Rapid cell-free antibody fragment production and binding analysis via fluorescence correlation.

IF 3.6 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods Pub Date : 2026-09-04 DOI:10.1016/j.ymeth.2026.08.007

Shakiba Nikfarjam, Chao Liu, Emma J Laurence, Adam N Laurence, Jennifer L Chlebek, Dante P Ricci, Steven A Hoang-Phou, Isabella M Carrano, Ted A Laurence, Matthew A Coleman

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引用次数: 0

Abstract

This study presents a workflow for rapid production and functional characterization of antibody fragments using an E. coli-based cell-free protein synthesis (CFPS) system. We quantified binding interactions by fluorescence correlation spectroscopy (FCS), which enabled determination of dissociation constants (K_D) between antibodies and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. As an initial validation, two conventionally expressed anti-RBD antibodies were analyzed to establish the reliability of the FCS-based binding measurements. To enable efficient cell-free production, strategies were developed to improve the solubility and yield of single-chain variable fragments (scFvs), including implementation of an established two-stage refolding workflow adapted for CFPS-derived proteins. This approach enabled recovery of functional scFvs from insoluble fractions. In addition, Fab fragments were successfully produced and characterized, with binding measurements confirming retention of antigen recognition. Together, these results demonstrate that CFPS can be combined with FCS to enable rapid, solution-phase evaluation of antibody fragment binding without reliance on conventional cell-based expression systems. This integrated platform provides a scalable approach for antibody screening and characterization, with potential applications in therapeutic antibody development and high-throughput discovery.

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快速无细胞抗体片段产生和荧光相关结合分析。

本研究提出了一种基于大肠杆菌的无细胞蛋白合成（CFPS）系统的抗体片段快速生产和功能表征的工作流程。我们通过荧光相关光谱（FCS）量化了结合相互作用，从而确定了抗体与SARS-CoV-2刺突蛋白受体结合域（RBD）之间的解离常数（KD）。作为初步验证，分析了两种常规表达的抗rbd抗体，以建立基于fcs的结合测量的可靠性。为了实现高效的无细胞生产，研究人员开发了提高单链可变片段（scFvs）的溶解度和产率的策略，包括适用于cfps衍生蛋白的两阶段重折叠工作流程的实施。这种方法可以从不溶性馏分中回收功能性scFvs。此外，Fab片段被成功生产和表征，结合测量确认抗原识别保留。总之，这些结果表明，CFPS可以与FCS结合，实现抗体片段结合的快速、溶液阶段评估，而不依赖于传统的基于细胞的表达系统。该集成平台为抗体筛选和表征提供了一种可扩展的方法，在治疗性抗体开发和高通量发现方面具有潜在的应用。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods 生物-生化研究方法

CiteScore

9.80

自引率

2.10%

发文量

222

审稿时长

11.3 weeks

期刊介绍： Methods focuses on rapidly developing techniques in the experimental biological and medical sciences. Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.