ELISA of Picogram Quantities of Aflatoxin M₁ in Urine and Milk

Pretreatment of milk or urine with a Sep-Pak C-18 reversed-phase cartridge permits detection by direct enzyme-linked immunosorbent assay (ELISA) of 10 to 25 pg/ml of aflatoxin M₁ added to samples. The samples were first loaded to the Sep-Pak cartridge followed by washing with 10% acetonitrile in water. Aflatoxin M₁ was eluted from the cartridge with 30% acetonitrile in water and then transferred to the methylene chloride. After the solvent was removed, the sample was redissolved in 50 μl of methanol, diluted with 0.45 ml of buffer, and finally subjected to the ELISA. The recovery of aflatoxin M₁ added to milk and urine in the range of 10 to 250 pg/ml was between 77 to 110%.