Development of chemiluminescent enzyme immunoassay for the determination of aflatoxin M₁ in milk products

In this study, an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for determining aflatoxin M₁ (AFM₁) in milk products has been developed. A luminol–hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The effects of several factors, including concentration and pH of phosphate buffer, dilution ratio of antibody and antigen and other relevant variables on the immunoassay, were studied and optimised by single-factor experiments. The developed method presented an IC₅₀ of 0.05 ng/mL, a detection limit of 0.01 ng/mL and a linear range from 0.018 to 0.13 ng/mL. This method has been successfully applied to the evaluation of AFM₁ in milk products, the recoveries ranging from 71.9% to 109.0%. A good correlation with the commercial available ELISA kit for AFM₁ (r = 0.9978) was obtained, indicating that the ic-CLEIA method developed can be used to determine AFM₁ in real samples.