A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.
A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.
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DOI：
10.1016/j.jim.2017.05.010
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发表时间：
2017-09
影响因子：
2.2
通讯作者：
Sather DN
中科院分区：
文献类型：
--
作者：
Carbonetti S;Oliver BG;Vigdorovich V;Dambrauskas N;Sack B;Bergl E;Kappe SHI;Sather DN
过去 40 年来，单克隆抗体技术使免疫学、传染病研究和现代医学取得了巨大进步。然而，许多单克隆抗体发现过程是劳动密集型和时间密集型、效率低且昂贵的。在这里，我们描述了一个优化的 mAb 发现平台，用于在小鼠系统中快速有效地分离、克隆和表征单克隆抗体。在此平台中，通过 FACS 分离来自免疫小鼠的抗原结合脾 B 细胞，并与 CD40L 阳性细胞共培养，以诱导增殖和 mAb 产生。共培养 12 天后，筛选细胞培养上清液中的抗原，并分离 IgG 阳性和 RNA 进行反转录。然后通过 PCR 扩增阳性孔 cDNA，并将所得扩增子克隆到独立于连接的表达载体中，然后直接用于转染 HEK293 细胞以产生重组抗体。生长 4 天后，可以使用生物层干涉测量法筛选条件培养基，进行抗原结合和亲和力测量。使用这种方法，我们能够分离出六种独特的、针对人类疟疾寄生虫恶性疟原虫抗原的功能性单克隆抗体。重要的是，该方法融合了几项重要的进步，避免了单细胞 PCR、限制性克隆和大规模蛋白质生产的需要，并且可以应用于多种蛋白质抗原。
Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40 years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12 days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4 days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens.
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影响因子：
2.2
作者：
Tiller, Thomas;Busse, Christian E.;Wardemann, Hedda
通讯作者：
Wardemann, Hedda
影响因子：
5.8
作者：
Vigdorovich, Vladimir;Oliver, Brian G.;Sather, D. Noah
通讯作者：
Sather, D. Noah
影响因子：
3.7
作者：
Smith SA;Crowe JE Jr
通讯作者：
Crowe JE Jr
影响因子：
3.8
作者：
Backliwal, Gaurav;Hildinger, Markus;Wurm, Florian M.
通讯作者：
Wurm, Florian M.
影响因子：
2.2
作者：
Tiller, Thomas;Meffre, Eric;Wardemann, Hedda
通讯作者：
Wardemann, Hedda