Isolating and engineering human antibodies using yeast surface display

被引：628

作者：

Chao, Ginger ^{[1
]}

Lau, Wai L. ^{[1
]}

Hackel, Benjamin J. ^{[1
]}

Sazinsky, Stephen L. ^{[1
]}

Lippow, Shaun M. ^{[1
]}

Wittrup, K. Dane ^{[1
]}

机构：

[1] MIT, Dept Chem Engn, Biol Engn Div, Cambridge, MA 02139 USA

来源：

NATURE PROTOCOLS | 2006年 / 1卷 / 02期

关键词：

D O I：

10.1038/nprot.2006.94

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

This protocol describes the process of isolating and engineering antibodies or proteins for increased affinity and stability using yeast surface display. Single-chain antibody fragments (scFvs) are first isolated from an existing nonimmune human library displayed on the yeast surface using magnetic-activated cell sorting selection followed by selection using flow cytometry. This enriched population is then mutagenized, and successive rounds of random mutagenesis and flow cytometry selection are done to attain desired scFv properties through directed evolution. Labeling strategies for weakly binding scFvs are also described, as well as procedures for characterizing and 'titrating' scFv clones displayed on yeast. The ultimate result of following this protocol is a panel of scFvs with increased stability and affinity for an antigen of interest.

引用

页码：755 / 768

页数：14

共 31 条

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