Fast-dissociating but highly specific antibodies are novel tools in biology, especially useful for multiplex super-resolution microscopy

STAR Protoc. 2021 Nov 18;2(4):100967. doi: 10.1016/j.xpro.2021.100967. eCollection 2021 Dec 17.


Fast-dissociating, highly specific monoclonal antibodies (FDSAs) are single-molecule imaging probes useful for many biological assays including consecutive, multiplexable super-resolution microscopy. We developed a screening assay to characterize the kinetics of antibody-antigen interactions using single-molecule microscopy and established a pipeline to identify FDSAs from thousands of monoclonal candidates. Provided here are detailed protocols to prepare multi-well glass-bottom plates necessary for our assay to identify hybridoma clones secreting FDSAs. Synthesis of fluorescently labeled Fab fragments (Fab probes) from FDSAs is also described. For complete details on the use and execution of this protocol, please refer to Miyoshi et al. (2021).

Keywords: Antibody; High Throughput Screening; Microscopy; Molecular/Chemical Probes; Protein Biochemistry; Single-molecule Assays.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal* / chemistry
  • Antibodies, Monoclonal* / metabolism
  • Female
  • Fluorescent Dyes* / chemistry
  • Fluorescent Dyes* / metabolism
  • Hybridomas / chemistry
  • Hybridomas / metabolism
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / metabolism
  • Mice
  • Mice, Inbred BALB C
  • Microscopy, Fluorescence / methods*
  • Single Molecule Imaging / methods*


  • Antibodies, Monoclonal
  • Fluorescent Dyes
  • Immunoglobulin Fab Fragments