Understanding How ELISA Methods Work to Detect Food Allergens

In order to produce quantitative results, a series of standards containing known amounts of the allergenic food protein is analyzed alongside the samples. The absorbance values for both the standards and samples are measured using a plate reader. When the absorbance values from the standards are plotted against the known concentrations, a standard curve can be developed (see Figure 1). The absorbances from the unknown samples can then be used to interpolate the amount of allergen present.

Interpreting Results from ELISA method

Understanding how to interpret the results from a food allergen ELISA method can be challenging, as a number of different factors can impact the method’s outputs.

Units and calibrators. Most commercial food allergen ELISAs report results in the concentration range of parts per million (ppm). The units of ppm indicate a concentration value for the analyte, which can also be expressed as mg analyte per kg product (mg/kg). Just using units of ppm or mg/kg does not, however, provide enough information for food allergen ELISAs. It is also important to know specifically what form of analyte the units are being expressed in. The most common analyte units for food allergen ELISAs are either whole commodity (e.g., ppm peanut, walnut, egg, etc.) or total protein (e.g., ppm peanut protein, walnut protein, egg protein, etc.). For some foods, however, there are commercial ELISA kits that express results on the basis of soluble protein from the allergenic food or a single protein analyte (e.g., ppm beta-lactoglobulin). In order to both understand the implications of a result from an ELISA and to compare results from different ELISA methods, it is crucial to have complete units expressed. The same sample analyzed by methods that use different units will have very different quantitative results, even if all other method conditions are similar.

Limit of detection, limit of quantification, and lower limit of applicability. As with many types of detection and quantification methods, food allergen ELISAs work only within a certain range of target analyte concentrations. The concentration below which a method is not able to distinguish a true positive from a true negative is known as the limit of detection (LOD). The LOD of a method, therefore, controls against false-positives arising from the food matrix and is generally estimated using a statistical evaluation of blank matrices. Because the LOD of a food allergen ELISA is highly dependent on the specific background food matrix being analyzed, it may not be as applicable across a diverse range of food products and ingredients as other method metrics.

The limit of quantification (LOQ) for a method is the lowest level at which a method can quantify an analyte with a specific level of precision (i.e., with a specific coefficient of variation, frequently 10 percent CV). The LOQ of a method as determined by statistical calculations is also dependent on the background matrix and may not represent an indication of the method performance across different food matrices. Method developers may therefore set a lower limit of applicability that better represents the performance of the method as the LOQ and establish that level by including it as the lowest positive value on the standard curve.

Selecting an Appropriate Method

One of the main considerations that needs to be accounted for when selecting a food allergen ELISA is whether the method detects the allergen-derived ingredient of concern. The ability to detect allergen-derived ingredients can depend on a number of factors. The first method characteristic that should be understood is what protein or groups of proteins the method is targeting—particularly important for allergenic foods from which the food industry produces ingredients containing different protein fractions.