2.2.2. cMID Calibration Curve Experiments

as the cELISA (Figure 4).

2.2.2. cMID Calibration Curve Experiments Once the optimal ratio of assay reagents of 2 µg·ml–1 of the coating antigen and 2.5 µg·ml–1 of mycotoxin-specific antibody in combination with 80 µg·ml–1 magnetic particles was determined based on the above-shown experiments, cMID calibration experiments for the detection of aflatoxin B1 were performed, see Figure 6A. For this, samples with serially diluted free AFB1 in the range of 0.006 ng·ml–1 to 50,000 ng·ml–1 were prepared. After adding biotinylated mAb to various dilutions of the analyte, a pre-incubation of one hour was done for a complete capturing of mycotoxins. Subsequently, the reaction mixture was applied onto AFB1-BSA coated and blocked columns. Afterward, 700 nm diameter magnetic particles functionalized with streptavidin at the above-determined optimum concentration of 80 µg·ml–1 were added. As shown in Figure 6A, the data show the expected reciprocal correlation of mycotoxin concentration and signal but with high variability of signals for dilutions in the low- ng·ml–1 range, which prevents a reliable determination of sensitivity parameters. We speculate that this observation could be caused by sterical hindrance within the polyethylene matrix due to the usage of big-sized particles, as can be seen in the standard deviation and data fluctuation with bead concentrations of 80 µg·ml–1 or higher (Figure 5). Based on these findings, the assay was repeated with the same parameters except the size of beads used. For this, tenfold smaller magnetic particles were used (70 nm) in order to avoid steric hindrance. As shown in Figure 6B, the use of smaller particles results in an approximately tenfold increased detection signal of roughly 600 mV with reduced variability. With this adapted assay procedure, IC50 Once the optimal ratio of assay reagents of 2 <sup>µ</sup>g·mL−<sup>1</sup> of the coating antigen and 2.5 <sup>µ</sup>g·mL−<sup>1</sup> of mycotoxin-specific antibody in combination with 80 <sup>µ</sup>g·mL−<sup>1</sup> magnetic particles was determined based on the above-shown experiments, cMID calibration experiments for the detection of aflatoxin B1 were performed, see Figure 6A. For this, samples with serially diluted free AFB1 in the range of 0.006 ng·mL−<sup>1</sup> to 50,000 ng·mL−<sup>1</sup> were prepared. After adding biotinylated mAb to various dilutions of the analyte, a pre-incubation of one hour was done for a complete capturing of mycotoxins. Subsequently, the reaction mixture was applied onto AFB1-BSA coated and blocked columns. Afterward, 700 nm diameter magnetic particles functionalized with streptavidin at the above-determined optimum concentration of 80 <sup>µ</sup>g·mL−<sup>1</sup> were added. As shown in Figure 6A, the data show the expected reciprocal correlation of mycotoxin concentration and signal but with high variability of signals for dilutions in the low- ng·mL−<sup>1</sup> range, which prevents a reliable determination of sensitivity parameters. We speculate that this observation could be caused by sterical hindrance within the polyethylene matrix due to the usage of big-sized particles, as can be seen in the standard deviation and data fluctuation with bead concentrations of 80 <sup>µ</sup>g·mL−<sup>1</sup> or higher (Figure 5). Based on these findings, the assay was repeated with the same parameters except the size of beads used. For this, tenfold smaller magnetic particles were used (70 nm) in order to avoid steric hindrance. As shown in Figure 6B, the use of smaller particles results in an approximately tenfold increased detection signal of roughly 600 mV with reduced variability. With this adapted assay procedure, IC<sup>50</sup> values of 5.4 ng·mL−<sup>1</sup> and a LOD of 1.1 ng·mL−<sup>1</sup> were determined, which is in the same sensitivity range as the cELISA (Figure 4).

values of 5.4 ng·ml–1 and a LOD of 1.1 ng·ml–1 were determined, which is in the same sensitivity range

**Figure 6.** cMID calibration curves with 2 µg·ml–1 AFB1-BSA conjugate coating and 2.5 µg·ml–1 biotinylated mAb in combination with (**A**) 700 nm streptavidin-functionalized magnetic particles, and (**B**) 70 nm streptavidin-functionalized magnetic particles with aflatoxin B1 competitor concentrations ranging from 0.006 ng·ml–1 to 500,000 ng·ml–1. LOD, limit of detection; IC50, half maximal inhibitory **Figure 6.** cMID calibration curves with 2 <sup>µ</sup>g·mL−<sup>1</sup> AFB1-BSA conjugate coating and 2.5 <sup>µ</sup>g·mL−<sup>1</sup> biotinylated mAb in combination with (**A**) 700 nm streptavidin-functionalized magnetic particles, and (**B**) 70 nm streptavidin-functionalized magnetic particles with aflatoxin B1 competitor concentrations ranging from 0.006 ng·mL−<sup>1</sup> to 500,000 ng·mL−<sup>1</sup> . LOD, limit of detection; IC50, half maximal inhibitory concentration. Each data point represents the mean ± SD (*n* = 2).
