High-throughput affinity profiling of antibodies against soluble and native membrane targets by digital flow cytometry
- 1University of Washington
Abstract
Binding affinity measurements are essential for optimizing and validating therapeutic and research antibodies, yet no high-throughput platform supports quantitative affinity measurements for both soluble and native membrane targets. Here we present a suite of digital flow cytometry (dFC) assays that determine equilibrium binding affinities by directly counting free and bound molecules in homogeneous mixtures without immobilization. The assays enable titration-free absolute KD measurements for fluorescent binding partners, affinity measurements for unlabeled antibodies against membrane receptors on cell-derived extracellular vesicles (EVs), and multiplexed assays for rapid ranking or screening of antibody variants. We demonstrate applications by quantifying the effects of degree-of-labeling on the affinity of antibody-dye conjugates, and of drug conjugation on therapeutic antibody binding to receptors on human cancer EVs. By combining single-molecule quantitation, adaptable assay designs, and high-throughput analysis, dFC provides a versatile platform for therapeutic antibody optimization and research antibody validation.
Related
- Protocol for FLOWMIST to profile autoreactivity of mouse monoclonal and serum antibodies by flow cytometry and microscopy
- Spectral flow cytometry for high-dimensional immune profiling: from technological advances to multimodal precision immunology
- High-Throughput Plasma Proteomic Profiling Identifies Novel Biomarker Candidates in Parkinson’s Disease with Cognitive Decline
- From background to foreground: secondary antibodies coupled to lipophilic ATTO dyes enable high-density membrane labeling in super-resolution and expansion microscopy
- Robust High-Throughput Flickering Spectroscopy for Measurements of Red Blood Cell Membrane Mechanics
BibTeX
@misc{cheng2026highthroughput,
title = {{High-throughput affinity profiling of antibodies against soluble and native membrane targets by digital flow cytometry}},
author = {Y. Cheng and S. Barcy and B. Fujimoto and W. Nelson and M. Zhao and D. T. Chiu},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.15.759441},
url = {https://www.biorxiv.org/content/10.64898/2026.10.15.759441v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - High-throughput affinity profiling of antibodies against soluble and native membrane targets by digital flow cytometry
AU - Y. Cheng
AU - S. Barcy
AU - B. Fujimoto
AU - W. Nelson
AU - M. Zhao
AU - D. T. Chiu
PY - 2026
DA - 2026/10/15
PB - bioRxiv
DO - 10.64898/2026.10.15.759441
UR - https://www.biorxiv.org/content/10.64898/2026.10.15.759441v1
AB - Binding affinity measurements are essential for optimizing and validating therapeutic and research antibodies, yet no high-throughput platform supports quantitative affinity measurements for both soluble and native membrane targets. Here we present a suite of digital flow cytometry (dFC) assays that determine equilibrium binding affinities by directly counting free and bound molecules in homogeneous mixtures without immobilization. The assays enable titration-free absolute KD measurements for fluorescent binding partners, affinity measurements for unlabeled antibodies against membrane receptors on cell-derived extracellular vesicles (EVs), and multiplexed assays for rapid ranking or screening of antibody variants. We demonstrate applications by quantifying the effects of degree-of-labeling on the affinity of antibody-dye conjugates, and of drug conjugation on therapeutic antibody binding to receptors on human cancer EVs. By combining single-molecule quantitation, adaptable assay designs, and high-throughput analysis, dFC provides a versatile platform for therapeutic antibody optimization and research antibody validation.
SN - 2692-8205
ER -