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Antibodies targeting G protein-coupled receptors (GPCRs) are important tools to study the biology and pharmacology of this important superfamily of cell surface signaling proteins. However, generating anti-GPCR antibodies is challenging, partly due to the high sequence homology of related GPCRs. Immunoassays built on suspension bead arrays (SBAs) can be applied to validate anti-GPCR antibodies for flow-based assay applications by enabling a multi-parallel assessment of on-target and off-target binding. In this chapter, we describe how to immobilize an antibody library to generate SBAs. We further describe how a library of engineered GPCRs can be combined with the multiplexed SBA assay to validate the specificity of the bead-bound anti-GPCR antibodies. The SBA-based approach presented here offers a versatile and robust tool for multiplexed characterization of antibody binding selectivity and off-target interactions, as well as for mapping GPCR epitopes involved in antibody binding.

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Highly Multiplexed Immunoassays for the Validation of Anti-GPCR Antibodies

  • 实验方案

  • 发表日期：

    2025年

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  • Annika Bendes,
  • Leo Dahl,
  • Thomas P. Sakmar,
  • Jochen M. Schwenk,
  • Ilana B. Kotliar

摘要

Antibodies targeting G protein-coupled receptors (GPCRs) are important tools to study the biology and pharmacology of this important superfamily of cell surface signaling proteins. However, generating anti-GPCR antibodies is challenging, partly due to the high sequence homology of related GPCRs. Immunoassays built on suspension bead arrays (SBAs) can be applied to validate anti-GPCR antibodies for flow-based assay applications by enabling a multi-parallel assessment of on-target and off-target binding. In this chapter, we describe how to immobilize an antibody library to generate SBAs. We further describe how a library of engineered GPCRs can be combined with the multiplexed SBA assay to validate the specificity of the bead-bound anti-GPCR antibodies. The SBA-based approach presented here offers a versatile and robust tool for multiplexed characterization of antibody binding selectivity and off-target interactions, as well as for mapping GPCR epitopes involved in antibody binding.