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The protocol describes isolating bnAbs from human B cells by sorting single SARS-CoV-2 spike-specific B cells from PBMCs, cloning monoclonal antibodies from single cells, screening for broadly neutralizing activity, and assessing neutralizing breadth and potency using a pseudovirus 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SARS-CoV-2 from human B cells  \nXueyan Huang, Maosheng Lu, Ling Li,   , Yucong Sun, Liwei Jiang, Teng Zuo  \n[jlw0531@cmpt.ac.cn](jlw0531@cmpt.ac.cn) (L.J.)  \n[zuot@cmpt.ac.cn](zuot@cmpt.ac.cn) (T.Z.)  \nHighlights  \nSteps for sorting single human B cells specific for SARSCoV-2 spike  \nInstructions for cloning monoclonal antibodies from single B cells  \nProcedures for screening broadly neutralizing monoclonal antibodies against SARS-CoV-2  \nSteps for measuring the neutralizing breadth and potency of monoclonal antibodies  \nHuang et al. , STAR Protocols 7, 104275  \nMarch 20, 2026 © 2025 The Author(s) . Published by Elsevier Inc.  \n[https://doi.org/10.1016/](https://doi.org/10.1016/)[ ](https://doi.org/10.1016/)[j.xpro.2025.104275](j.xpro.2025.104275)  \nll  \nOPEN ACCESS  \nProtocol  \nProtocol to isolate broadly neutralizing monoclonal antibodies against SARS-CoV-2 from human B cells  \nXueyan Huang,1,2 Maosheng Lu, 1,2 Ling Li, 1 Zuowei Wang,1 Yunjian Li, 1,2 Xixian Chen, 1,2 Yi Sun, 1,2 Ruiping Du, 1,2 Rongrong Qin, 1,2 Lin Hu, 1,2 Yucong Sun, 1,2 Liwei Jiang,1,3,* and Teng Zuo 1,4,*  \n1Laboratory of Immunoengineering, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China  \n2University of Science and Technology of China, Hefei 230026, China  \n3Technical contact  \n4Lead contact  \n*Correspondence: [jlw0531@cmpt.ac.cn](jlw0531@cmpt.ac.cn) (L.J.), [zuot@cmpt.ac.cn](zuot@cmpt.ac.cn) (T.Z.) [https://doi.org/10.1016/j.xpro.2025.104275](https://doi.org/10.1016/j.xpro.2025.104275)  \nSUMMARY  \nBroadly neutralizing monoclonal antibodies (bnAbs) against SARS-CoV-2 are crucial for developing therapeutics and vaccines that confer broad protection against SARS-CoV-2 variants and related sarbecoviruses. Here, we present a protocol to isolate bnAbs against SARS-CoV-2 from human B cells. We describe steps for sorting single spike-specific B cells from human peripheral blood mononuclear cells, cloning monoclonal antibodies (mAbs) from single B cells, screening mAbs with broadly neutralizing activity, and measuring the neutralizing potency  \nof bnAbs against a panel of pseudoviruses.  \nFor complete details on the use and execution of this protocol, please refer to  \nChen et al.1  \nBEFORE YOU BEGIN  \nThis protocol describes the detailed steps for isolating bnAbs against SARS-CoV-2 from peripheral blood mononuclear cells (PBMCs) of individuals exposed to SARS-CoV-2 by vaccination or infection. Overall, this protocol includes four major steps: Single-cell sorting of spike-specific B cell (Steps 1-4); Single-cell RT-PCR and mAb cloning (Steps 5-11); Screening for bnAbs(Steps 12- 16); Measurement of neutralizing breadth and potency (Steps 17-20) . Although this protocol is built for isolating bnAbs against SARS-CoV-2, it can be adapted for developing human mAbs against other antigens.  \nInnovation  \nThe current protocol is modified from the protocols published previously.2 ,3 One major modification is that we directly assemble the second-round PCR products with linearized expression vectors (Steps 8-10) . Another major modification is that we perform functional screening of the mAbs before we sequence them (Steps 12-16) . In addition, we provide the detailed steps for neutralization assay based on pseudovirus.  \nInstitutional permissions  \nAs human blood samples are used to conduct this protocol, researchers need to acquire approvals from their ethics committee. Data shown in this protocol were obtained under the approval of Ethics Committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences (Approval Number:  \nYXLL-2023-47) .  \nSTAR Protocols 7, 104275, March 20, 2026 © 2025 The Author(s) . Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license ([http://creativecommons.org/licenses/by-nc-nd/4.0/](http://creativecommons.org/license","cbCaihLjILH7aF4D","https://ap.wps.com/l/cbCaihLjILH7aF4D","pdf",7593719,26,"English","# SUMMARY\n## BEFORE YOU BEGIN\n### Innovation\n### Institutional permissions\n# Isolation of human PBMCs\n## Timing: 3 h\n### 1. Blood collection\n### 2. Isolation of plasma and PBMCs","[{\"question\":\"What is the overall workflow of this protocol?\",\"answer\":\"It includes four major parts: single-cell sorting of spike-specific B cells, single-cell RT-PCR and mAb cloning, screening for bnAbs, and measurement of neutralizing breadth and potency using pseudoviruses.\"},{\"question\":\"Which source material is used to isolate the antibodies?\",\"answer\":\"The protocol uses peripheral blood mononuclear cells (PBMCs) from individuals exposed to SARS-CoV-2 by vaccination or infection.\"},{\"question\":\"How are bnAbs activity and potency evaluated?\",\"answer\":\"The protocol screens monoclonal antibodies for broadly neutralizing activity and measures neutralizing breadth and potency against a panel of pseudoviruses.\"}]","Protocol to isolate broadly neutralizing monoclonal antibodies against SARS-CoV-2 from human B cells | PDF",1790658832,66]