期刊
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
卷 411, 期 6, 页码 1287-1295出版社
SPRINGER HEIDELBERG
DOI: 10.1007/s00216-018-01563-7
关键词
Variable domains of heavy-chain antibody; IgG3a; VHH-AP; One-step ELISA; Triazophos
资金
- Key Project of Inter-Governmental International Scientific and Technological Innovation Cooperation [2016YFE0108900]
- National Key Research and Development Program of China [2016YFD0800606]
- National Institute of Environmental Health Sciences Superfund Research Program [P42ES04699]
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 6.6ng/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 (R-2=0.99). The VHH-AP fusion protein shows potential for the analysis of triazophos in various matrices.
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