Antibody Microarray Immunoassay for Simultaneous Quantification of Multiple Mycotoxins in Corn Samples
Xian Zhang,
Zuohuan Wang,
Yun Fang,
Renjie Sun,
Tong Cao,
Narayan Paudyal,
Weihuan Fang,
Houhui Song
Affiliations
Xian Zhang
China-Australian Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, College of Animal Science and Technology, Zhejiang A&F University, Lin’an 311300, Zhejiang, China
Zuohuan Wang
Zhejiang University Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou 310058, Zhejiang, China
Yun Fang
Technic Center of Zhejiang Entry-Exit Inspection and Quarantine Bureau, 126 Fuchun Road, Hangzhou 310012, Zhejiang, China
Renjie Sun
Zhejiang University Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou 310058, Zhejiang, China
Tong Cao
Zhejiang University Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou 310058, Zhejiang, China
Narayan Paudyal
Zhejiang University Institute of Preventive Veterinary Medicine and Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, 388 Yuhangtang Road, Hangzhou 310058, Zhejiang, China
Weihuan Fang
China-Australian Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, College of Animal Science and Technology, Zhejiang A&F University, Lin’an 311300, Zhejiang, China
Houhui Song
China-Australian Joint Laboratory for Animal Health Big Data Analytics, Zhejiang Provincial Engineering Laboratory for Animal Health Inspection and Internet Technology, College of Animal Science and Technology, Zhejiang A&F University, Lin’an 311300, Zhejiang, China
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.
