Data published in 2016 by iFAAM (Integrated Approaches to Food Allergen and Allergy Management) revealed that 60% of recalls or withdrawals within the food industry were the result of allergen notifications. Not only does this figure highlight the importance of allergen management, but it also suggests that many sites are struggling to succeed in this area.

In an effort to tackle this problem, a growing number of factories are reevaluating their approach to allergen control – eager to enhance their operation and stay abreast of the latest technology.

As a system that can NOT be used for the direct detection of allergens, you may be surprised to learn that the A3 methodology is being incorporated into allergen management programs across the world – helping sites to reduce costs, fill the gaps of their current system, make life easier for their operatives, and secure more accurate results.

The reason for this is fairly simple: Proper Cleaning = Reduced Risk of Allergen Cross-Contact

In this article, we will take a closer look at the science behind this tried-and-tested approach.


Plus, before reading any further, you can also follow the link below to watch a webinar on Hygiene Monitoring in High Care Environments. Hosted by Klipspringer Director Alex Carlyon, our in-house A3 specialist Radek Tameczka, and scientific advisor Dr. Stephan Speidel, this webinar explores the limitations of conventional hygiene monitoring methods and provides real-world examples of A3 verification at High Care production sites.

How do you ensure the cleans at your site are effective?

This involves a two step process:

Cleaning Validation

At this point, you assess the effectiveness of your site’s Sanitation Standard Operating Procedure (SSOP). Essentially, you prove the cleaning methods performed at your factory are effective in removing allergens and reducing the risk of cross contamination.

Depending on the applications and risk levels at your site, this process will typically be carried out by Lateral Flow Immunoassays or ELISA tests, as these methodologies target specific allergens.

Cleaning Verification

Once the validation process has taken place, you will need to find a suitable method for verifying your cleaning program. This will typically occur on a daily basis, so it is important to find a quick, easy-to-use, and low-cost approach that doesn’t compromise on accuracy.

Again, a lot will rely on the applications and risk levels of a site, but the most popular options for daily verification are visual inspection, ATP tests, protein-specific swabs, and the A3 system.

What are the different testing methods?

Allergen-Specific Testing

A survey carried out by Food Safety Magazine revealed that 18% of food production sites rely entirely on allergen-specific tests. With examples including ELISA, PCT, and Mass Spectrometry, these methods are highly sensitive and specific. However, they are also unsuited to a busy production space. Instead, they often require specialist equipment, on-site laboratories, and highly trained personnel. ELISA testing, for example, involves complex calculations using standard curves, and Mass Spectrometry involves the analysis of large quantities of data.

These limitations have led many sites to incorporate rapid testing methods into their allergen control program. This doesn’t always involve stepping away from the highly specific tests mentioned above, with sites often turning to rapid testing as a means of reducing the frequency of use for the more complex methodologies.

Although sites have the option of using a mix of both complex and rapid testing options, the survey also revealed that 13% of sites rely on a combination of rapid tests, 36% rely on ATP testing, and 17% rely on protein-specific tests. This means that 66% of participating sites rely solely on rapid testing methods such as protein swabs, Lateral Flow Immunoassays, and ATP tests.

Protein-Specific Testing

When someone is allergic to a certain food, they will experience an allergic reaction if their immune system recognises the relevant proteins.

This helps to explain why such a large percentage of sites use protein-specific tests for their allergen management. However, this approach also comes with limitations, as protein-specific tests are qualitative and can only generate non-specific, non-numerical results. Sites will also see a reduced efficiency when protein swabs are tasked with detecting a substance that has low levels of residual protein contamination.

Click the button below for an in depth look at the benefits and limitations of protein-specific tests.

Lateral Flow Immunoassays

Lateral Flow Immunoassays are another popular choice for allergen testing. Designed with the detection of specific substances in mind, this approach is similar to the more complex methodologies mentioned above. However, it generates rapid results and doesn’t require any expensive or complex equipment.

The fact that Lateral Flow Tests detect specific substances is incredibly useful; however, this can also be a drawback for sites that are unable to find a LFI that targets the allergen they handle. This is also a common issue with ELISA testing.

Another limitation of LFIs is that due to their specificity, they are unable to assess several allergens simultaneously. In fact, if there is more than one allergen to assess, sites will have to cover the cost of multiple LFIs for all the different allergens at play.

If you would like to further explore the role of Lateral Flow Tests, it is worth noting that we have launched a new range of Rapid Allergen Test Kits for: Egg (Ovalbumin), Casein, Gluten, Soya, Peanut, Crustacean, and Buckwheat.

The kits within this range offer the following benefits:

  • Minimum accuracy rate of 0.5ppm
  • Simple, two-step process (eliminates Extraction Buffer Solution)
  • Assay time of 10 minutes
  • Stored at 2-30°C (no need for fridge)
  • Lifespan of 12 months

ATP Tests

Despite being unable to directly detect specific allergens, the survey revealed that ATP testing was the most popular methodology for food production sites. This is due to an understanding that if the concentration of surface-bound ATP is reduced below a defined value, a surface can be declared clean and free of allergens.

