抗体文库的噬菌体展示筛选：对选定结合物的筛选。

Phage Display Selection of Antibody Libraries: Screening of Selected Binders.

作者信息

Peng Haiyong, Rader Christoph

机构信息

Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, Florida 33458, USA.

Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, University of Florida, Jupiter, Florida 33458, USA

出版信息

Cold Spring Harb Protoc. 2024 Oct 15. doi: 10.1101/pdb.prot108603.

DOI:10.1101/pdb.prot108603

PMID:39406527

Abstract

Phage display selection of antibody libraries is a powerful method for generating and evolving monoclonal antibodies. The pComb3 phagemid family of phage display vectors facilitates the mining of antibody libraries in Fab format from human and nonhuman antibody repertoires. Here, we describe the screening for monoclonal Fab binders after selection of a polyclonal pool of Fab binders to an antigen of interest, with the goal of identifying and sequencing monoclonal antibodies that bind the antigen with high affinity and specificity. The screening cascade involves a phage ELISA, followed by a crude Fab ELISA and DNA fingerprinting and sequencing. The protocol outlines phage and crude Fab ELISAs using purified antigen immobilized on microplates, native antigen expressed on eukaryotic cells, or both.

摘要

噬菌体展示抗体文库筛选是一种生成和进化单克隆抗体的强大方法。pComb3噬菌体展示载体的噬菌粒家族有助于从人源和非人源抗体库中挖掘Fab格式的抗体文库。在此，我们描述了在选择针对感兴趣抗原的多克隆Fab结合物池后筛选单克隆Fab结合物的方法，目的是鉴定和测序以高亲和力和特异性结合该抗原的单克隆抗体。筛选级联包括噬菌体ELISA，随后是粗制Fab ELISA以及DNA指纹图谱和测序。该方案概述了使用固定在微孔板上的纯化抗原、真核细胞上表达的天然抗原或两者进行噬菌体和粗制Fab ELISA的方法。