Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology , 4259 Nagatsuta-cho, Midori-ku, Yokohama, Kanagawa 226-8503, Japan.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Anal Chem. 2017 Oct 17;89(20):10783-10789. doi: 10.1021/acs.analchem.7b02047. Epub 2017 Oct 3.
Claudins (CLs) are membrane proteins found in tight junctions and play a major role in establishing the intercellular barrier. However, some CLs are abnormally overexpressed on tumor cells and are valid clinical biomarkers for cancer diagnosis. Here, we constructed antibody Fab fragment-based Quenchbodies (Q-bodies) as effective and reliable fluorescent sensors for detecting and visualizing CLs on live tumor cells. The variable region genes for anti-CL1 and anti-CL4 antibodies were used to express recombinant Fab fragments, and clones recognizing CL4 with high affinity were selected for making Q-bodies. When two fluorescent dyes were conjugated to the N-terminal tags attached to the Fab, the fluorescent signal was significantly increased after adding nanomolar-levels of purified CL4. Moreover, addition of the Q-body to CL4-expressing cells including CL4-positive cancer cells led to a clear fluorescence signal with low background, even without washing steps. Our findings suggested that such Q-bodies would serve as a potent tool for specifically illuminating membrane targets expressed on cancer cells, both in vitro and in vivo.
紧密连接蛋白（CLs）是存在于紧密连接中的膜蛋白，在建立细胞间屏障方面发挥着重要作用。然而，一些 CLs 在肿瘤细胞上异常过表达，是癌症诊断的有效临床生物标志物。在这里，我们构建了基于抗体 Fab 片段的 Quenchbody（Q-body），作为用于检测和可视化活肿瘤细胞上 CLs 的有效且可靠的荧光传感器。使用针对 CL1 和 CL4 抗体的可变区基因来表达重组 Fab 片段，并选择对 CL4 具有高亲和力的克隆来制备 Q-body。当将两种荧光染料缀合到附着在 Fab 上的 N 端标签上时，加入纳摩尔级别的纯化 CL4 后，荧光信号显著增加。此外，将 Q-body 添加到表达 CL4 的细胞（包括 CL4 阳性癌细胞）中，即使没有洗涤步骤，也会导致清晰的荧光信号和低背景。我们的研究结果表明，这种 Q-body 将成为一种有力的工具，可用于特异性地照亮癌细胞上表达的膜靶标，无论是在体外还是体内。
