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


ABSTRACT: 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.

SUBMITTER: Miyoshi T 

PROVIDER: S-EPMC7904085 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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

Miyoshi Takushi T   Zhang Qianli Q   Miyake Takafumi T   Watanabe Shin S   Ohnishi Hiroe H   Chen Jiji J   Vishwasrao Harshad D HD   Chakraborty Oisorjo O   Belyantseva Inna A IA   Perrin Benjamin J BJ   Shroff Hari H   Friedman Thomas B TB   Omori Koichi K   Watanabe Naoki N  

Cell reports 20210201 5


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 f  ...[more]

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