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A method for rapid nanobody screening with no bias of the library diversity
iScience ( IF 4.6 ) Pub Date : 2024-01-18 , DOI: 10.1016/j.isci.2024.108966 Zhiqing Tao 1, 2 , Xiaoling Zhao 3, 4 , Huan Wang 5 , Juan Zhang 3 , Guosheng Jiang 5 , Bin Yu 1, 2 , Yihao Chen 1, 2 , Mingjun Zhu 1, 2 , Junli Long 1, 2 , Lei Yin 6 , Xu Zhang 1, 2 , Maili Liu 1, 2, 7 , Lichun He 1, 2
iScience ( IF 4.6 ) Pub Date : 2024-01-18 , DOI: 10.1016/j.isci.2024.108966 Zhiqing Tao 1, 2 , Xiaoling Zhao 3, 4 , Huan Wang 5 , Juan Zhang 3 , Guosheng Jiang 5 , Bin Yu 1, 2 , Yihao Chen 1, 2 , Mingjun Zhu 1, 2 , Junli Long 1, 2 , Lei Yin 6 , Xu Zhang 1, 2 , Maili Liu 1, 2, 7 , Lichun He 1, 2
Affiliation
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Nanobody, referred to the variable domain of heavy-chain-only antibodies, has several advantages such as small size and feasible 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.
中文翻译：
一种不影响文库多样性的快速纳米抗体筛选方法
纳米抗体是指仅重链抗体的可变域，具有体积小、表达可行等优点，使其在科学研究和治疗方面具有广阔的前景。传统的纳米抗体筛选和表达方法常常需要亚克隆到表达载体中以及扩增引起的多样性损失。在这里，我们开发了一种同时筛选和表达的集成方法。克隆纳米抗体文库并在培养基中秘密表达。通过 1-3 轮稀释和遵循泊松分布的再生来分离靶标特异性纳米抗体。这确保了阳性克隆不会被剔除，每轮稀释中阳性克隆的数量都会增加 10 倍以上。最终，我们直接从免疫文库中分离出 5 个针对死亡结构域受体 5 的纳米抗体和针对激烈火球菌 DNA 聚合酶的 5 纳米抗体。值得注意的是，我们的方法无需专用仪器即可进行纳米抗体筛选，证明了其在各种生物医学应用的常规单克隆纳米抗体生产中的广泛适用性。
更新日期：2024-01-18
中文翻译：
一种不影响文库多样性的快速纳米抗体筛选方法
纳米抗体是指仅重链抗体的可变域，具有体积小、表达可行等优点，使其在科学研究和治疗方面具有广阔的前景。传统的纳米抗体筛选和表达方法常常需要亚克隆到表达载体中以及扩增引起的多样性损失。在这里，我们开发了一种同时筛选和表达的集成方法。克隆纳米抗体文库并在培养基中秘密表达。通过 1-3 轮稀释和遵循泊松分布的再生来分离靶标特异性纳米抗体。这确保了阳性克隆不会被剔除，每轮稀释中阳性克隆的数量都会增加 10 倍以上。最终，我们直接从免疫文库中分离出 5 个针对死亡结构域受体 5 的纳米抗体和针对激烈火球菌 DNA 聚合酶的 5 纳米抗体。值得注意的是，我们的方法无需专用仪器即可进行纳米抗体筛选，证明了其在各种生物医学应用的常规单克隆纳米抗体生产中的广泛适用性。
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