This is a common misconception.

Appendix 10 of the FDA’s guidance for Cleaning and Sanitation For the Control of Allergens actually advises against a total reliance on ATP testing for cleaning verification in relation to allergens. The reason given is that ATP is not present in all foods and is not a specific indicator for allergens.

Consequently, sites that are relying solely on ATP testing could be working with surfaces and equipment that are still contaminated with organic residue, and most worryingly, allergens.

The A3 System

In response to the limitations of ATP testing, Kikkoman Biochemifa launched a new hygiene monitoring technology in 2017: the A3 system. As with ATP tests, this system does not directly test for allergens. However, unlike conventional ATP tests, it is able to detect all organic residue. This means that it can help to monitor the risks of allergen contamination.

As the A3 system is a more recent technology, you can find an explanation of how the system works and how it can support your allergen control program below.

What is the A3 system and how does it work?

The A3 system consists of the A3 Lumitester Smart unit and Lucipac Swabs. The unit itself is used in a similar way to conventional ATP tests. Lightweight, app based, handheld, and autocalibrated, all you need to do is run your choice of swab across the relevant test point, insert it into the meter, and wait around ten seconds for a result. Before this, you will have worked with our Technical Support Team to establish your bespoke pass/fail limits, and it is these limits that allow the unit to determine if the surface has passed or if it is contaminated and in need of further cleaning.

It’s the Lucipac Swabs that really set the A3 system apart from conventional ATP tests. This is because they introduce recycling enzymes that allow for conversion between all three adenosine molecules: ATP, ADP, and AMP. As a result, the readings you receive relate to the total organic residue on a surface. This will be the case even after the degradation of ATP has taken place.

Depending on your application, there are three types of swabs to choose from:  Surface Swabs, Water Swabs, or Pre-moistened Surface Swabs.

How could the A3 system support allergen management at your site?

Reliable Results

The A3 system generates accurate and reliable results under a wide range of conditions – even after the organic residue it is testing for has been exposed to processes such as cleaning, heating, blanching, and fermentation. This is not the case for all rapid testing options.

The graph below offers a comparison of the A3 system and protein swabs when detecting raw fish and meat residue on a stainless steel surface.

The orange markers reflect the four points at which the testing was carried out. The first test was taken before the surface was washed, the second after a fifteen second rinse with cold water, the third after a fifteen second rinse with 50°C water, and the fourth after the surface was cleaned with detergent and a sponge.

The benchmark value of 200 RLU was put in place for the A3 system, and it was only after the final test that the reading fell below this value and the surface was confirmed to be clean. In contrast, the protein swab was only sensitive enough to detect residue before the first wash and detected only a trace of meat after the second.

Here we can see that the results of protein-specific tests are compromised if the denaturation of proteins occurs through processes such as thermal processing or exposure to chemical agents. In contrast, the A3 system continued to generate accurate readings throughout.

Heightened Sensitivity - Part One

The following diagram offers a comparison of the A3 system, protein swabs, ATP tests, and Lateral Flow Immunoassays when detecting peanut residue.

To generate these results, 10g portions of peanuts were mixed with 90ml of sterile distilled water and homogenised to form 10-fold dilutions, before 10-fold serial dilutions were carried out.

With 100 RLU as the established action level, the calculated concentration equivalents are displayed below. The concentration of total protein was also evaluated by the ELISA and Bradford assays to produce the theoretical dilution factors that are plotted with the blue and red Xs.

The results of this experiment revealed that:

  • The ATP tests could detect samples between a 10¹-fold and 10²-fold dilution
  • The protein swab test could detect samples up to a 10²-fold dilution
  • The Lateral Flow Immunoassays could detect samples up to a 10⁵-fold dilution
  • The A3 system could detect samples between a 10⁵-fold and 10⁶-fold dilution

As you can see, only the Lateral Flow Immunoassays and the A3 system were sensitive enough to detect the 10ppm (parts per million) levels of peanut protein calculated by the ELISA and Bradford assays, with the A3 system proving to be the most sensitive of the four methodologies.

Heightened Sensitivity - Part Two

The graphs below show the different levels of Luminescence Intensity (RLU) registered by both the A3 system and conventional ATP tests when testing the same surfaces. As you will see, the surfaces were tested for a number of different allergens:

In all eight instances, the readings generated by the A3 system were more than double the readings generated by the ATP tests. If we use the example of raw shrimp, we can see that the conventional ATP test generated an RLU value of well below 100,000, whereas the A3 system generated a reading of around 450,000 RLU.

Building on this data, it is clear that depending on the pass/fail limits of a site, there is every chance that the ATP tests would be incorrectly passing surfaces or equipment that are in need of further cleaning.

