Development of a one-step immunoassay for triazophos using camel single-domain antibody–alkaline phosphatase fusion protein
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Triazophos is mainly used in Asian and African countries for the control of insects in agricultural production. Camelid variable domains of heavy-chain antibodies (VHHs) show great promise in monitoring environmental chemicals such as pesticides. To improve the rate of success in the generation of VHHs against triazophos, genes specifically encoding VHH fragments from the unique allotype IgG3a of an immunized Camelus bactrianus were amplified by using a pair of novel primers and introduced to construct a diverse VHH library. Five out of seven isolated positive clones, including the VHH T1 with the highest affinity to triazophos, were derived from the allotype IgG3a. A one-step enzyme-linked immunosorbent assay (ELISA) using VHH T1 genetically fused with alkaline phosphatase (AP) had a half-maximum inhibition concentration of 6.6 ng/mL for triazophos. This assay showed negligible cross-reactivity with a list of important organophosphate pesticides (< 0.1%). The average recoveries of triazophos from water, soil, and apple samples determined by the one-step ELISA ranged from 83 to 108%, having a good correlation with those by a gas chromatography mass spectrometry (R2 = 0.99). The VHH-AP fusion protein shows potential for the analysis of triazophos in various matrices.
KeywordsVariable domains of heavy-chain antibody IgG3a VHH-AP One-step ELISA Triazophos
The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
This work was supported in part by the Key Project of Inter-Governmental International Scientific and Technological Innovation Cooperation (2016YFE0108900), the National Key Research and Development Program of China (2016YFD0800606), and the National Institute of Environmental Health Sciences Superfund Research Program (P42ES04699), USA.
Compliance with ethical standards
The animal experiments were approved by the China Agricultural University Animal Care and Use Committee. All procedures performed in this research involving Bactrian camels were in accordance with the ethical standards of the China Agricultural University Animal Care and Use Committee. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Conflict of interest
The authors declare that they have no conflict of interest.
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