Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast

Ling Ding, Mark Azam, Yu Huei Lin, James Sheridan, Shuanghong Wei, Gigi Gupta, Rakesh K. Singh, Michelle H. Pauling, Waihei Chu, Antares Tran, Nai Xuan Yu, Jiefeng Hu, Wei Wang, Hao Long, Dong Xiang, Li Zhu, Shao Bing Hua

Research output: Contribution to journalArticle

1 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1957-1966
Number of pages10
JournalProtein Science
Volume19
Issue number10
DOIs
StatePublished - Oct 2010

Keywords

  • Affinity maturation
  • Antibody library
  • Human scFv
  • Interleukin-8
  • Two-hybrid

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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    Ding, L., Azam, M., Lin, Y. H., Sheridan, J., Wei, S., Gupta, G., Singh, R. K., Pauling, M. H., Chu, W., Tran, A., Yu, N. X., Hu, J., Wang, W., Long, H., Xiang, D., Zhu, L., & Hua, S. B. (2010). Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast. Protein Science, 19(10), 1957-1966. https://doi.org/10.1002/pro.484