A method for rapid nanobody screening with no bias of the library diversity
Zhiqing Tao,
Xiaoling Zhao,
Huan Wang,
Juan Zhang,
Guosheng Jiang,
Bin Yu,
Yihao Chen,
Mingjun Zhu,
Junli Long,
Lei Yin,
Xu Zhang,
Maili Liu,
Lichun He
Affiliations
Zhiqing Tao
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
Xiaoling Zhao
Department of Reproductive Medicine, General Hospital of Central Theater Command of the People’s Liberation Army, Wuhan, Hubei 430061, China; Qinhe Life Science Ltd, Wuhan 430000, China
Huan Wang
School of Life Science and Technology, Weifang Medical University, Weifang, Shandong 261053, China
Juan Zhang
Department of Reproductive Medicine, General Hospital of Central Theater Command of the People’s Liberation Army, Wuhan, Hubei 430061, China
Guosheng Jiang
School of Life Science and Technology, Weifang Medical University, Weifang, Shandong 261053, China
Bin Yu
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
Yihao Chen
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
Mingjun Zhu
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
Junli Long
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
Lei Yin
State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Department of Clinical Oncology, Renmin Hospital of Wuhan University, Wuhan University, Wuhan, Hubei Province 430072, China
Xu Zhang
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China
Maili Liu
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China; Optics Valley Laboratory, Hubei 430074, China
Lichun He
State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics, National Center for Magnetic Resonance in Wuhan, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071, China; University of Chinese Academy of Sciences, Beijing 100049, China; Corresponding author
Summary: Nanobody, referred to the variable domain of heavy-chain-only antibodies, has several advantages such as small size and feasible Escherichia coli expression, making them promising for scientific research and therapies. Conventional nanobody screening and expression methods often suffer from the need for subcloning into expression vectors and amplification-induced diversity loss. Here, we developed an integrated method for simultaneous screening and expression. Nanobody libraries were cloned and secretly expressed in the culture medium. Target-specific nanobodies were isolated through 1–3 rounds of dilution and regrowth following the Poisson distribution. This ensured no dismissal of positive clones, with populations of positive clones increasing over 10-fold in each dilution round. Ultimately, we isolated 5 nanobodies against death domain receptor 5 and 5 against Pyrococcus furiosus DNA polymerase directly from their immunized libraries. Notably, our approach enables nanobody screening without specialized instruments, demonstrating broad applicability in routine monoclonal nanobody production for diverse biomedical applications.
