*4.6. Optimization of cMID*

After coating and blocking of immunofiltration columns, 500 µL samples of biotinylated antibody, diluted in PBS to various final concentrations between 0.3 <sup>µ</sup>g·mL−<sup>1</sup> and 10 <sup>µ</sup>g·mL−<sup>1</sup> , were applied and incubated for one hour at room temperature. Afterward, a washing step was performed by rinsing two times 750 <sup>µ</sup>L PBS through the column. Subsequently, for checkerboard titration, 500 <sup>µ</sup>L of 180 <sup>µ</sup>g·mL−<sup>1</sup> 700 nm magnetic beads suspension in PBS (pH 7.4) was added and flushed through by gravity flow. For bead response analysis, various concentrations of 700 nm magnetic beads were applied. Another washing step, as described above, was performed. For readout, columns were inserted into the portable magnetic reader and the measuring signal in mV was detected, as previously described in Rettcher et al. (2015) [26].
