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Updated: Nov 25, 2025

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

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Methods to Validate Binding and Kinetics of "Proximity-Inducing" Covalent Immune-Recruiting Molecules.

Eden Kapcan^1,2,3, Benjamin Lake^1,2,3, Zi Yang^1,2,3

¹McMaster Immunology Research Center (MIRC), McMaster University, Hamilton, Ontario, Canada.

Current Protocols in Chemical Biology

|December 16, 2020

Summary

This study introduces new biophysical and analytical methods, including biolayer interferometry and flow cytometry, to characterize covalent immune recruiters (CIRs) and their antibody interactions. These assays enable precise assessment of target selectivity and covalent labeling kinetics for advanced chemical biology research.

Keywords:

covalent antibody recruitment immune oncology molecular glues proximity-induced reaction synthetic immune modulation

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Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Published on: August 4, 2015

Area of Science:

Chemical Biology
Biophysical Chemistry
Immunology

Background:

Covalent inhibitors and chemoproteomic probes are crucial in translational research.
Characterizing their interactions requires robust biophysical and analytical methods.
Covalent Immune Recruiters (CIRs) are emerging tools in tumor immuno-oncology.

Purpose of the Study:

To describe biolayer interferometry (BLI), flow cytometry, and fluorescence assays for characterizing CIR:antibody interactions.
To enable assessment of target selectivity and differentiation between noncovalent binding and covalent adduct formation.
To provide methods for analyzing CIR-antibody binding and covalent-labeling kinetics.

Main Methods:

Biolayer interferometry (BLI) for 'on-probe' and 'in-solution' kinetic analysis of CIRs.
Fluorescence SDS-PAGE to monitor antibody labeling kinetics with CIRs.
Flow cytometry-based antibody-dependent cellular phagocytosis (ADCP) assay for target-specific immune activation and binding discernment.

Main Results:

Demonstrated high-sensitivity measurement of CIR labeling activity using BLI.
Successfully monitored antibody labeling with a fluorescent handle via SDS-PAGE.
Validated target-specific immune activation and differentiated binding from covalent adduct formation using ADCP assays.

Conclusions:

The described BLI, fluorescence SDS-PAGE, and flow cytometry methods provide robust characterization of covalent tagging technologies.
These assays are essential for assessing selectivity and kinetics of novel covalent inhibitors and probes.
The developed protocols support the advancement of CIR technology and similar small molecule or protein-based systems in chemical biology and immuno-oncology.