Sensitive Aflatoxin M1 Detection in Milk by ELISA: Investigation of Different Assay Configurations

Area of Science:

• Food Science and Technology
• Analytical Chemistry
• Toxicology

Background:

• Aflatoxin M1 (AFM1) contamination in dairy products poses significant health risks, including hepatotoxicity and carcinogenicity.
• Strict regulatory limits exist for AFM1 in milk and dairy products, necessitating sensitive and rapid detection methods.
• Existing immunoassay methods for AFM1 detection may lack the required sensitivity or speed for routine food industry application.

Purpose of the Study:

• To investigate and optimize competitive enzyme immunoassay configurations for sensitive and rapid AFM1 determination in milk.
• To develop a highly sensitive assay with a short assay duration suitable for food industry screening.
• To evaluate the analytical performance of the optimized immunoassay for AFM1 detection.

Main Methods:

• Comparison of different competitive enzyme immunoassay configurations for AFM1 detection.
• Utilized a configuration involving anti-AFM1 antibody, biotinylated AFM1-BSA conjugate, and secondary antibody-coated microwells.
• Validated assay performance including detection limit, dynamic range, repeatability, accuracy, and assay time.

Main Results:

• The optimized assay configuration achieved a six-fold lower detection limit compared to other tested configurations.
• A detection limit of 5.0 pg/mL and a dynamic range up to 2.0 ng/mL were obtained.
• The assay demonstrated excellent repeatability (intra-assay CV < 3.2%, inter-assay CV < 6.5%), accuracy (87.5–108% recovery), and a rapid assay time of 1.5 hours.

Conclusions:

• The developed competitive enzyme immunoassay offers high sensitivity and a short analysis time for AFM1 detection in milk.
• The assay's analytical characteristics make it suitable for routine use in the food industry for ensuring dairy product safety.
• The optimized immunoassay configuration holds potential for improving the detection sensitivity of other low-molecular-weight analytes.