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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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-01-01
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| Series: | Scientific Reports |
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| Online Access: | https://doi.org/10.1038/s41598-024-82288-9 |
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| author | Eilyn R. Lacy Rupesh Nanjunda Scott L. Klakamp Deborah Kwok Jennifer F. Nemeth Gordon D. Powers H. Hugo Caicedo Steven A. Jacobs |
| author_facet | Eilyn R. Lacy Rupesh Nanjunda Scott L. Klakamp Deborah Kwok Jennifer F. Nemeth Gordon D. Powers H. Hugo Caicedo Steven A. Jacobs |
| author_sort | Eilyn R. Lacy |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-73671aa4ce83428f8cb5c158d1ce3094 |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-73671aa4ce83428f8cb5c158d1ce30942025-02-02T12:22:11ZengNature PortfolioScientific Reports2045-23222025-01-0115111510.1038/s41598-024-82288-9A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteinsEilyn R. Lacy0Rupesh Nanjunda1Scott L. Klakamp2Deborah Kwok3Jennifer F. Nemeth4Gordon D. Powers5H. Hugo Caicedo6Steven A. Jacobs7Johnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryJohnson & Johnson, Therapeutics DiscoveryAbstract 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.https://doi.org/10.1038/s41598-024-82288-9AffinityCell affinitySPRMSDMSD-CATBispecific antibodies |
| spellingShingle | Eilyn R. Lacy Rupesh Nanjunda Scott L. Klakamp Deborah Kwok Jennifer F. Nemeth Gordon D. Powers H. Hugo Caicedo Steven A. Jacobs A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins Scientific Reports Affinity Cell affinity SPR MSD MSD-CAT Bispecific antibodies |
| title | A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins |
| title_full | A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins |
| title_fullStr | A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins |
| title_full_unstemmed | A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins |
| title_short | A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins |
| title_sort | novel label free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins |
| topic | Affinity Cell affinity SPR MSD MSD-CAT Bispecific antibodies |
| url | https://doi.org/10.1038/s41598-024-82288-9 |
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