5 Fundamentals of Allergen Analysis #4: Matrix effects during allergen analysis

Allergen analysis is a powerful and indispensable tool to inform, confirm and evaluate every allergen management plan. Nevertheless, particular methods do come with their own sets of challenges. A particularly wide-ranging obstacle is the so-called “matrix effect”.

From an analytical point of view, a sample could be described as comprised of two components: the analyte (the allergen, in our case) and the matrix, which is everything in the sample that is not the analyte. The composition and physicochemical characteristics of the matrix can profoundly affect the response of the method of analysis, leading to an under- (suppression) or overestimation (enhancement) of the allergen in the sample.

The matrix can affect LOD and LOQ

One of the most commonly overseen aspects of the matrix effect is that it alters both the limit of detection and limit of quantification of the method. Therefore, unless the kit provider has already validated the method for the matrix we are analyzing, the first thing to do is to check the LOD of the method in this matrix and to evaluate the matrix effect. This can be done by the method of standard addition (for quantitative methods), or by evaluating the ability of the method to recover defined amounts of an added allergen (in the case of qualitative tests).

The matrix and the sample extraction

The matrix can also affect other aspects of the method of analysis without directly contributing to the response itself. For example, the pH of the matrix and the presence of certain compounds can disturb the conformation of the allergen or cause precipitation when in the solution. A common example of this sort of interference is that caused by the high content of polyphenols in samples like chocolate or certain grains. If the allergen of interest is rich in proline residues or –SH groups (as is the case for milk proteins like casein and β-lactoglobulin, respectively) the allergens tend to aggregate during extraction and are thus underestimated. For these cases, the addition of a different protein (that itself does not interfere in the detection) also rich in proline or –SH groups is recommended. The interaction of the polyphenols with the extra protein prevents the allergen from reacting, normally improving the recovery of the method. There are also some commercially available polymers to help with this issue.

High sugars or fat content can also affect the efficiency of the extraction procedure before the analysis. The formation of very viscous solutions or colloids tends to interfere with extraction efficiency. In these cases, different approaches should be considered, such as warming up the sample before extraction, the extension of the extraction time or the centrifugation of the sample after this step. In some cases, further optimization of the method could require collaboration with the kit developer.

Immunoassays are the most commonly used method in allergen detection in food. Unfortunately, they are vulnerable to low pH levels or high concentrations of alcohols, which could directly disrupt the structure of the detection antibody. In these cases, it is always useful to check the pH and to adjust it to the level for which the kit was validated. For alcohol content, the simple dilution of the sample is usually enough. One caveat: you will need to recalculate the LOD of the method (which will increase) to account for this extra dilution step.

As you can see, allergen analysis is more than just picking a kit off the shelf and using it! In the next issue, we will discuss more challenges that surround allergen analysis.