Development Of Enzyme-Linked Immunosorbent Assay For Cow’s Milk Allergens

Casein, β-lactoglobulin and a-lactalbumin are important food allergens in milk, and can cause allergic reactions, especially in infants and children. In this study, indirect competitive enzyme-linked immunoassays (ELISA) was established for the rapid detection of milk allergens.By immunizing rabbits and mice with milk proteins and β-lactoglobulin, the polyclonal antibodies against those proteins were obtained. The parameters of ELISA, including the concentration of coating-antigen, dilution of antibody, and pH of protein dilution buffer were optimized, and indirect competitive ELISAs were established.The sensitivity (IC50) of the indirect competitive ELISA for β-lactoglobulin was3.08±0.24μg/mL. Cross-reactivities against casein was1.59%, no cross reaction was observed with soy, sesame, peanuts, eggs, etc. some common allergens, showing good specificity of the established method.The sensitivity (IC50) of indirect competitive ELISA for total milk protein was3.19±0.17μg/mL. The method can effectively detect casein, β-lactoglobulin, a-lactalbumin and the sensitivity (IC50) were3.52±0.46μg/mL,3.91Π0.84p.g/mL and16.27Π0.06μg/mL, respectively. No cross-reaction was observed with several other common allergens, such as soy, sesame, peanuts, eggs, etc., indicating good specificity of the ELISA. Reasonable intra-plate coefficient variance (0.25%-8.53%) and inter-plate coefficient variance (0.75%-11.72%) showing high repeatability of the established indirect competitive ELISA.The recoveries of total milk proteins from bread and biscuits were81.79-93.49%and81.17%-89.30%, respectively, with coefficient of variation (CV) less than15%. The results indicate that the established indirect competitive ELISA is suitable for the rapid detection of milk allergens in foods.