Improved Sensitivity of Allergen Detection by Immunoaffinity LC-MS/MS Using Ovalbumin as a Case Study

Abstract

Food allergies are caused by severe hypersensitivity to specific food allergens such as the egg protein ovalbumin. It is therefore important to test food products for the presence of allergens to protect allergic people from accidental ingestion. For egg detection, ELISA is the only reasonable commercially available test format, although the recognition of target allergens can be affected by food processing, which may lead to false negative results. Current mass spectrometry-based detection methods may overcome this issue, but these approaches are often less sensitive. Here we combined the advantages of antibody-based and MS-based methods by developing an immunoaffinity LC-MS/MS technique to detect the common egg allergen Gal d 2. We investigated the principal functionality of this method with incurred cookie material containing whole egg powder. We found that the new method matched easily the sensitivity of egg specific ELISA tests. Further western blot experiments indicated that this strategy may be unaffected by food processing, providing an important alternative strategy for the detection and quantification of allergens in food.

Detection of Allergenic Proteins in Foodstuffs: Advantages of the Innovative Multiplex Allergen Microarray-Based Immunoassay Compared to Conventional Methods

“…This procedure often allows the use of not-so-mild extraction conditions, which can include extreme pH and the presence of denaturing and reducing agents. This aspect can represent an advantage because these harsh conditions allow more efficient extraction of, for example, denatured and otherwise insoluble proteins [ 72 ]. Therefore, the extracts for mass spectrometry analysis can contain more protein components (including otherwise insoluble or not extracted molecules), compared to those prepared for other types of investigations, such as immunochemical methods.…”

Food allergen analysis: A review of current gaps and the potential to fill them by matrix‐assisted laser desorption/ionization

“…Studies applying LC–MS/MS to the analysis of food allergens have proliferated and include: on seven allergens in flour and bread (Heick et al., 2011), cow's milk protein in cookies (Cordeiro et al., 2021), on coconut protein (Piredda et al., 2022), milk in meat‐based foods (Bianco et al., 2022), caprine and bovine milk allergens (Nelis et al., 2023), flaxseed storage proteins (Bueno‐Díaz et al., 2022), ovalbumin (Röder et al., 2021), egg milk and peanut in foods (Xiong et al., 2021), egg and milk in samples (labelled no milk and egg) (Fan et al., 2023), nuts and peanuts hidden in bakery products (Luparelli et al., 2023), piscine gelatine (Grundy et al., 2023), crustacean tropomyosin and arginine kinase in food matrix (Li et al., 2022), and multiallergens in chocolate (Henrottin et al., 2023). The publication of the AOAC First action 2017.17, “Detection and quantitation of selected food allergens by LC‐MS/MS” (New et al., 2020) and of BS EN 17644:2022 “Foodstuffs‐Detection of food allergens by liquid chromatography‐ mass spectrometry (LC‐MS) methods—General considerations” (European Committee for Standardization, 2022) has aided comparable robust, rugged (Burns et al., 2009), and reproducible results to be obtained by different analysts, instrumentation, and laboratories.…”

Analytical Methods for Allergen Control in Food Processing

“…MS is gaining more confidence in allergen detection and quantification. Either by itself or coupled with other methods such as immunoaffinity, for example, as seen in [ 20 ], would enhance the performance of MS by improving method sensitivity. Similarly, combining a competitive ELISA and MS allowed for the quantification of gluten in dried yeast and yeast-containing products, as demonstrated in [ 21 ].…”