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.

Global analysis of VHHs framework regions with a structural alphabet

“…In addition the VHHs are interesting alternative to the use of monoclonal antibodies for therapy as shown by recent studies of their use against the Dengue virus [19], H5N1 influenza [20], viral infection [21], aflatoxins in agro-products [22], head and neck cancers [23], vascular endothelial growth factor implicated in cancers [24], venom therapy [25] and Plasmodium knowlesi malaria vector [26]. Phase II clinical trials are currently underway to evaluate the efficacy and toxicity of a therapeutic Nanobody TM in patients with thrombotic thrombocytopenic purpura [27] showing recent promising results [28].…”

Advances in Visual Immunoassays for Sensitive Detection of Mycotoxins in Food—A Review

“…Currently, various immunoassays involving enzyme-linked immunosorbent assay (ELISA) [32,33], lateral flow immunoassay (LFI) [34][35][36][37] have been demonstrated as an excellent platform for discrimination of mycotoxins. Among them, ELISA and LFI served as the representative visual immunoassay, have attracted continuous interest due to their advantages of simple, and on-sites for rapid screening mycotoxins.…”

Development of an Anti-Idiotypic VHH Antibody and Toxin-Free Enzyme Immunoassay for Ochratoxin A in Cereals

“…In view of these advantages, VHH antibodies are more suitable for the development of alternative reagents for immunoassay. The anti-id nanobody is used as a surrogate for antigens and standards have been applied to the toxic-free green immunoassays for aflatoxins [21,39], further prompting us to study its use as an alternative to other mycotoxins. However, there are no reports on a toxin-free competitive ELISA for analyzing OTA by using anti-id VHH antibody as a surrogate standard.…”