Characterization of an Incurred Food Sample Containing Milk Proteins for Inter-Kit Evaluation in External Quality Assessment Programs

Abstract

Food allergies, including cow’s milk allergy, remain important food safety and regulatory science issues, particularly in children. Reliable allergen labeling and surveillance depend on analytical methods that perform appropriately for processed foods, in which allergenic proteins may be denatured, aggregated, or otherwise modified during manufacturing. External quality assessment (EQA) programs, also referred to as proficiency testing, are essential for evaluating routine food allergen testing. However, EQA materials prepared by spiking food matrices may not adequately represent incurred allergens in commercially processed foods. In this study, we characterized an incurred food sample containing trace levels of milk proteins prepared from commercially available processed foods. We then evaluated its homogeneity, dilution linearity, and inter-kit comparability as preliminary requirements for its possible use in EQA-related studies. A cookie product containing milk protein at a concentration close to the Japanese labeling reference level was diluted with purified water to prepare the incurred sample. The sample showed good homogeneity and acceptable dilution linearity when analyzed using a casein-specific enzyme-linked immunosorbent assay (ELISA) kit. In contrast, dilution-corrected values increased monotonically when analyzed using ELISA kits targeting both casein and β-lactoglobulin, indicating that the tested dilution range did not meet the predefined linearity criterion. These findings indicate that incurred samples can reveal kit-dependent analytical behavior. Because no matched spiked control, multi-laboratory trial, or independent lot was evaluated, this study should be regarded as a material-characterization and inter-kit evaluation study rather than as a full EQA validation.