Challenges in Allergen Testing - Spiking and Recoveries

Photo Romer Labs

Adrian Rogers discusses the basics on detecting allergens in food – from finding the right test kit to methods for precise validation.

When I started developing immunoassays for the detection of allergens in food, the first thing that struck me was the wide range of different food types or matrices that the assays had to work with. Coming from a medical immunoassay background, there was a limited number of different matrices to work with. In my case, this was blood serum. With food there is an almost infinite range of different sample types, each with their own specific properties.

How do I choose the right test kit?

So how do we ensure that the test kit produced is suitable for use with such a diverse and challenging range of samples? This is where sample validation comes in. The process involves adding a known amount of an allergen of interest to our matrix (spike) and then trying to get that allergen back out again (recovery).
An important thing to remember is that, as the name implies, immunoassays use biological components (antibodies) to achieve the detection of the allergenic proteins of interest. As with all biological systems, the kits are sensitive to extremes.
In the case of foods, the kits may not work as they should in the presence of strong acid or alkali, high salt, high fat, etc. Many of these extremes can be countered during the extraction process. Kits therefore use a buffered system to cope with changes in pH and the addition of the buffer to the sample helps reduce and dilute some of the other problems such as salt and fat.

Picture Adrian Rogers

About the author

Adrian Rogers has been with Romer Labs for 6 years in his role as a Senior Research Scientist. He is responsible for research and development within Romer's allergen competence centre based in the UK.
Before joining Romer Labs, Adrian was an R&D Scientist involved in the development of ELISA and Lateral Flow immunoassays for the detection of food allergens. Adrian is a microbiologist by training and has 15 years experience in the development of immunoassays, 13 years of which have been spent developing test kits for the detection of food allergens.
Over the years Adrian has been involved in a number of food allergy projects including EuroPrevall, an EU funded multidisciplinary integrated project which investigated the prevalence of food allergy across Europe. He is currently a member of the University of Manchester's Food and Health Network allergy cluster and co-ordinates Romer Labs’ contribution to the “Innovate UK Knowledge Transfer Project”, with the University of Manchester looking at improving soya allergen analysis.

Is my recovery acceptable?

When it comes to the recovery of a known amount of allergen from a sample matrix, what is deemed acceptable? Before answering this, we need to define where we are starting from. Is it an incurred sample or a spiked one?
Incurred samples are defined as samples in which a known amount of the food allergen has been incorporated during processing, mimicking as closely as possible the actual conditions under which the sample matrix would normally be manufactured.
The subject of incurred samples will be discussed in more depth in a subsequent issue of Spot On. In this article, I will concentrate on outlining a more accessible method of spiking a known amount of allergen into a matrix as received from the supplier or manufacturer and measuring its recovery (see box text ‘Reference guideline’ for guidelines on recovery).
With regard to recovery, the guidance states that: “Ideal percent recovery levels would range from 80 to 120%. Recovery levels are affected by both the efficiency of the extraction step and the ELISA procedure. “With ELISA methods for food allergens, this level of recovery is not always possible, particularly when certain difficult matrixes are analysed. In addition, the recovery from incurred samples can be substantially different from those obtained using spiked samples. For this reason, recoveries between 50 and 150% will be considered acceptable so long as they can be shown to be consistent.
The guidelines were published in 2010 by the Association of Analytical Communities (AOAC) with particular reference to quantitative ELISA (Enzyme Linked Immunosorbent Assay) methods. Many of the key points are also applicable to qualitative or semi-quantitative LFD (Lateral Flow Device) methods.

The "science" behind spiking

When we receive or encounter a new food type that has not been tested before, we will undertake spike recovery validation to ensure it works as it should with our test kits. We will spike in at three different levels of allergen – low, medium and high – to cover the range of detection of the assay.
The low allergen spike will be close to the Lower Limit of Quantitation, LLOQ, of the ELISA (in this case the lowest value calibrator above 0 ppm) or close to the Limit of Detection, LOD, of a lateral flow device. The medium spike will be in the middle of the ELISA calibration curve, and the high spike will be at or near the Upper Limit of Quantitation, ULOQ (the highest ppm value calibrator). The sample is extracted and tested in accordance with the product insert supplied with the kit.
So for example, if we spike 5 ppm of almond into chocolate, we would expect to see a recovery of 4 ppm to 6ppm. If the result is outside of this range, then there are steps that can be taken to help improve the recovery. From experience, chocolate is one of the most challenging food matrices to test – it is full of tannins and other polyphenols which can bind to any allergenic protein that may be present and form insoluble complexes which are difficult to extract.
Such difficulties can be overcome by adding extra protein to the extraction buffer. The excess protein binds to the polyphenols and makes the allergens available for extraction. My protein of choice is fish gelatine, although other material such as milk powder can be used to improve the extraction efficiency from high polyphenol containing foods. If using milk powder, be careful not to contaminate your laboratory space, especially if you are carrying out milk allergen testing.
Lateral Flow Devices, or strips or dipsticks as they are sometimes referred to, can be validated for spike recovery in a similar way to an allergen ELISA test kit. The thing to be aware of when choosing a high spike level is that although LFDs are capable of detecting very high ppm levels, you can actually overload the device by adding too much allergen. This can occur in amounts greater than 1% of the allergenic food. (See box for more information on this topic)

Maintaining quality and test precision

It may be necessary for a kit manufacturer to work closely with customers who routinely test challenging food matrixes. It is important to verify that the kit is working as it should and to the customer’s satisfaction. This can be achieved, as detailed above, by undertaking allergen spike recovery experiments into the problematic matrix.
In some cases it may be desirable to modify or change the standard kit method to meet the demands of the sample and/or the customer; this should always be undertaken with the guidance of the kit manufacture to ensure the quality and reproducibility of the test kit.

The Hook Effect

The Hook Effect

Overloading the device can lead to a false negative result. This process is referred to as the “hook effect”. The hook effect does not pose a problem in day-to-day testing using the strips.
In fact from my experience, it is only usually encountered when you are trying to verify if the LFDs are working correctly by testing 100% of the allergenic food. By doing so, the amount of allergen present exceeds the finite amount of the colored labelling material, often colloidal gold or colored latex coupled to the detection antibody.
The excess unlabelled allergen migrates along the membrane quicker than the heavier color-labelled allergen, saturating all the binding sites on the capture antibodies immobilised on the membrane surface. When the color-labelled allergen arrives, no binding sites remain, so it simply continues on to the wicking pad at the end of the test device. Since no binding sites were available, the color-labelled allergen cannot create the colored test line that would normally represent a positive result.

Reference guideline:
In this article, reference is made to this accepted published guidance with regard to assay validation: Validation Procedures for Quantitative Allergen ELISA Methods: Community Guidance and Best Practices Abbot et al Journal of AOAC International Vol 93, No 2, 2010