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	Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting
	Sun, Shuang1,2; Yang, Xiao3; Wang, Haifeng4; Zhao, Yun1; Lin, Yan1; Ye, Chen1; Fang, Xiangdong3; Hang, Haiying1,5
刊名	Applied microbiology and biotechnology
	2016-07-01
卷号	100期号:13页码:5977-5988
关键词	Coiled coiled heterodimeric variable domain (ccfv) Anchored periplasmic expression (apex) Flow cytometry Affinity maturation Precision sorting
ISSN号	0175-7598
DOI	10.1007/s00253-016-7472-1
通讯作者	Hang, haiying(hh91@ibp.ac.cn)
英文摘要	Recombination of antibody light and heavy chain libraries greatly increases the size of a two-chain paired antibody library, thus easing the construction of large antibody libraries. here, light and heavy chain variable domains paired by a coiled coil were applied to a bacterial inner membrane display system. however, the probability of the correct pairing of light and heavy chains through random recombination after each round of flow cytometric sorting and cloning was very low in the presence of mostly unmatched light and heavy chain genes, resulting in inefficient enrichment; a target antibody clone in the ratio of 1:100,000 negative control spheroplasts was unable to be enriched by six rounds of sorting and cloning by a conventional sorting strategy (sorting the top 1 %). by just sorting the top 0.000025 % of spheroplasts, we succeeded in enriching the target antibody clone mixed with negative control spheroplasts in a ratio of 1:10(8) by just one round of sorting and cloning. furthermore, using this gating strategy, we efficiently enriched for an antibody clone with an affinity slightly better than the parent antibody clone from mixed spheroplasts which were present in the ratio of 1 better affinity clone to 10 parent clones to 10(6) negative control clones after just two rounds of sorting and cloning, suggesting that this gating strategy is highly sensitive in distinguishing between clones with a small difference in affinity and also enriching for clones with a higher affinity. taken together, the combination of the display of a two-chain paired antibody library and the use of stringent gating has significantly increased the efficiency of the antibody maturation system.
WOS关键词	FUNCTIONAL GABA(B) RECEPTOR ; YEAST-CELL-SURFACE ; ESCHERICHIA-COLI ; RIBOSOME DISPLAY ; MAMMALIAN-CELLS ; SCFV ANTIBODIES ; LIBRARY ; SELECTION ; DNA ; ELECTROTRANSFORMATION
WOS研究方向	Biotechnology & Applied Microbiology
WOS类目	Biotechnology & Applied Microbiology
语种	英语
出版者	SPRINGER
WOS记录号	WOS:000378725700027
内容类型	期刊论文
URI标识	http://www.corc.org.cn/handle/1471x/2374639
专题	中国科学院大学
通讯作者	Hang, Haiying
作者单位	1.Chinese Acad Sci, Inst Biophys, Key Lab Prot & Peptide Pharmaceut, Natl Lab Biomacromol, Beijing 100101, Peoples R China 2.Univ Chinese Acad Sci, Beijing 100039, Peoples R China 3.Chinese Acad Sci, Beijing Inst Genom, CAS Key Lab Genome Sci & Informat, Beijing 100101, Peoples R China 4.North China Univ Sci & Technol, Coll Life Sci, Tangshan 063000, Hebei Province, Peoples R China 5.Chinese Acad Sci, Inst Biophys, Beijing 100101, Peoples R China
推荐引用方式 GB/T 7714	Sun, Shuang,Yang, Xiao,Wang, Haifeng,et al. Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting[J]. Applied microbiology and biotechnology,2016,100(13):5977-5988.
APA	Sun, Shuang.,Yang, Xiao.,Wang, Haifeng.,Zhao, Yun.,Lin, Yan.,...&Hang, Haiying.(2016).Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.Applied microbiology and biotechnology,100(13),5977-5988.
MLA	Sun, Shuang,et al."Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting".Applied microbiology and biotechnology 100.13(2016):5977-5988.

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