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Bendes A, Dahl L, Sakmar TP, Schwenk JM, Kotliar IB
Methods in molecular biology (Clifton, N.J.) 2937 (-) 189-199 [2025-07-02; online 2025-07-02]
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.
PubMed 40593421
DOI 10.1007/978-1-0716-4591-8_11
Crossref 10.1007/978-1-0716-4591-8_11