Protocol to generate fast-dissociating recombinant antibody fragments for multiplexed super-resolution microscopy

Area of Science:

• Biotechnology
• Microscopy
• Molecular Biology

Background:

• Multiplexed high-density label super-resolution microscopy, such as imaging by integrating exchangeable single-molecule localization (IRIS), is crucial for visualizing cellular structures.
• IRIS requires fast-dissociating binders for precise molecular localization.

Purpose of the Study:

• To develop a protocol for generating antibody-based probes for IRIS microscopy.
• To accelerate binder dissociation rates while maintaining specificity for improved super-resolution imaging.

Main Methods:

• Retrieving antibody sequences from public databases.
• Performing site-directed mutagenesis at the base of complementarity-determining region loops.
• Constructing, purifying, and evaluating recombinant antibody probes.

Main Results:

• Successfully generated antibody-based IRIS probes from existing sequences.
• Achieved accelerated dissociation rates of the probes.
• Maintained high binding specificity of the generated probes.

Conclusions:

• The developed protocol provides a method for creating effective antibody-based IRIS probes.
• This advancement facilitates enhanced resolution and molecular distribution analysis in cellular and tissue imaging.