Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.
Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.
复制标题
DOI：
10.1038/srep11333
复制
发表时间：
2015-06-10
影响因子：
4.6
通讯作者：
Sawasaki T
中科院分区：
文献类型：
--
作者：
Takeda H;Ogasawara T;Ozawa T;Muraguchi A;Jih PJ;Morishita R;Uchigashima M;Watanabe M;Fujimoto T;Iwasaki T;Endo Y;Sawasaki T
G蛋白偶联受体（GPCRs）是重要的药物作用靶点之一，抗GPCRs单克隆抗体（mAb）是GPCRs功能分析的重要工具。然而，由于重组GPCR抗原的制备困难，以及缺乏有效的mAb筛选方法，因此开发GPCR特异性mAb非常困难。在这里，我们描述了一种新的方法，用于生产单克隆抗体对GPCR使用两个原始的方法，双层透析法和生物素化脂质体为基础的相互作用试验（BiLIA），这两个都是使用小麦无细胞蛋白质合成系统和脂质体技术开发的。采用双层透析法成功合成了各种GPCR，其质量和数量均满足免疫要求。为了筛选特异性单克隆抗体，我们设计了BiLIA检测抗体与脂质体上膜蛋白之间的相互作用。BiLIA防止GPCR变性，然后优选选择构象敏感性抗体。使用这种方法，我们成功地获得了针对DRD 1、GHSR、PTGER 1和T1R1的mAb。对于DRD 1 mAb，获得了36种小鼠mAb和6种兔mAb，其以高亲和力特异性识别天然DRD 1。其中，一半的mAb是构象敏感的mAb，并且两个mAb识别DRD1的胞外环2。这些结果表明，该方法可用于GPCR mAb的生产。
G-protein-coupled receptors (GPCRs) are one of the most important drug targets, and anti-GPCR monoclonal antibody (mAb) is an essential tool for functional analysis of GPCRs. However, it is very difficult to develop GPCR-specific mAbs due to difficulties in production of recombinant GPCR antigens, and lack of efficient mAb screening method. Here we describe a novel approach for the production of mAbs against GPCR using two original methods, bilayer-dialysis method and biotinylated liposome-based interaction assay (BiLIA), both of which are developed using wheat cell-free protein synthesis system and liposome technology. Using bilayer-dialysis method, various GPCRs were successfully synthesized with quality and quantity sufficient for immunization. For selection of specific mAb, we designed BiLIA that detects interaction between antibody and membrane protein on liposome. BiLIA prevented denaturation of GPCR, and then preferably selected conformation-sensitive antibodies. Using this approach, we successfully obtained mAbs against DRD1, GHSR, PTGER1 and T1R1. With respect to DRD1 mAb, 36 mouse mAbs and 6 rabbit mAbs were obtained which specifically recognized native DRD1 with high affinity. Among them, half of the mAbs were conformation-sensitive mAb, and two mAbs recognized extracellular loop 2 of DRD1. These results indicated that this approach is useful for GPCR mAb production.
登录
查看更多内容
影响因子：
6.4
作者：
Asada H;Uemura T;Yurugi-Kobayashi T;Shiroishi M;Shimamura T;Tsujimoto H;Ito K;Sugawara T;Nakane T;Nomura N;Murata T;Haga T;Iwata S;Kobayashi T
通讯作者：
Kobayashi T
影响因子：
64.8
作者：
Hino, Tomoya;Arakawa, Takatoshi;Iwanari, Hiroko;Yurugi-Kobayashi, Takami;Ikeda-Suno, Chiyo;Nakada-Nakura, Yoshiko;Kusano-Arai, Osamu;Weyand, Simone;Shimamura, Tatsuro;Nomura, Norimichi;Cameron, Alexander D.;Kobayashi, Takuya;Hamakubo, Takao;Iwata, So;Murata, Takeshi
通讯作者：
Murata, Takeshi
DOI：
10.1073/pnas.0804766105
发表时间：
2008-10-14
影响因子：
11.1
作者：
Kaiser, Liselotte;Graveland-Bikker, Johanna;Zhang, Shuguang
通讯作者：
Zhang, Shuguang
影响因子：
4.7
作者：
Miura, Eriko;Fukaya, Masahiro;Watanabe, Masahiko
通讯作者：
Watanabe, Masahiko
影响因子：
3.5
作者：
Nozawa A;Ogasawara T;Matsunaga S;Iwasaki T;Sawasaki T;Endo Y
通讯作者：
Endo Y