Development of two immunoassay formats to detect β-lactoglobulin:: Influence of heat treatment on β-lactoglobulin immunoreactivity and assay applicability in processed food

Milk proteins are commonly used as ingredients in the food industry because of their functional properties, but they can cause severe reactions in milk-allergic individuals. In this work, two enzyme-linked immunosorbent assay (ELISA) formats were developed to detect bovine P-lactoglobulin. The indirect competitive ELISA involved the use of anti-P-lactoglobulin antisera, and the sandwich ELISA involved the use of specific antibodies isolated using a P-lactoglobulin immunosorbent material. The effect of heat treatment on immunoreactivity of the protein in buffer and in milk was determined with both assays. The amount of immunoreactive protein in buffer and in milk decreased as determined by the sandwich ELISA, whereas the amount increased when measuring with the competitive ELISA. Several food products, including meat, bakery products, sauces, and snacks, were analyzed. With both assays, 10 of 11 products in which the ingredient list included the terms "powdered milk" or "milk proteins" contained P-lactoglobulin. However, the P-lactoglobulin concentration in these products obtained with the competitive ELISA were much higher than those obtained with the sandwich ELISA. These differences could be explained by the fact that the determination of P-lactoglobulin concentration by immuroassay is influenced by differences in antibody recognition of the protein present in highly processed foods. Therefore, the antigen-binding properties of antibodies used in a particular immuroassay are important for a correct interpretation of results obtained in food processed at high temperature.