Abstract
Enzyme-linked immunosorbent assay (ELISA) test kits have been widely used for the determination of mycotoxins in agricultural products and foods, however, this test uses toxin standards with high toxicity and carcinogenicity that seriously threaten human health. In this work, the anti-idiotypic nanobody VHH 2-24 was first developed and then, using it as a surrogate standard, a toxin-free enzyme immunoassay for ochratoxin A (OTA) was established. The IC50 value of the VHH 2-24 surrogate standard-based ELISA was 0.097 µg/mL, with a linear range of 0.027–0.653 µg/mL. The average recoveries were tested by spike-and-recovery experiments, and ranged from 81.8% to 105.0%. The accuracy of the developed ELISA for detecting OTA was further verified by using the high performance liquid chromatography (HPLC) method, and an excellent correlation was observed. In summary, the toxin-free ELISA established in this study proves the latent use of the anti-idiotypic VHH as a surrogate calibrator for other mycotoxins and highly toxic small molecule analysis to improve assay properties for highly sensitive analyte determination in agricultural products.
Highlights
Ochratoxin A (OTA) is a secondary metabolite produced by the genus Aspergillus and Penicillium, especially A. ochraceus and P. verrucosum [1,2]
Ten corn samples naturally contaminated with OTA were selected and the levels of OTA were determined by the newly developed Enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) methods
We developed a toxin-free, sensitive and selective ELISA for the analysis of OTA
Summary
Ochratoxin A (OTA) is a secondary metabolite produced by the genus Aspergillus and Penicillium, especially A. ochraceus and P. verrucosum [1,2]. Immunoassays based on the specific binding of the antigens and antibodies have the advantages of rapid sample preparation, selectivity, high sensitivity and cost effectiveness [17,18]. Numerous studies have reported the superiority of developing toxin substitutes for immunoassays, mainly involving aflatoxins, deoxynivalenol, ochratoxin A, fumonisins, zearalenone and citrinin [21,22,23,24,25,26] These alternatives can be obtained by the preparation of anti-idiotypic antibodies. Compared with traditional recombinant antibodies, nanobodies have better solubility, high thermal stability and high tolerance to organic solvents [38] In view of these advantages, VHH antibodies are more suitable for the development of alternative reagents for immunoassay. The obtained VHH 2-24 was used as the surrogate standard to develop a toxic-free indirect competitive enzyme-linked immunosorbent assay for the detection of OTA in agricultural products
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