*4.4. Preparation of Immunofiltration Columns*

The equilibration of immunofiltration columns was done, as described by Rettcher et al. (2015) [26]. In brief, after degassing in 96% (v/v) ethanol in a desiccator at –0.8 bar pressure, columns were washed sequentially with 750 µL 50% (v/v) ethanol-water, 750 µL water and twice 750 µL coupling buffer. Afterward, for coupling of aflatoxin B1-BSA conjugate to the matrix, the conjugate was applied to the column in gravity flow diluted in 500 µL coupling buffer and incubated for one hour at room temperature in dark surrounding. For checkerboard titration, a coating concentration, as shown in Figure A2, was used. For the bead-response curve, a coating concentration of 2 <sup>µ</sup>g·mL−<sup>1</sup> was applied, as well as for cMID assays. Subsequent washing of the columns was performed twice with 750 µL PBS. Remaining binding sites were blocked by applying twice 750 µL of MID-BP. After the first 750 µL flushed through the column by gravity flow, an incubation time of 5 min was set. After the second time, columns were incubated for further 30 min after they were washed again twice with PBS.

After equilibration or blocking, columns can be stored in the coupling buffer or PBS, respectively, at 4 ◦C for at least 14 days. In this study, a maximum storage time of one day was used.
