Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples

We developed and tested a prototype of an antibody microarray immunoassay for simultaneous quantitative detection of four typical mycotoxins (aflatoxin B₁, ochratoxin A, zearalenone, and fumonisin B₁) in corn samples. The test kit consisted of a nitrocellulose membrane layered with immobilized monoclonal antibodies against mycotoxins. During the assay, the mycotoxin-protein conjugates were biotinylated. The signal detection was enhanced by a combination of the biotin-streptavidin system and enhanced chemiluminescence (ECL). This improved the sensitivity of the assay. Under the optimized conditions, four calibration curves with goodness of fit (R² > 0.98) were plotted. The results showed that the detection limits for aflatoxin B₁, ochratoxin A, zearalenone, and fumonisin B₁ were 0.21, 0.19, 0.09, and 0.24 ng/mL, with detection ranges of 0.47⁻55.69, 0.48⁻127.11, 0.22⁻31.36, and 0.56⁻92.57 ng/mL, respectively. The limit of detection (LOD) of this antibody microarray for aflatoxin B₁, ochratoxin A, zearalenone, and fumonisin B₁ in corn was 5.25, 4.75, 2.25, and 6 μg/kg, respectively. The recovery rates from the spiked samples were between 79.2% and 113.4%, with coefficient of variation <10%. The results of the analysis of commercial samples for mycotoxins using this new assay and the liquid chromatography-tandem mass spectrometry (LC-MS/MS) were comparable and in good agreement. This assay could also be modified for the simultaneous detection of other multiple mycotoxins, as well as low-weight analytes, hazardous to human health.