2.2.1. Development of Competitive Magnetic Immunodetection

2.2.1. Development of Competitive Magnetic Immunodetection In analogy to the development of the cELISA, a similar strategy was used for establishing suitable cMID conditions regarding AFB1-BSA coating and suitable antibody concentrations. For this purpose, equilibrated polyethylene filters were coated with AFB1-BSA conjugate concentrations ranging from 0.5 µg·ml–1 up to 10 µg·ml–1 with one column per condition. Remaining binding sites were blocked with a BSA solution. Then aflatoxin B1-specific biotinylated monoclonal antibody AFB1\_002 in concentrations ranging from 0.3 µg·ml–1 up to 10 µg·ml–1 were applied onto these columns after pre-incubation with 180 µg·ml–1 of 700 nm superparamagnetic streptavidin-functionalized particles (Figure A2). After rinsing the columns with PBS by gravity flow, the columns were inserted into the handheld magnetic readout device and superparamagnetic In analogy to the development of the cELISA, a similar strategy was used for establishing suitable cMID conditions regarding AFB1-BSA coating and suitable antibody concentrations. For this purpose, equilibrated polyethylene filters were coated with AFB1-BSA conjugate concentrations ranging from 0.5 <sup>µ</sup>g·mL−<sup>1</sup> up to 10 <sup>µ</sup>g·mL−<sup>1</sup> with one column per condition. Remaining binding sites were blocked with a BSA solution. Then aflatoxin B1-specific biotinylated monoclonal antibody AFB1\_002 in concentrations ranging from 0.3 <sup>µ</sup>g·mL−<sup>1</sup> up to 10 <sup>µ</sup>g·mL−<sup>1</sup> were applied onto these columns after pre-incubation with 180 <sup>µ</sup>g·mL−<sup>1</sup> of 700 nm superparamagnetic streptavidin-functionalized particles (Figure A2). After rinsing the columns with PBS by gravity flow, the columns were inserted into the handheld magnetic readout device and superparamagnetic particles were excited by frequency mixing technology resulting in a response signal in the millivolt (mV) range after amplification.

particles were excited by frequency mixing technology resulting in a response signal in the millivolt (mV) range after amplification. Especially in the range of low antibody concentrations between 0.3 µg·ml–1 and up to 1.3 µg·ml-1, a saturation of measurement signal was reached at higher coating concentrations. In contrast, using higher antibody concentrations, no clear saturation of signal was observed even at higher coating concentrations. Furthermore, using antibody concentrations of 5 µg·ml–1 or 10 µg·ml–1 resulted in high and erratic signal variability. As explained in Section 2.1.1, the lowest possible antibody concentration should be used for obtaining the highest possible sensitivity. On the other hand, reducing the antibody concentrations leads to a reduction of possible binding sites for magnetic particles and, as a consequence, to a reduction in measuring signal, which may limit the dynamic range as well as the overall robustness of the assay. To obtain the highest possible measuring signal in combination with Especially in the range of low antibody concentrations between 0.3 <sup>µ</sup>g·mL−<sup>1</sup> and up to 1.3 <sup>µ</sup>g·mL−<sup>1</sup> , a saturation of measurement signal was reached at higher coating concentrations. In contrast, using higher antibody concentrations, no clear saturation of signal was observed even at higher coating concentrations. Furthermore, using antibody concentrations of 5 <sup>µ</sup>g·mL−<sup>1</sup> or 10 <sup>µ</sup>g·mL−<sup>1</sup> resulted in high and erratic signal variability. As explained in Section 2.1.1, the lowest possible antibody concentration should be used for obtaining the highest possible sensitivity. On the other hand, reducing the antibody concentrations leads to a reduction of possible binding sites for magnetic particles and, as a consequence, to a reduction in measuring signal, which may limit the dynamic range as well as the overall robustness of the assay. To obtain the highest possible measuring signal in combination with the highest possible sensitivity, 2 <sup>µ</sup>g·mL−<sup>1</sup> coating in combination with 2.5 <sup>µ</sup>g·mL−<sup>1</sup> biotinylated antibody were used.

the highest possible sensitivity, 2 µg·ml–1 coating in combination with 2.5 µg·ml–1 biotinylated antibody were used. As shown in Figure A2, the measuring signal increased with higher antibody concentrations. Therefore, it can be assumed that at the used concentrations of 180 µg·ml–1 there is still an excess of magnetic particles when using 2.5 µg·ml–1 biotinylated mAb. To test this hypothesis, different As shown in Figure A2, the measuring signal increased with higher antibody concentrations. Therefore, it can be assumed that at the used concentrations of 180 <sup>µ</sup>g·mL−<sup>1</sup> there is still an excess of magnetic particles when using 2.5 <sup>µ</sup>g·mL−<sup>1</sup> biotinylated mAb. To test this hypothesis, different amounts of 700 nm magnetic particles ranging from 2.5 <sup>µ</sup>g·mL−<sup>1</sup> to 180 <sup>µ</sup>g·mL−<sup>1</sup> were flushed through immunofiltration columns coated with 2 <sup>µ</sup>g·mL−<sup>1</sup> AFB1-BSA conjugate after applying 2.5 <sup>µ</sup>g·mL−<sup>1</sup>

amounts of 700 nm magnetic particles ranging from 2.5 µg·ml–1 to 180 µg·ml–1 were flushed through

biotinylated antibody to the matrix (Figure 5). The expected saturation of measuring signal was observed when using more than 80 <sup>µ</sup>g·mL−<sup>1</sup> magnetic particles. Adding 80 <sup>µ</sup>g·mL−<sup>1</sup> resulted in 41.2 mV ± 5.5 mV, in comparison to higher bead concentrations with averaged signals of 47.4 mV ± 4.1 mV. For further experiments, 80 <sup>µ</sup>g·mL−<sup>1</sup> magnetic particle suspension was used. biotinylated antibody to the matrix (Figure 5). The expected saturation of measuring signal was observed when using more than 80 µg·ml–1 magnetic particles. Adding 80 µg·ml–1 resulted in 41.2 mV ± 5.5 mV, in comparison to higher bead concentrations with averaged signals of 47.4 mV ± 4.1 mV. For further experiments, 80 µg·ml–1 magnetic particle suspension was used.

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immunofiltration columns coated with 2 µg·ml–1 AFB1-BSA conjugate after applying 2.5 µg·ml–1

**Figure 5.** Dose-dependent measuring signal of 700 nm streptavidin-functionalized magnetic particles after applying 2.5 µg·ml–1 biotinylated AFB1\_002 monoclonal antibody onto 2 µg·ml–1 AFB1-BSA coated immunofiltration columns. Each data point represents mean ± SD (n = 2). **Figure 5.** Dose-dependent measuring signal of 700 nm streptavidin-functionalized magnetic particles after applying 2.5 <sup>µ</sup>g·mL−<sup>1</sup> biotinylated AFB1\_002 monoclonal antibody onto 2 <sup>µ</sup>g·mL−<sup>1</sup> AFB1-BSA coated immunofiltration columns. Each data point represents mean ± SD (*n* = 2).
