*4.8. Data Analysis*

For competitive ELISA as well as for competitive magnetic immunodetection and data analysis, a Hill Slope fit was done with GraphPad Prism 8.0.0. The following formulas were used to determine the LOD on the signal and on the concentration scale:

$$\text{Signal}\_{\text{Limit of detection}} = \text{Average}\_{\text{Sample without Component}} - \text{3x SD}\_{\text{Sample without Component}} \tag{1}$$

$$\text{Concentration}\_{\text{Limit of Detention}} = \left( \sqrt[\text{HainiumSignal} - \text{Lowest Signal}} - 1 \right) \times 10 \text{C}\_{50} \text{ ng} \cdot \text{mL}^{-1} \tag{2}$$

**Author Contributions:** Conceptualization, F.S. and J.P.; methodology, J.P.; validation, J.P.; formal analysis, J.P.;

ܔ܉ܖܑ܁ ܜܛ܍ܟܗۺ − ܔ܉ܖܑ܁ ܕܝܕܑܠ܉ۻ

*Toxins* **2020**, *12*, x FOR PEER REVIEW 13 of 15

**Author Contributions:** Conceptualization, F.S. and J.P.; methodology, J.P.; validation, J.P.; formal analysis, J.P.; resources, F.S., H.S. S.S. and H.-J.K.; investigation, J.P.; data curation, J.P.; writing—original draft preparation, J.P.; writing—review and editing, F.S., H.S., H.-J.K. and S.S.; visualization, J.P.; supervision, F.S. and H.S.; project administration, F.S., H.S. and S.S.; funding acquisition, F.S. All authors have read and agreed to the published version of the manuscript. resources, F.S., H.S. S.S. and H.J.K.; investigation, J.P.; data curation, J.P.; writing—original draft preparation, J.P.; writing—review and editing, F.S., H.S., H.J.K. and S.S.; visualization, J.P.; supervision, F.S. and H.S.; project administration, F.S., H.S. and S.S.; funding acquisition, F.S. **Funding:** This research was funded by the land North Rhine-Westphalia and the European Regional

**Funding:** This research was funded by the land North Rhine-Westphalia and the European Regional Development Fund (ERDF; German: EFRE) under grant number EFRE-0801298, reference number LS-2-1-014 and The APC was funded by Fraunhofer IME. Development Fund (ERDF; German: EFRE) under grant number EFRE-0801298, reference number LS-2-1-014 and The APC was funded by Fraunhofer IME. **Acknowledgments:** The authors would like to thank Dr. Stefan Rasche for his helpful advices and support given

**Acknowledgments:** The authors would like to thank Stefan Rasche for his helpful advices and support given in discussions. in discussions. **Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Appendix A Appendix A** 

**Figure A1.** ELISA checkerboard titration determining suitable aflatoxin B1-BSA (AFB1-BSA) coating concentrations ranging from 0.1 µg·ml–1 to 5 µg·ml–1 and monoclonal antibody (mAb) AFB1\_002 concentrations ranging from 19.5 ng·ml–1 to 1250 ng·ml–1. Absorbance was measured at 405 nm by indirect readout after using a mouse-specific secondary antibody conjugated to horseradish **Figure A1.** ELISA checkerboard titration determining suitable aflatoxin B1-BSA (AFB1-BSA) coating concentrations ranging from 0.1 <sup>µ</sup>g·mL−<sup>1</sup> to 5 <sup>µ</sup>g·mL−<sup>1</sup> and monoclonal antibody (mAb) AFB1\_002 concentrations ranging from 19.5 ng·mL−<sup>1</sup> to 1250 ng·mL−<sup>1</sup> . Absorbance was measured at 405 nm by indirect readout after using a mouse-specific secondary antibody conjugated to horseradish peroxidase and application of respective substrate (*n* = 1).

peroxidase and application of respective substrate (*n* = 1).

**Figure A2.** Magnetic immunodetection checkerboard titration determining suitable aflatoxin B1-BSA (AFB1-BSA) coating concentrations and aflatoxin B1-specific biotinylated monoclonal antibody AFB1\_002 concentrations. The readout was done by detecting 700 nm streptavidin-functionalized magnetic particles coupled to biotinylated AFB1\_002 using FMMD (*n* = 1). **Figure A2.** Magnetic immunodetection checkerboard titration determining suitable aflatoxin B1-BSA (AFB1-BSA) coating concentrations and aflatoxin B1-specific biotinylated monoclonal antibody AFB1\_002 concentrations. The readout was done by detecting 700 nm streptavidin-functionalized magnetic particles coupled to biotinylated AFB1\_002 using FMMD (*n* = 1).
