the effect of critical changes.

The concentration of cross contact allergens in a final product may be validated using analytical testing. It should be noted that this is not a mandatory part of this procedure, but that it may be useful in some circumstances.

Consult a skilled analyst to ensure that the correct methodologies are used and to ensure that the units of measure are appropriate to use with the VITAL Action Levels Grid.

Where the concentration of allergens identified by analytical testing is greater than found during the VITAL risk assessment, consider reviewing the risk assessment for other factors. Consideration should be given to the type of analysis, nature and form of the allergen. It is important to consider the nature of food processing as some processes may reduce the amount of detectable allergen. Further to this, where quantitative measure is done by both analytical and physical measures, in order to consider the situation of greater risk, the greater of the two should be taken. Where analytical results are significantly higher than would be expected based on calculation, consideration should be given to other possible contributing factors.

Finished product testing may be used to verify results from cross contact allergens identified in a VITAL risk assessment. When comparing analytical test results with concentrations calculated from the VITAL risk assessment, it is important to ensure that the units of measure are comparable. The VITAL Action Levels Grid uses concentration (parts per million [ppm]) of total protein. Analytical results use a range of units of measure. Check with the analyst as some kits provide conversion factors to assist in standardising units of measure.

• National Association of Testing Authorities (NATA)
• AOAC INTERNATIONAL

Allergen Testing – Special Interest Group (AT-SIG)  

• AT-SIG Briefing Note – Undeclared peanuts in pesto – analytical testing considerations (7 Feb 2020)