title: Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast.
abstract: We have developed a technology for rapidly generating novel and fully human antibodies by simply using the antigen DNA. A human single-chain variable fragment (scFv) antibody library was constructed in a yeast two-hybrid vector with high complexity. After cloning cDNA encoding the mature sequence of human interleukin-8 (hIL8) into the yeast two-hybrid system vector, we have screened the human scFv antibody library and obtained three distinct scFv clones that could specifically bind to hIL8. One clone was chosen for further improvement by a novel affinity maturation process using the error-prone PCR of the scFv sequence followed by additional rounds of yeast two-hybrid screening. The scFv antibodies of both primary and affinity-matured scFv clones were expressed in E. coli. All purified scFvs showed specific binding to hIL8 in reciprocal coimmunoprecipitation and ELISA assays. All scFvs, as well as a fully human IgG antibody converted from one of the scFv clones and expressed in the mammalian cells, were able to effectively inhibit hIL8 in neutrophil chemotaxis assays. The technology described can generate fully human antibodies with high efficiency and low cost.
authors: Department of Medical Oncology, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
authors: Ling
authors: L
authors: Ding
authors: Mark
authors: M
authors: Azam
authors: Yu-Huei
authors: YH
authors: Lin
authors: James
authors: J
authors: Sheridan
authors: Shuanghong
authors: S
authors: Wei
authors: Gigi
authors: G
authors: Gupta
authors: Rakesh K
authors: RK
authors: Singh
authors: Michelle H
authors: MH
authors: Pauling
authors: Waihei
authors: W
authors: Chu
authors: Antares
authors: A
authors: Tran
authors: Nai-Xuan
authors: NX
authors: Yu
authors: Jiefeng
authors: J
authors: Hu
authors: Wei
authors: W
authors: Wang
authors: Hao
authors: H
authors: Long
authors: Dong
authors: D
authors: Xiang
authors: Li
authors: L
authors: Zhu
authors: Shao-Bing
authors: SB
authors: Hua
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D000915:Antibody Affinity / Q000276:immunology; D002465:Cell Movement / Q000187:drug effects; D002634:Chemotaxis, Leukocyte / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D019151:Peptide Library; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D016209:Interleukin-8; D019151:Peptide Library; D057127:Single-Chain Antibodies