A rapid immunochemical strategy for aflatoxin M1 monitoring in donor human milk

Human milk is widely recognized as the optimal food for infants, providing essential nutrients and immunological protection. However, when unavailable, donor human milk from milk banks is the preferred alternative, especially for preterm or vulnerable infants. While microbiological safety in donor milk is routinely addressed through pasteurization, the presence of chemical contaminants remains largely overlooked. Aflatoxin M1 (AFM1), a toxic metabolite of aflatoxin B1, is one of the most harmful and commonly encountered mycotoxins in milk. Although AFM1 is strictly regulated in bovine milk within the European Union, it is not currently monitored in human milk. In this study, we report the development and validation of a highly sensitive and robust enzyme-linked immunosorbent assay for the quantification of AFM1 in human milk. A specific monoclonal antibody was produced and used in an optimized immunoassay format requiring only a minimal sample preparation consisting of centrifugation and buffer dilution. The assay demonstrated high sensitivity (the limit of detection and the limit of quantification were 0.017 and 0.023 ng/mL, respectively), rapid analysis time (< 60 min), and good accuracy, with recoveries ranging from 85 % to 111 % within the 0.025-0.1 ng/mL AFM1 concentration range. This is the first validated immunoassay for AFM1 detection in donor human milk at levels compliant with EU regulations for infant food, offering a cost-effective solution for routine screening of AFM1 in donor milk, thereby enhancing chemical safety monitoring practices within human milk banks.