Understanding How ELISA Methods Work to Detect Food Allergens

Undeclared food allergens are a significant food safety hazard, and manufacturers need to have practices, processes, and controls in place to prevent the presence of undeclared major food allergens in their products. The detection and quantification of food allergen residues is an important capability for robust food allergen control, and methods capable of detecting and quantifying proteins from allergenic foods can be used in a number of ways. Food manufacturers can use allergen detection methods to assess various aspects of allergen control plans, including cleaning procedures, supply chain controls, and overall allergen management. In addition, manufacturers may need to rely on food allergen detection methods to confirm an alleged instance of undeclared food allergens in a product and conduct root-cause analyses. Food allergen detection methods are also used by regulatory authorities to investigate the presence of undeclared major food allergens in products on the marketplace, either as part of research studies or as enforcement actions.

Understanding how food allergen methods work, how to select the appropriate method for a particular application, how results from these methods are interpreted, and what potential issues may arise with the methods is critical for food manufacturers when implementing allergen control plans or navigating potential allergen recalls.

How Methods Work

Currently, the detection and quantification of food allergens in finished food products is primarily conducted using enzyme-linked immunosorbent assays (ELISAs). ELISAs detect proteins from allergenic foods by using antibodies that specifically recognize the food proteins of interest. Method developers produce these allergen-specific antibodies in laboratory animals by exposing them to the food or protein target of interest. After an immune response has been developed, antibodies can be collected, screened for specificity and affinity, and subsequently used in an ELISA.

Most ELISAs utilized for food allergen detection use a sandwich ELISA format. In a sandwich ELISA, one source of allergen-specific antibody is coated onto the surface of microwells, generally in a 96-well plate format. After coating with the antibodies (also referred to as capture antibodies), the wells are coated with a blocking agent to prevent any non-specific binding of components from the sample. In commercial allergen ELISA kits, pre-coated and blocked wells are provided as one of the kit reagents.
When conducting a sandwich ELISA method, an extract from the sample of interest or method controls is then added to individual microwells. During an incubation period, any proteins present in the sample from the target allergenic food will bind to the antibodies present on the surface of the microwell. The region on the protein that is recognized by the antibody is known as an epitope.

Figure 1. Quantification using a standard curve.

Following incubation, the wells will be washed thoroughly to remove any unbound sample components. A second allergen-specific antibody will then be added to the wells and will bind to target proteins already captured in the microwells, forming an antibody sandwich with the target protein in the middle. In commercial assays, this second antibody will have an enzyme attached (or conjugated) to it, and the second antibody is therefore commonly referred to as the conjugate antibody. Following another washing step to remove unbound conjugate antibody, the substrate for the conjugated enzyme will be added to the wells. The enzyme present in the microwell will convert the substrate to a specific color product, indicating the presence of an intact antibody sandwich and therefore the presence of the food allergen target.

The amount of color generated in the microwell will depend on the amount of enzyme present, which in turn depends on the amount of target allergen protein present. This relationship between color intensity and amount of target allergen protein can be used to quantify the amount of target allergen in a sample.