Scholarlink

<p>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 (K<sub>D</sub>) and receptor density within the same experiment.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

A novel label-free method to determine equilibrium dissociation constants of antibodies binding to cell surface proteins

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年1月27日

查看全文
  • Eilyn R. Lacy,
  • Rupesh Nanjunda,
  • Scott L. Klakamp,
  • Deborah Kwok,
  • Jennifer F. Nemeth,
  • Gordon D. Powers,
  • H. Hugo Caicedo,
  • Steven A. Jacobs

摘要

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.