☆ 4.7 Article

Highly specific nanobody against herbicide 2,4-dichlorophenoxyacetic acid for monitoring of its contamination in environmental water

SCIENCE OF THE TOTAL ENVIRONMENT (2021)

Journal

SCIENCE OF THE TOTAL ENVIRONMENT

Volume 753, Issue -, Pages -

Publisher

ELSEVIER

DOI: 10.1016/j.scitotenv.2020.141950

Keywords

Surface water monitoring; Antibody specificity; Nanobody isolation; FLEIA based on nanobody-AP

Funding

National Institute of Environmental Health Science Superfund Research Program [P42ES004699]
National Institute of Health RIVER Award [R35 ES030443-01]
National Academy of Sciences
USAID (NAS) [2000009144]

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Abstract

The study successfully developed a nanobody-based immunoassay for monitoring trace levels of 2,4-D in the environment. Utilizing a step-wise strategy from hapten design to nanobody-AP fusion production, the research achieved successful results, demonstrating the method as a convenient and practical tool for monitoring 2,4-D residuals.

2,4-dichlorophenoxyacetic acid (2,4-D), a widely used herbicide, is a small organic chemical pollutant in the environment. To develop a nanobody-based immunoassay for monitoring trace levels of 2,4-D, a step-wise strategy for the generation of nanobodies highly specific against this small chemical was employed. Firstly, we synthesized three novel haptens mimicking 2,4-D and assessed their influence on the sensitivity and specificity of the existing antibody-based assay. Polyclonal antibodies (pAb) from rabbits showed good sensitivity and moderate specificity for 2,4-D, pAb from llama based on selected haptens showed similar performance when compared to those from rabbits. Secondly, nanobodies derived from llama were generated for 2,4-D by an effective procedure, including serum monitoring and one-step library construction. One nanobody, NB3-9, exhibited good sensitivity against 2,4-D (IC50 = 29.2 ng/mL) had better specificity than the rabbit pAb#1518, with no cross-reactivities against the 2,4-D analogs tested. Thirdly, one-step fluorescent enzyme immunoassay (FLEIA) for 2,4-D based on a nanobody-alkaline phosphatase (AP) fusion was developed with IC50 of 1.9 ng/mL and a linear range of 0.4-8.6 ng/mL. Environmental water samples were analyzed by FLEIA and LC-MS/MS for comparison, and the results were consistent between both methods. Therefore, the proposed step-wise strategy from hapten design to nanobody-AP fusion production was successfully conducted, and the resulting nanobody based FLEIA was demonstrated as a convenient tool to monitor 2,4-D residuals in the environment. (C) 2020 Elsevier B.V. All rights reserved.

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Precise isolation and structural origin of an ultra-specific nanobody against chemical compound

Huijuan Yang, Natalia Vasylieva, Jiaxin Wang, Zhenfeng Li, Wenbo Duan, Shuang Chen, Kai Wen, Hui Meng, Xuezhi Yu, Jianzhong Shen, Bruce D. Hammock, Zhanhui Wang

Summary: Highly specific antibodies are essential for accurate immunoassays in environmental monitoring. This study demonstrates that nanobodies possess higher specificity than conventional antibodies due to their unique structural features. A specific nanobody (H1-17) was isolated using an immune phage display library and showed no cross-reactivity with other analogs. The structural specificity of H1-17 was compared to a conventional antibody using modeling and mutagenesis validation, revealing the key role of a noncanonical disulfide bond and mutual-checking hydrogen bonds. Immunoassays with zero false positive rate were developed using nanobodies for precise chemical compound detection.

JOURNAL OF HAZARDOUS MATERIALS (2023)