Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing
Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing
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DOI：
10.1074/jbc.m115.676635
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发表时间：
2015-10-30
影响因子：
4.8
通讯作者：
Whitehead, Timothy A.
中科院分区：
文献类型：
--
作者：
Kowalsky, Caitlin A.;Faber, Matthew S.;Whitehead, Timothy A.
了解中和和非中和抗原表位在人类病原体上的精确位置，可以更好地了解抗体功效的结构基础，这将加快疫苗、预防措施和治疗方法的合理设计。然而，充分利用来自单细胞技术和抗体库测序的丰富信息，需要开发一种高通量、廉价的方法来绘制抗体-抗原相互作用的构象表位。在这里，我们展示了这样一种结合了全面诱变、细胞表面展示和DNA深度测序的方法。我们开发了分析方程来确定表位位置，并通过绘制针对TNF，百日咳毒素和癌症靶点TROP2的不同抗体的精细表位来显示方法的有效性。在这三种情况下，实验确定的构象表位与先前的实验数据集一致，证实了实验管道的可靠性。一旦综合文库生成，精细的构象表位图可以以每天4个的速度制备。
Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.