title: A toxin-free enzyme-linked immunosorbent assay for the analysis of aflatoxins based on a VHH surrogate standard.
abstract: A toxin-free enzyme-linked immunosorbent assay (ELISA) for aflatoxins was developed using an anti-idiotype nanobody VHH 2-5 as surrogate standard. Anti-idiotype nanobody VHH 2-5 was generated by immunizing an alpaca with anti-aflatoxin monoclonal antibody 1C11. This assay was used to detect aflatoxins in agro-products after a simple extraction with 75 % methanol/H2O. Aflatoxin concentration was calculated by a two-step approach: the concentration of VHH 2-5 was first obtained by a four-parameter logistic regression from the detected absorbance value at 450 nm, and then converted to aflatoxin concentration by a linear equation. The assay exhibits a limit of detection (LOD) of 0.015 ng mL(-1), which is better than or comparable with conventional immunoassays. The performance of our VHH surrogate-based ELISA was further validated with a high-performance liquid chromatography (HPLC) method for total aflatoxins determination in 20 naturally contaminated peanut samples, displaying a good correlation (R (2) = 0.988). In conclusion, the proposed assay represents a first example applying an anti-idiotype VHH antibody as a standard surrogate in ELISA. With the advantages of high stability and ease of production, the VHH antibody-based standard surrogate can be extended in the future to immunoassays for other highly toxic compounds. Graphical Abstract ᅟ.
authors.affiliation: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, China.|College of Food Science and Engineering, Northwest A&F University, 28 Xinong Road, Yangling, Xianyang, Shaanxi, 712100, China.
authors.forename: Yanru
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, China. peiwuli@oilcrops.cn.|Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, Hubei, 430062, China. peiwuli@oilcrops.cn.|Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan, Hubei, 430062, China. peiwuli@oilcrops.cn.|Laboratory of Risk Assessment for Oilseeds Products, Wuhan, Ministry of Agriculture, Wuhan, Hubei, 430062, China. peiwuli@oilcrops.cn.|Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan, Hubei, 430062, China. peiwuli@oilcrops.cn.
authors.forename: Peiwu
authors.initials: P
authors.lastname: Li
authors.affiliation: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, China. zhangqi521x@126.com.|Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan, Hubei, 430062, China. zhangqi521x@126.com.|Laboratory of Risk Assessment for Oilseeds Products, Wuhan, Ministry of Agriculture, Wuhan, Hubei, 430062, China. zhangqi521x@126.com.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, China.|Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, Hubei, 430062, China.|Laboratory of Risk Assessment for Oilseeds Products, Wuhan, Ministry of Agriculture, Wuhan, Hubei, 430062, China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Hu
authors.affiliation: Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan, 430062, China.|Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, Hubei, 430062, China.|Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan, Hubei, 430062, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000348:Aflatoxins / Q000032:analysis* / Q000276:immunology; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology; D000917:Antibody Formation; D010367:Arachis / Q000737:chemistry* / Q000276:immunology; D002161:Camelids, New World; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005504:Food Analysis / Q000379:methods*; D005506:Food Contamination / Q000032:analysis; D057230:Limit of Detection; D061905:Single-Domain Antibodies / Q000737:chemistry* / Q000276:immunology
chemical_list: D000348:Aflatoxins; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D061905:Single-Domain Antibodies
keywords: Aflatoxin; Immunoassay; Nanobody; Surrogate; Toxin-free