Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells
Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells
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DOI：
10.1021/acs.bioconjchem.5b00613
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发表时间：
2016-01-01
影响因子：
4.7
通讯作者：
Ramakrishnan, Ramesh
中科院分区：
文献类型：
--
作者：
Gong, Haibiao;Holcomb, Ilona;Ramakrishnan, Ramesh
核酸序列的多样性使得基因组学研究能够以高度多重的形式进行。由于多重蛋白质检测仍然是一个挑战，因此使用基因组学工具用于此目的将是有用的。这可以通过将特异性寡核苷酸与抗体缀合来实现。在缀合有多核苷酸的抗体与其靶标结合后，蛋白质水平可以转化为寡核苷酸水平。在这篇报告中，我们描述了一种简单的方法来制备的寡核苷酸偶联抗体，并讨论了这种方法在寡核苷酸延伸反应（OER）的多重蛋白质检测的应用。缀合基于应变促进的炔-叠氮化物环加成（无Cu点击反应），其中抗体用二苯并环辛炔（DBCO）部分活化，随后与叠氮化物修饰的寡核苷酸共价连接。在功能测试中，优化了反应条件和纯化工艺，以达到最大产率和最佳性能。OER测定采用针对每种蛋白质靶标开发的一对抗体结合剂（两种抗体，每种抗体与其自身的寡核苷酸缀合）。这两种寡核苷酸在其3 '末端含有独特的六碱基互补区域，以允许通过DNA合成酶退火和延伸以形成DNA模板。预扩增后，通过qPCR检测DNA模板。将不同的寡核苷酸序列分配给不同的抗体结合剂，以实现多重蛋白质检测。当使用重组蛋白进行测试时，一些抗体结合剂，如对CSTB、MET、EpCAM和CASP 3特异的那些，具有5 - 6个对数的动态范围。抗体结合剂也以多重形式用于OER测定，并且结合剂成功地检测了细胞裂解物中以及与C1系统组合的单细胞中的蛋白质靶标。这种基于点击反应的抗体缀合程序具有成本效益，需要最少的动手时间，并且非常适合开发负担得起的多重蛋白质测定，这提供了加速蛋白质组学研究的潜力。
The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this methods application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and is well-suited for the development of affordable multiplex protein assays, which provides the potential to accelerate proteomics research.