Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.
Cell Rep
; 34(5): 108708, 2021 02 02.
Article
em En
| MEDLINE
| ID: mdl-33535030
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.
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Base de dados:
MEDLINE
Assunto principal:
Imagem Individual de Molécula
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Hibridomas
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Microscopia
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Anticorpos
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article