Proceedings for Annual Meeting of The Japanese Pharmacological Society
Online ISSN : 2435-4953
The 96th Annual Meeting of the Japanese Pharmacological Society
Session ID : 96_2-B-SS02-6
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Development of fast-dissociating recombinant antibody probes for multiplexed super-resolution molecular mapping
*Qianli ZhangAkitoshi MiyamotoTakao ArimoriJunichi TakagiNaoki Watanabe
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CONFERENCE PROCEEDINGS OPEN ACCESS

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Abstract

We report a mutagenesis strategy that can effectively increase the dissociation rate of antibodies by orders of magnitude without compromising the binding specificity. Single-molecule localization microscopy greatly surpasses the diffraction limit of conventional optical microscopy. The imaging fidelity and labelling density, however, are limited by spatial interference between bulky antibodies in a confined resolved area. IRIS has overcome the problem using exchangeable probes that transiently bind to endogenous targets. In our previous research, generation of fast-dissociating IRIS probes has been challenging. In the present study, we have developed a new mutagenesis strategy that make it feasible to generate IRIS probes from the repository of off-the-shelf antibodies. We successfully generated dozens of IRIS probes and demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections. Our fast-dissociating probes achieved 4-fold higher label density than conventional super-resolution approaches. Thus, IRIS could visualize the feature of synaptic components with higher fidelity. In addition, the mutagenesis strategy will provide more applications for high affinity antibodies developed in pharmaceutical research, such as super-resolution imaging based disease diagnosis and biomarker identification.

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