A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins
Abstract Solution-based affinity assays are used for the selection and characterization of proteins that could be developed into therapeutic molecules. However, these assays have limitations for cell-surface proteins as in most cases their purification requires detergent solubilization and are unlik...
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| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-01-01
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| Series: | Scientific Reports |
| Subjects: | |
| Online Access: | https://doi.org/10.1038/s41598-024-82288-9 |
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| Summary: | Abstract Solution-based affinity assays are used for the selection and characterization of proteins that could be developed into therapeutic molecules. However, these assays have limitations for cell-surface proteins as in most cases their purification requires detergent solubilization and are unlikely to assume conformations in solution that resemble their native states in cell membranes. This report describes a novel electrochemiluminescence-based method, called MSD-CAT, for the affinity analysis of antibodies binding to cell-surface receptors. MSD-CAT was used to evaluate the binding of monoclonal antibodies, Fab fragments, and bispecific antibodies targeting the cell-surface receptor interleukin 3 receptor alpha (CD123) and the results were compared to data obtained using surface plasmon resonance (SPR). The data showed that MSD-CAT can be successfully applied to determine binding affinity on cells in a label free format and without the need for laborious solubilization procedures to generate recombinant antigen. In addition, this method has the potential for high-throughput application while enabling simultaneous determination of equilibrium dissociation constant (KD) and receptor density within the same experiment. |
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| ISSN: | 2045-2322 |