用于验证抗G蛋白偶联受体（GPCR）抗体的高度多重免疫测定法。

Highly Multiplexed Immunoassays for the Validation of Anti-GPCR Antibodies.

作者信息

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

机构信息

Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Solna, Sweden.

Laboratory of Chemical Biology and Signal Transduction, The Rockefeller University, New York, NY, USA.

出版信息

Methods Mol Biol. 2025;2937:189-199. doi: 10.1007/978-1-0716-4591-8_11.

DOI:10.1007/978-1-0716-4591-8_11

PMID:40593421

Abstract

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.

摘要

靶向G蛋白偶联受体（GPCR）的抗体是研究这一重要的细胞表面信号蛋白超家族生物学和药理学的重要工具。然而，产生抗GPCR抗体具有挑战性，部分原因是相关GPCR的序列同源性高。基于悬浮微珠阵列（SBA）构建的免疫测定可用于通过对靶标和非靶标结合进行多平行评估，来验证用于基于流式细胞术的检测应用的抗GPCR抗体。在本章中，我们描述了如何固定抗体文库以生成SBA。我们还描述了如何将工程化GPCR文库与多重SBA检测相结合，以验证与微珠结合的抗GPCR抗体的特异性。本文介绍的基于SBA的方法为抗体结合选择性和非靶标相互作用的多重表征，以及绘制参与抗体结合的GPCR表位提供了一种通用且强大的工具。