Sampling and preparation
A total of 678 raw milk and 438 heat treated milk samples were
analyzed during the period of 2013e2014 including all seasons.
Milk powders (n ¼ 64), white cheeses (n ¼ 47), hard cheeses
(n ¼ 27), yoghurts (n ¼ 56), ice creams (n ¼ 21) and infant formulas
(n ¼ 33) were also collected, making a total of 322 milk products
investigated for the presence of AFM1. All samples were stored at
2e8 C or frozen at 20 C until further analysis. Milk and milk
products samples were prepared according to the ELISA kit manufacturer's
instructions.
2.2. Aflatoxin analysis by ELISA (procedure and method validation)
Determination of AFM1 was done by Enzyme Linked Immunosorbent
Assay (ELISA) method using standard validated commercial
kit (Code MA418, Tecna, Italy). The assay was performed in
plastic microwells which have been coated with anti-AFM1 antibodies.
AFM1 standard solutions and samples were added to the
microwells. During the first incubation, free AFM1 molecules are
bound to the anti-AFM1 antibodies. Any unbound substance was
then removed in a washing step. A second incubation was