*4.3. cELISA Procedure*

For competitive ELISA procedure, AFB1-BSA conjugate was diluted in coupling buffer and plated with 100 µL per well onto a high binding 96-well microtiter plate. As mentioned in Section 4.2, all incubation steps were performed at room temperature for one hour in the dark. After the washing step with PBS-T, each well was blocked with EBP and incubated. Meanwhile, pre-incubation of free aflatoxin B1with the monoclonal anti-aflatoxin B1 antibody was prepared. For this, 75 µL of a serial dilution of aflatoxin B1 in PBS was prepared, and then 75 µL of antibody solution diluted in PBS was added and incubated. After washing the blocked assay plate three times with PBS-T, 100 µL of pre-incubated samples were transferred to each well, respectively. After incubation, the plate was washed three times with PBS-T. Subsequently, 100 µL of conjugated goat anti-mouse IgG-HRPO secondary antibody, diluted 1:10,000 in PBS was added to each well and incubated. After washing three times with PBS-T, <sup>100</sup> <sup>µ</sup>L of 1 mg·mL−<sup>1</sup> ABTS substrate in ABTS buffer was added, and absorbance was measured at 405 nm after 15 min incubation in the dark.
