A1 Refereed original research article in a scientific journal
Multiplexed Affinity Characterization of Protein Binders Directly from a Crude Cell Lysate by Covalent Capture on Suspension Bead Arrays
Authors: Huovinen Tuomas, Lindenburg Laurens, Minter Ralph, Hollfelder Florian
Publisher: AMER CHEMICAL SOC
Publication year: 2021
Journal: Analytical Chemistry
Journal name in source: ANALYTICAL CHEMISTRY
Journal acronym: ANAL CHEM
Volume: 93
Issue: 4
First page : 2166
Last page: 2173
Number of pages: 8
ISSN: 0003-2700
DOI: https://doi.org/10.1021/acs.analchem.0c03992
Abstract
The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (K-d) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.
The precise determination of affinity and specificity is a crucial step in the development of new protein reagents for therapy and diagnostics. Paradoxically, the selection of protein binders, e.g., antibody fragments, from large combinatorial repertoires is a rapid process compared to the subsequent characterization of selected clones. Here we demonstrate the use of suspension bead arrays (SBA) in combination with flow cytometry to facilitate the post-selection analysis of binder affinities. The array is designed to capture the proteins of interest (POIs) covalently on the surface of superparamagnetic color-coded microbeads directly from expression cell lysate, based on SpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated by analyzing the affinities of a typical phage display output, i.e., clones consisting of single-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and 24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that the equilibrium dissociation constants (K-d) obtained from multiplexed SBA assays correlate well with experiments performed on a larger scale, while the antigen consumption was reduced >100-fold compared to the conventional 96-well plate format. Protein screening and characterization by SBAs is a rapid and reagent-saving analytical format for combinatorial protein engineering to address specificity maturation and cross-reactivity profiling of antibodies.
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