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Development of a Quenchbody for the Detection and Imaging of the Cancer-Related Tight-Junction-Associated Membrane Protein Claudin

Jeong, Hee-Jin, Kawamura, Takuya, Iida, Manami, Kawahigashi, Yumi, Takigawa, Mutsumi, Ohmuro-Matsuyama, Yuki, Chung, Chan-I, Dong, Jinhua, Kondoh, Masuo, Ueda, Hiroshi
Analytical chemistry 2017 v.89 no.20 pp. 10783-10789
antibodies, biomarkers, clones, fluorescence, fluorescent dyes, genes, image analysis, membrane proteins, neoplasm cells, neoplasms, tight junctions, washing
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.