Protocol to isolate broadly neutralizing monoclonal antibodies against SARS-CoV-2 from human B cells

Sep 29, 2026

Uploaded by Grenda

Broadly neutralizing monoclonal antibodies (bnAbs) against SARS-CoV-2 are vital for therapeutics and vaccines providing broad protection against SARS-CoV-2 variants and related sarbecoviruses. 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 panel.

LanguageEnglish

CategoryHealthcare

FormatPDF

Pages26

Reading Time1 h 6 min

AccessFree to download

SUMMARY

BEFORE YOU BEGIN

Innovation

Institutional permissions

Isolation of human PBMCs

Timing: 3 h

1. Blood collection

2. Isolation of plasma and PBMCs

ll OPEN ACCESS Protocol Protocol to isolate broadly neutralizing monoclonal antibodies against SARS-CoV-2 from human B cells Xueyan Huang, Maosheng Lu, Ling Li, , Yucong Sun, Liwei Jiang, Teng Zuo [jlw0531@cmpt.ac.cn](jlw0531@cmpt.ac.cn) (L.J.) [zuot@cmpt.ac.cn](zuot@cmpt.ac.cn) (T.Z.) Highlights Steps for sorting single human B cells specific for SARSCoV-2 spike Instructions for cloning monoclonal antibodies from single B cells Procedures for screening broadly neutralizing monoclonal antibodies against SARS-CoV-2 Steps for measuring the neutralizing breadth and potency of monoclonal antibodies Huang et al. , STAR Protocols 7, 104275 March 20, 2026 © 2025 The Author(s) . Published by Elsevier Inc. [https://doi.org/10.1016/](https://doi.org/10.1016/)[ ](https://doi.org/10.1016/)[j.xpro.2025.104275](j.xpro.2025.104275) ll OPEN ACCESS Protocol Protocol to isolate broadly neutralizing monoclonal antibodies against SARS-CoV-2 from human B cells Xueyan 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,* 1Laboratory of Immunoengineering, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China 2University of Science and Technology of China, Hefei 230026, China 3Technical contact 4Lead contact *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) SUMMARY Broadly 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 of bnAbs against a panel of pseudoviruses. For complete details on the use and execution of this protocol, please refer to Chen et al.1 BEFORE YOU BEGIN This 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. Innovation The 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. Institutional permissions As 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: YXLL-2023-47) . STAR 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

FAQs

What is the overall workflow of this protocol?

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.

Which source material is used to isolate the antibodies?

The protocol uses peripheral blood mononuclear cells (PBMCs) from individuals exposed to SARS-CoV-2 by vaccination or infection.

How are bnAbs activity and potency evaluated?

The protocol screens monoclonal antibodies for broadly neutralizing activity and measures neutralizing breadth and potency against a panel of pseudoviruses.

Sep 29, 2026

Uploaded by Grenda