Min Junseon, Song Eun Kyung, Kim Hansol, Kim Kyoung Taek, Park Tae Joo, Kang Sebyung
Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 689-798, Korea.
Department of Chemistry, Seoul National University, Seoul, 151-747, Korea.
Sci Rep. 2016 Apr 11;6:24159. doi: 10.1038/srep24159.
We construct a novel recombinant secondary antibody mimic, GST-ABD, which can bind to the Fc regions of target-bound primary antibodies and acquire multiple HRPs simultaneously. We produce it in tenth of mg quantities with a bacterial overexpression system and simple purification procedures, significantly reducing the manufacturing cost and time without the use of animals. GST-ABD is effectively conjugated with 3 HRPs per molecule on an average and selectively bind to the Fc region of primary antibodies derived from three different species (mouse, rabbit, and rat). HRP-conjugated GST-ABD (HRP-GST-ABD) is successfully used as an alternative to secondary antibodies to amplify target-specific signals in both ELISA and immunohistochemistry regardless of the target molecules and origin of primary antibodies used. GST-ABD also successfully serves as an anchoring adaptor on the surface of GSH-coated plates for immobilizing antigen-capturing antibodies in an orientation-controlled manner for sandwich-type indirect ELISA through simple molecular recognition without any complicated chemical modification.
我们构建了一种新型重组二级抗体模拟物GST-ABD，它可以与靶标结合的一级抗体的Fc区域结合，并同时获得多个辣根过氧化物酶（HRP）。我们通过细菌过表达系统和简单的纯化程序以毫克数量的十分之一来生产它，在不使用动物的情况下显著降低了制造成本和时间。GST-ABD平均每个分子可有效结合3个HRP，并选择性地结合源自三种不同物种（小鼠、兔子和大鼠）的一级抗体的Fc区域。无论所用一级抗体的靶标分子和来源如何，HRP偶联的GST-ABD（HRP-GST-ABD）都成功用作二级抗体的替代品，以在酶联免疫吸附测定（ELISA）和免疫组织化学中放大靶标特异性信号。GST-ABD还成功地作为谷胱甘肽（GSH）包被板表面的锚定衔接子，通过简单的分子识别以定向控制的方式固定抗原捕获抗体，用于夹心型间接ELISA，而无需任何复杂的化学修饰。
