Immunoassay Platform for Casein Antigens in Hydrolyzed Casein-Based Infant Formula

Hypoallergenic formulas are the sole source of nutrition for infants that are either allergic to milk proteins or at risk of developing allergies. Strategies to provide nutritional sustenance, while preventing allergic reactions, include designing formulas based on extensively hydrolyzed casein which is presumably devoid of antigenic epitopes. Assays devoted to the assessment of antigenic protein motifs are crucial to verify the absence of relevant antigens in formulas and the raw materials used in their preparation. Evaluation of commercial immunoassay kits intended for the detection of milk proteins in foods led to the conclusion that a specific assay for extensively hydrolyzed casein-based formulas was necessary to improve allergen recoveries and assay consistency. The purpose of this investigation was to develop a reproducible path, from the generation of antibodies to the pre-validation of immunoassays optimized for the analysis of hydrolyzed casein-based infant formula. We prepared purified antisera from sheep immunized with bovine acid-precipitated casein to establish a platform consisting of a slot blot immunoassay and an enzyme-linked immunosorbent assay. Results indicate that sheep can reliably produce antibodies against epitopes in the casein fraction of bovine milk, thus providing a quantitative reagent that binds to immobilized casein in different formats. The limits of detection and quantitation for standard solutions for the enzyme-linked immunoassay were 0.8 and 2.5 ppm, respectively. The limit of detection in the extensively hydrolyzed casein-based formula was 0.5 ppm and the limit of quantitation 1.4 ppm. This account describes two reproducible immunoassays that are accessible to any laboratory or manufacturing setting and do not require proprietary ingredients or undisclosed extraction solutions. While these tests were developed to quantitate casein in hypoallergenic formula matrices, an application of the slot blot immunoassay to assess residual casein on manufacturing surfaces is also described in the present account.