Abstract
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.
Highlights
Mycotoxins, the secondary metabolites produced by fungi such as Aspergillus, Fusarium, and Penicillium, often contaminate agricultural produce during harvest, storage, or processing [1].The most frequently encountered and studied mycotoxins in cereal grains and feeds are aflatoxins, ochratoxins, zearalenone, and fumonisins [2]
The results obtained in this study confirmed that the analytical method developed in this study, targeting different mycotoxins for simultaneous detection, is possible and workable
An antibody microarray strategy based on direct competition and the biotin-streptavidin signal amplification system for the simultaneous detection of four mycotoxins, is implemented
Summary
Mycotoxins, the secondary metabolites produced by fungi such as Aspergillus, Fusarium, and Penicillium, often contaminate agricultural produce during harvest, storage, or processing [1].The most frequently encountered and studied mycotoxins in cereal grains and feeds are aflatoxins, ochratoxins, zearalenone, and fumonisins [2]. The major toxicities of these mycotoxins include carcinogenicity (aflatoxin B1 , AFB1 ), potentially carcinogenic and nephrotoxic agents (ochratoxin A, OTA), estrogenic and reproductive toxicity (zearalenone, ZEN), and equine leucoencephalomalacia and porcine pulmonary edema (fumonisin B1 , FB1 ) [3]. Their widespread distribution has become a major concern in food and feed safety, and simultaneous occurrence of multiple mycotoxins is a common phenomenon. Considering the serious health risk of mycotoxins, many countries and regulatory authorities, such as the Joint Food and Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA), have defined the maximum residue limit (MRL)
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