2.1.2. cELISA Calibration Curve Experiments

To identify the optimal assay parameters, calibration experiments were performed with the previously most promising combinations of AFB1-BSA coating and aflatoxin B1-specific monoclonal antibody AFB1\_002 concentrations (Figure 3). Free aflatoxin B1 was used as a competitor, dilutions ranging from 0.006 ng·mL−<sup>1</sup> to 50,000 ng·mL−<sup>1</sup> . The combination of 0.2 µg·mL−<sup>1</sup> coating and 150 ng·mL−<sup>1</sup> antibody resulted in the highest sensitivity in combination with stable data behavior. Corresponding IC<sup>50</sup> and Limit of Detection (LOD) values of all four combinatorial experiments are shown in Table 1. Although antibody concentrations below 75 ng·mL−<sup>1</sup> should theoretically lead to a higher sensitivity, this was actually not observed due to divergent measuring values and thus a non-reliable assay procedure under these conditions (compare 0.4 µg·mL−<sup>1</sup> coating and 75 ng·mL−<sup>1</sup> mAb). Furthermore, reducing the amount of mAb resulted in a twofold increase of readout time from approximately 10 min to more than 20 min (data not shown).

**Figure 3.** Competitive ELISA-based calibration curves of different pairs of aflatoxin B1-BSA coating and aflatoxin B1-specific monoclonal antibody AFB1\_002 concentrations pairs. As a competitor, free aflatoxin B1 in dilutions ranging from 0.006 ng·mL−<sup>1</sup> up to 50,000 ng·mL−<sup>1</sup> in sample buffer was used. The indirect readout was done at 405 nm after the application of mouse-specific secondary antibody conjugated with horseradish peroxidase and respective substrate. Each data point represents the mean ± SD (*n* = 3).


**Table 1.** Sensitivity values of different cELISA-based calibration measurements with different pairs of aflatoxin B1-BSA coating and aflatoxin B1-specific monoclonal antibodies.

Considering the reliability of the developed cELISA, a further repetition of the best-paired concentration setting, as shown in Table 1, was done. Results yielded in an averaged IC<sup>50</sup> value of 3.79 ng·mL−<sup>1</sup> and an averaged LOD of 0.39 ng·mL−<sup>1</sup> , as shown in Figure 4. Those sensitivity values obtained by our ELISA setup were used as a reference for competitive magnetic immunodetection development.

**Figure 4.** Averaged calibration curve with 0.2 µg·ml–1 AFB1-BSA conjugate coating and 150 ng·ml–1 mAb with aflatoxin B1 competitor concentrations ranging from 0.006 ng·ml–1 to 500,000 ng·ml–1. Measuring signal was achieved by indirect readout at 405 nm with mouse-specific secondary antibody conjugated to horseradish peroxidase and respective substrate LOD: limit of detection; IC50: half maximal inhibitory concentration. Each data point represents the mean ± SD (*n* = 12). **Figure 4.** Averaged calibration curve with 0.2 <sup>µ</sup>g·mL−<sup>1</sup> AFB1-BSA conjugate coating and 150 ng·mL−<sup>1</sup> mAb with aflatoxin B1 competitor concentrations ranging from 0.006 ng·mL−<sup>1</sup> to 500,000 ng·mL−<sup>1</sup> . Measuring signal was achieved by indirect readout at 405 nm with mouse-specific secondary antibody conjugated to horseradish peroxidase and respective substrate LOD: limit of detection; IC50: half maximal inhibitory concentration. Each data point represents the mean ± SD (*n* = 12).