The video below offers a comparison of the A3 system and three of the leading ATP models when testing a surface contaminated with Mustard – one of the 14 major allergens.

Similar tests were also carried out on surfaces contaminated with peanut butter, hazelnut, soy beans, shrimp, scallops, sardines, yoghurt, cheese, chocolate, ice cream, and bread.

As you will see, despite the surfaces being contaminated to the point that they would fail a visual inspection, only the A3 system generated a fail result with every test. In contrast, the ATP systems were unable to detect ADP and AMP, so regularly passed surfaces that were in need of further cleaning.

Hygiena EnSure Touch

Lumitester A3 Smart System

3M Clean-Trace System

Neogen AccuPoint®

Mustard

0 RLU

Passed

4813 RLU

Failed

26 RLU

Passed

22 RLU

Passed

Peanut Butter

0 RLU

Passed

968 RLU

Failed

26 RLU

Passed

30 RLU

Passed

Hazelnut

7 RLU

Passed

13511 RLU

Failed

29 RLU

Passed

43 RLU

Passed

Soy beans

7 RLU

Passed

9917 RLU

Failed

27 RLU

Passed

43 RLU

Passed

Shrimp

34413 RLU

Passed

87294 RLU

Failed

80018 RLU

Passed

6153 RLU

Passed

Scallops

1127 RLU

Failed

737543 RLU

Failed

1127 RLU

Failed

14 RLU

Passed

Sardines

0 RLU

Passed

999999 RLU

Failed

44 RLU

Passed

27 RLU

Passed

Yoghurt

2820 RLU

Failed

38078 RLU

Failed

21394 RLU

Failed

718 RLU

Failed

Cheese

0 RLU

Passed

24282 RLU

Failed

23 RLU

Passed

11 RLU

Passed

Chocolate

375 RLU 

Failed

8979 RLU

Failed

153 RLU

Caution

109 RLU

Passed

Ice cream

682 RLU

Failed

41882 RLU

Failed

1526 RLU

Failed

6 RLU

Passed

Bread

0 RLU

Passed

1128 RLU

Failed

20 RLU

Passed

46 RLU

Passed

Strengthen your Allergen Control Program

As previously mentioned, although the A3 system does NOT directly test for allergens, its ability to detect all organic residue does mean that it can support the other testing carried out at your site.

Whilst conventional ATP tests could pass a surface only for your allergen-specific tests to fail it, the A3 system will help you to accurately predict the results of your next round of testing. This is particularly useful if your site relies on tests that take a long time to generate results and are often carried out off site.

This graph depicts the correlation between results generated by the A3 system and an ELISA test for milk protein. The readings were taken throughout the cleaning process.

  • Square: before washing
  • Triangle: after first wash
  • Circle: after final wash

The ELISA test produced a reading of 10-40 μg before the first wash and 0.1 μgo or less after the final wash. Mirroring this trajectory, the A3 test indicated 10³ to 2 x 10⁵ RLU before washing and less than 100 RLU after the final wash.

Case Study: McCain Foods

One of the key motivating factors behind McCain Foods undergoing an A3 trial was the fact that its Scarborough site was in the process of revalidating its entire cleaning regime in support of effective allergen management. Due to growing demand from consumers, the company had been steadily developing a series of gluten-free products and wanted to ensure its approach to Hygiene Monitoring was accurate and reliable.

During the validation process, McCain Foods decided to carry out testing before cleaning. These tests included rapid-tests, allergen-specific tests and ELISA tests. Post-clean, visual checks were also carried out. If the surface passed, conventional ATP tests were used, then if the surface passed, an allergen-specific test was used, and finally, if the surface passed, a sample was sent away for ELISA testing.

During this process, an interim technical person identified a series of confusing results generated by the conventional ATP tests. These conventional tests indicated that a blanched potato didn’t contain any ATP. They also passed surfaces that were failing visual checks. In contrast, the A3 system was able to accurately detect organic residue – failing surfaces that the ATP tests had been passing.

Over a six-to-eight-month period, McCain Foods found that if a surface passed an A3 test it would go on to pass a gluten swab and then an ELISA test. During this period, the company didn’t see a single allergen fail following an A3 swab pass. This gave McCains the confidence to reduce the amount of allergen swabbing and ELISA testing carried out, saving money and speeding up the process of releasing their product line. 

We hope that you now have a greater understanding of the relationship between allergen testing and the A3 system. As we have made clear throughout this article, the system itself does not test for specific allergens; however, it could play a major role in your allergen control program.

If you would like further guidance in this area or would be interested in learning more about the A3 system mentioned in this article, you can contact us on 01473461800 or sales@klipspringer.com. Alternatively, you can fill out the contact form below and one of our friendly team members will be in touch. 

If you would like to learn more about the A3 system, the Klipspringer team would be happy to help. Share your details below to arrange a free consultation.