Miyoshi, TakushiZhang, QianliMiyake, TakafumiWatanabe, ShinOhnishi, HiroeChen, JijiVishwasrao, Harshad D.Chakraborty, OisorjoBelyantseva, Inna A.Perrin, Benjamin J.Shroff, HariFriedman, Thomas B.Omori, KoichiWatanabe, Naoki2022-05-202022-05-202021-02-02Miyoshi T, Zhang Q, Miyake T, et al. Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures. Cell Rep. 2021;34(5):108708. doi:10.1016/j.celrep.2021.108708https://hdl.handle.net/1805/29106Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.en-USPublisher PolicyF-actin turnoverFab fragment probesTIRF microscopydiSPIMEspinFast-dissociating antibodyHair cellsLight-sheet microscopySingle-molecule microscopyStereociliaSuper-resolution microscopyArticle