Validated Sandwich Enzyme-Linked Immunosorbent Assay for Casein and Its Application to Retail and Milk-Allergic Complaint Foods

Abstract

Cows' milk is a commonly allergenic food. Cross-contamination of milk proteins into nondairy, kosher-pareve foods prepared on shared processing equipment can cause severe, life-threatening reactions in milk-allergic individuals. A sandwich-type enzyme-linked immunosorbent assay (ELISA; 96-well plate format) was developed for the detection of undeclared casein in foods. Rabbit anti-casein antibodies were used as the capture reagent. Food samples and standards were ground, extracted in 0.01 M phosphate-buffered saline, clarified by centrifugation, and added to the wells. Goat anti-casein antibodies were employed as the detector antibody, and the amount of antibody bound was determined with a commercial rabbit anti-goat immunoglobulin conjugated to alkaline phosphatase, with subsequent substrate reaction. Antibodies developed were specific to casein, with no cross-reaction observed with 30 foods and food ingredients. Non-milk-containing products such as fruit juices, fruit juice bars, sorbets, and dark and pareve-labeled chocolate were purchased from June 2002 through June 2003. In addition, samples allegedly causing eight milk-allergic consumer complaints were analyzed. The ELISA had a detection limit of less than 0.5 ppm of casein. The casein content in the analyzed foods ranged from less than 0.5 ppm to more than 40,000 ppm casein; undeclared casein residues were found in all of the samples implicated in allergic reactions. The levels of milk contamination in some of the other surveyed products could also be hazardous for milk-allergic consumers. This ELISA method provides a useful quality control tool for the food industry and could also be used as a validation of kosher-pareve status.

Evaluation of indirect competitive and double antibody sandwich ELISA tests to determine β-lactoglobulin and ovomucoid in model processed foods

“…European Legislation, such as Directive 2003/89/EC and amendments (European Commission, 2003) requires that all ingredients added to food products have to be labelled and demands the obligatory declaration of those liable to cause allergies or intolerances in which milk and egg and products thereof are included. Furthermore, the inadvertent presence of allergens results from practices in the food industry such as the use of contaminated raw materials, inadequate cleaning of shared equipment or other sources of cross-contact (Hefle & Lambrecht, 2004). Therefore, reliable detection methods for food allergens are necessary to ensure its compliance with the food labelling and to improve consumer protection.…”

“…These techniques are mainly based on the interaction between the allergenic protein and the specific antibodies obtained against it. At present, only the ELISA technique is used in the routine analysis due to its high precision, simple handling and good potential for standardisation (de Luis, Pérez, Sánchez, Lavilla, & Calvo, 2007;Hefle & Lambrecht, 2004;Mariager, Solve, Eriksen, & Brogren, 1994;Poms et al, 2004).…”

Development and evaluation of two ELISA formats for the detection of β-lactoglobulin in model processed and commercial foods

“…However, Hefle and Lambrecht (2004) when analysing non-milk contained foods found that all dark chocolate samples and 18% of sorbets, fruit juice bars and drinks samples analyzed contained undeclared casein.…”

Rocket immunoelectrophoresis and ELISA as complementary methods for the detection of casein in foods?

“…Similarly, the RIE method can be used to confirm the casein analyses from an indirect ELISA recently developed in our laboratory; with a LOD of B/1 mg kg (1 (Sletten et al unpublished data). A recently developed sandwich ELISA has also been described, with a claimed detection limit of 0.5 mg kg (1 (Hefle & Lambrecht 2004).…”