Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Qian Wu; Xiaodong Wang; Yong Gu; Xiao Zhang; Yao Qin; Heng Chen; Xinyu Xu; Tao Yang; Mei Zhang

doi:10.1007/s11427-016-5077-7

Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library

Sci China Life Sci. 2016 Jul;59(7):686-93. doi: 10.1007/s11427-016-5077-7. Epub 2016 Jun 7.

Authors

Qian Wu¹, Xiaodong Wang¹, Yong Gu¹, Xiao Zhang², Yao Qin¹, Heng Chen¹, Xinyu Xu¹, Tao Yang¹, Mei Zhang³

Affiliations

¹ Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
² Key Laboratory of Antibody Technique, Ministry of Health, Nanjing Medical University, Nanjing, 210029, China.
³ Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China. zhangmei@njmu.edu.cn.

PMID: 27270580
DOI: 10.1007/s11427-016-5077-7

Abstract

Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1D was constructed and single-chain antibodies against ZnT8 were screened and identified. Human T1D single-chain variable fragment (scFv) phage display library consists of approximately 1×10(8) clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F' and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325Trp mutation. The specificity to human islet cells of these scFvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1D phage display antibody library and identified two ZnT8-specific scFv clones, C27 and C22. These ZnT8-specific scFvs are potential agents in immunodiagnostic and immunotherapy of T1D.

Keywords: Zinc transporter 8 (ZnT8); phage display; single-chain variable fragment (scFv); type 1 diabetes (T1D).

MeSH terms

Bacteriophages / genetics*
Cation Transport Proteins / analysis*
Cation Transport Proteins / immunology
Diabetes Mellitus, Type 1 / immunology*
Electrophoresis, Polyacrylamide Gel
Humans
Recombinant Proteins / analysis
Recombinant Proteins / immunology
Single-Chain Antibodies / genetics
Single-Chain Antibodies / immunology*
Zinc Transporter 8

Substances

Cation Transport Proteins
Recombinant Proteins
SLC30A8 protein, human
Single-Chain Antibodies
Zinc Transporter 8