{"title":"Phage Display Selection of Antibody Libraries: Screening of Selected Binders.","authors":"Haiyong Peng, Christoph Rader","doi":"10.1101/pdb.prot108603","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":10496,"journal":{"name":"Cold Spring Harbor protocols","volume":" ","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cold Spring Harbor protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1101/pdb.prot108603","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
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.
噬菌体展示选择抗体库是产生和进化单克隆抗体的一种有效方法。噬菌体展示载体 pComb3 phagemid 系列有助于从人类和非人类抗体库中挖掘 Fab 格式的抗体库。在这里，我们介绍了在筛选出与感兴趣的抗原结合的多克隆 Fab 结合体后筛选单克隆 Fab 结合体的方法，目的是鉴定和测序与抗原结合具有高亲和力和特异性的单克隆抗体。筛选步骤包括噬菌体酶联免疫吸附试验、粗Fab酶联免疫吸附试验、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、表达在真核细胞上的原生抗原或两者的噬菌体和粗Fab ELISA。
Cold Spring Harbor protocolsBiochemistry, Genetics and Molecular Biology-Biochemistry, Genetics and Molecular Biology (all)
CiteScore
3.00
自引率
0.00%
发文量
163
期刊介绍：
Cold Spring Harbor Laboratory is renowned for its teaching of biomedical research techniques. For decades, participants in its celebrated, hands-on courses and users of its laboratory manuals have gained access to the most authoritative and reliable methods in molecular and cellular biology. Now that access has moved online. Cold Spring Harbor Protocols is an interdisciplinary journal providing a definitive source of research methods in cell, developmental and molecular biology, genetics, bioinformatics, protein science, computational biology, immunology, neuroscience and imaging. Each monthly issue details multiple essential methods—a mix of cutting-edge and well-established techniques.
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