Abstract
Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL−1 and a limit of detection of 0.059 ng mL−1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.
Highlights
Ochratoxin A (OTA), a secondary mold metabolite produced by Aspergillus and Penicillium, is one of the most frequently reported mycotoxins in various types of food such as cereal products, dried fruits, and coffee [1]
The results show the reliability of Nb-alkaline phosphatase (AP)-based one-step enzyme-linked immunosorbent assay (ELISA) for the detection of OTA in rice
The anti-OTA nanobody Nb28 fused with alkaline phosphatase (Nb28-AP) served as the primary antibody and reporter molecule
Summary
Ochratoxin A (OTA), a secondary mold metabolite produced by Aspergillus and Penicillium, is one of the most frequently reported mycotoxins in various types of food such as cereal products, dried fruits, and coffee [1]. OTA can induce multiple toxic effects to humans and animals, and was categorized as group 2B (possible carcinogens to humans) by the International Agency for Research on Cancer [2,3,4]. A recent study has provided evidence for the mechanism of OTA carcinogenesis in humans [5]. Considering the toxicities of OTA, most countries have set regulations for it in different food products. The maximum level is 10 μg kg−1 in dried vine fruit, 5 μg kg−1 in unprocessed cereals, and 3 μg kg−1 in cereal products set by the European Union [6]. Adequate techniques for OTA screening in agricultural commodities are indispensable
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