A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.
A covalent and cleavable antibody-DNA conjugation strategy for sensitive protein detection via immuno-PCR.
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DOI：
10.1038/srep22675
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发表时间：
2016-03-07
影响因子：
4.6
通讯作者：
Mulder KW
中科院分区：
文献类型：
--
作者：
van Buggenum JA;Gerlach JP;Eising S;Schoonen L;van Eijl RA;Tanis SE;Hogeweg M;Hubner NC;van Hest JC;Bonger KM;Mulder KW
免疫PCR通过使用抗体-DNA缀合物将基于特异性抗体的蛋白质检测与基于PCR的定量灵敏度结合起来。这种缀合物的生产取决于两种生物分子的快速有效缀合策略的可用性。在这里，我们提出了一种方法来生产可切割的抗体-DNA缀合物，采用四嗪和反式环辛烯（TCO）之间的逆电子需求狄尔斯-阿尔德反应的快速动力学。我们的战略包括三个步骤。首先，用化学可裂解的NHS-s-s-四嗪官能化抗体。随后，通过酶促添加N3-dATP并偶联至反式-环辛烯-PEG 12-二苯并环辛炔（TCO-PEG 12-DBCO），用TCO官能化双链DNA。最后，通过以1：2的低摩尔比混合官能化抗体和dsDNA快速且有效地获得缀合物。此外，引入化学可裂解的二硫化物接头有助于免疫染色后dsDNA的释放和灵敏检测。我们在免疫PCR中显示了对人表皮干细胞标志物ITGA 6和ITGB 1以及分化标志物转氨酶1（TGM 1）的特异性和敏感性蛋白检测。我们预计，化学裂解的抗体-DNA结合物的生产将为多重免疫PCR实验和免疫测序方法的发展提供坚实的基础。
Immuno-PCR combines specific antibody-based protein detection with the sensitivity of PCR-based quantification through the use of antibody-DNA conjugates. The production of such conjugates depends on the availability of quick and efficient conjugation strategies for the two biomolecules. Here, we present an approach to produce cleavable antibody-DNA conjugates, employing the fast kinetics of the inverse electron-demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO). Our strategy consists of three steps. First, antibodies are functionalized with chemically cleavable NHS-s-s-tetrazine. Subsequently, double-stranded DNA is functionalized with TCO by enzymatic addition of N3-dATP and coupling to trans-Cyclooctene-PEG12-Dibenzocyclooctyne (TCO-PEG12-DBCO). Finally, conjugates are quickly and efficiently obtained by mixing the functionalized antibodies and dsDNA at low molar ratios of 1:2. In addition, introduction of a chemically cleavable disulphide linker facilitates release and sensitive detection of the dsDNA after immuno-staining. We show specific and sensitive protein detection in immuno-PCR for human epidermal stem cell markers, ITGA6 and ITGB1, and the differentiation marker Transglutaminase 1 (TGM1). We anticipate that the production of chemically cleavable antibody-DNA conjugates will provide a solid basis for the development of multiplexed immuno-PCR experiments and immuno-sequencing methodologies.