title: Antibody affinity maturation through combining display of two-chain paired antibody and precision flow cytometric sorting.
abstract: 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.
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.|University of Chinese Academy of Sciences, Beijing, 100039, China.
authors.forename: Shuang
authors.initials: S
authors.lastname: Sun
authors.affiliation: CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Yang
authors.affiliation: College of Life Sciences, North China University of Science and Technology, Tangshan, Hebei Province, 063000, China.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Wang
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Ye
authors.affiliation: CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Fang
authors.affiliation: Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. hh91@ibp.ac.cn.|Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. hh91@ibp.ac.cn.
authors.forename: Haiying
authors.initials: H
authors.lastname: Hang
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D005434:Flow Cytometry / Q000379:methods*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
chemical_list: D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains
keywords: Affinity maturation; Anchored periplasmic expression (APEx); Coiled coiled heterodimeric variable domain (ccFv); Flow cytometry; Precision sorting