A Microfluidic Chip for Screening and Sequencing of Monoclonal Antibody at a Single-Cell Level
A Microfluidic Chip for Screening and Sequencing of Monoclonal Antibody at a Single-Cell Level
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用于单细胞水平单克隆抗体筛选和测序的微流控芯片
DOI：
10.1021/acs.analchem.1c00918
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发表时间：
2021
影响因子：
7.4
通讯作者：
Wei Zewen
中科院分区：
文献类型：
--
作者：
Zhang Weikai;Li Qin;Jia Fei;Hu Zhiyuan;Wei Zewen
抗体的重链和轻链的配对决定了单克隆抗体（mAb）的特异性。获得编码配对重链和轻链可变区（VH：VL）的基因至关重要，但在传统方法中，这是一个劳动密集型和成本密集型的过程。新兴的微流控芯片为我们提供了一个通过对单个靶细胞进行测序来直接获得天然配对的VH：VL基因的门户。该研究提出了一种新的方法，其中用于从单细胞获得天然配对的VH：VL基因的所有处理步骤在单个微流控芯片中完成，而不是多个离散的装置。微流控芯片在单细胞水平上进行单细胞捕获/抗体特异性的原位荧光检查/细胞裂解/基因扩增。通过从单个杂交瘤细胞中有效获得抗CD 45 mAb的配对VH：VL基因的概念验证，已证明微流控芯片能够捕获/筛选/裂解单个抗体分泌细胞并进行片上逆转录-聚合酶链反应。所提出的方法实现了显著改善的细胞损失/人力/时间成本，更重要的是，天然VH：VL基因配对的确定性，这是抗体发现有效性的最决定性因素之一。
The pairing of heavy and light chains of an antibody decides the specificity of monoclonal antibodies (mAbs). Acquisition of the genes encoding variable regions of paired heavy and light chains (VH:VL) is crucial, but it is a labor- and cost-intensive process in traditional methods. The emerging microfluidic chips have brought us to a portal of directly acquiring natively paired VH:VLgenes by sequencing single target cells. This study presents a novel method in which all processing steps for acquiring natively paired VH:VLgenes from single cells are finished in a single microfluidic chip, not multiple discrete devices. The microfluidic chip performs single-cell trapping/in situfluorescence examination of antibody specificity/cell lysis/gene amplification all at a single-cell level. By a proof-of-concept validation of efficiently acquiring paired VH:VLgenes of anti-CD45 mAbs from single hybridoma cells, the microfluidic chip has been proved capable of trapping/screening/lysing single antibody-secreting cells and performing an on-chip reverse transcription-polymerase chain reaction. The presented method has realized remarkably improved cell loss/human labor/time cost, and more importantly, determinacy of native VH:VLgene pairing, which is one of the most decisive factors of effectiveness for antibody discovery.