*4.2. Optimization of ELISA*

The most suitable aflatoxin B1-BSA coating concentrations for a competitive ELISA protocol in combination with appropriate anti-aflatoxin B1 monoclonal antibody concentrations were determined by checkerboard titration. Throughout the following protocol, all incubation steps were performed at room temperature for one hour in the dark. For coating varying concentrations of AFB1-BSA, the antigen was diluted in a coupling buffer, and 100 µL per well was added to a 96-well highbinding microtiter plate (Greiner Bio-One) and incubated as mentioned above. After washing each well thrice with PBS-T, all wells were blocked using 200 µL of EBP and incubated. Then, the plate was washed again, and concentrations of monoclonal antibody ranging from 19.5 ng·mL−<sup>1</sup> up to 1250 ng·mL−<sup>1</sup> , diluted in PBS, were added. After another washing step, as described above, 100 µL of secondary antibody goat anti-mouse IgG-HRPO, diluted 1:10,000 in PBS was added to each well and incubated. Prior to readout with 100 <sup>µ</sup>L of 1 mg·mL−<sup>1</sup> ATBS substrate in ABTS buffer, the plate was washed again. Absorption was measured at 405 nm after 10 min incubation in the dark.
