2.3.1. Sample preparation
Eight millilitres of milk was defatted by centrifugation at 3500 g and 10 C for 10 min. The upper cream layer was removed,and the defatted supernatant was subjected to the ELISA test for
AFM1.
2.3.2. Test procedure
One-hundred millilitres of the standard solutions or the defatted samples were added in wells and incubated for 30 min at room temperature (20e25 C) in the dark. The liquidwas then sucked out of the wells, and all the wells were filled with 250 mL of washing buffer before sucking out the liquid again. The washing procedure was repeated twice. Next, 100 mL of the diluted enzyme conjugate was added in the wells and incubated for 15 min at room temperature in the dark. This washing procedure was repeated two more times. Then, 100 mL of chromogenwas added to each well and incubated for 15 min at room temperature in the dark. Lastly,100 mL of the stop solution was added to each well, and the absorbance was measured at 450 nm by a microplate reader