2.1.1. ELISA Checkerboard Titration Test

In order to determine optimal aflatoxin B1-BSA (AFB1-BSA) coating concentrations and the appropriate amount of aflatoxin B1-specific monoclonal antibody to obtain the highest possible sensitivity, a checkerboard titration test was performed. As shown in Figure A1, AFB1-BSA coating concentrations ranging from 0.1 µg·mL−<sup>1</sup> up to 5 µg·mL−<sup>1</sup> in combination with aflatoxin B1-specific monoclonal antibody (mAb) AFB1\_002 concentrations ranging from 19.5 ng·mL−<sup>1</sup> up to 1250 ng·mL−<sup>1</sup> were tested in a 96-well plate. Increasing signals were achieved with antibody concentrations above 19.5 ng·mL−<sup>1</sup> and coating concentrations up to 0.6 µg·mL−<sup>1</sup> . Coating of AFB1-BSA with higher amounts than 0.6 µg·mL−<sup>1</sup> resulted in saturated readout signals at all antibody concentrations that did not further increase. Considering sensitivity, the lowest possible amount of antibody should be used with coating concentrations resulting in highest possible signals. As a consequence, combinations of 75 ng·mL−<sup>1</sup> or 150 ng·mL−<sup>1</sup> antibody with 0.2 µg·mL−<sup>1</sup> and 0.4 µg·mL−<sup>1</sup> coating of AFB1, respectively, seem to be best suited for further experiments.
