Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures

Fast-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 t...

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Bibliographic Details
Published in:Cell reports (Cambridge) 2021-02, Vol.34 (5), p.108708-108708, Article 108708
Main Authors: Miyoshi, Takushi, Zhang, Qianli, Miyake, Takafumi, Watanabe, Shin, Ohnishi, Hiroe, Chen, Jiji, Vishwasrao, Harshad D., Chakraborty, Oisorjo, Belyantseva, Inna A., Perrin, Benjamin J., Shroff, Hari, Friedman, Thomas B., Omori, Koichi, Watanabe, Naoki
Format: Article
Language:English
Subjects:
Animals
Antibodies - metabolism
Cell Culture Techniques
diSPIM
espin
F-actin turnover
Fab fragment probes
fast-dissociating antibody
hair cells
Humans
Hybridomas - metabolism
light-sheet microscopy
Mice
Microscopy - methods
Single Molecule Imaging - methods
single-molecule microscopy
stereocilia
super-resolution microscopy
TIRF microscopy
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Summary:Fast-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. [Display omitted] •Antibody screening assay is developed using semi-automated single-molecule microscopy•Pipeline is established to identify fast-dissociating, highly specific antibodies•Fast-dissociating Fab probes are useful tools for multiplex super-resolution imaging Based on single-molecule microscopy of antibody-antigen interaction, Miyoshi et al. demonstrate that fast dissociation can be a property of highly specific antibodies. Fab probes synthesized from these antibodies are useful imaging probes for multiplex super-resolution microscopy and could detect rapid turnover of actin crosslinkers in dense F-actin cores of stereocilia.
ISSN:2211-1247
2211-1247
DOI:10.1016/j.celrep.2021.108708