The AFM1 quantification was carried out via a previously
described ELISA protocol (Anfossi et al., 2008) as follows: 60 mL of
AFM1 standard solutions or sample extracts were added to the
same amount of the diluted anti-AFM1 antiserum and incubated in
non-coated wells for 50 min. One hundred microlitres of the
mixture were transferred into coated wells (functionalised with an
AFM1-BSA conjugate) and incubated for 15 min. After washing,
100 mL of the diluted anti-rabbit antibody (conjugate with the
peroxidase) was incubated in wells for 15 min. Colour development
was obtained by a 20 min incubation with the TMB solution (100 mL
per well), followed by the addition of 50 mL of a stop solution.
Finally, absorbance was recorded at 450 nm