Screening and identification of human ZnT8-specific single-chain variable fragment (scFv) from type 1 diabetes phage display library.
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.
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Qian
Q
Wu
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Xiaodong
X
Wang
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Yong
Y
Gu
Key Laboratory of Antibody Technique, Ministry of Health, Nanjing Medical University, Nanjing, 210029, China.
Xiao
X
Zhang
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Yao
Y
Qin
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Heng
H
Chen
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Xinyu
X
Xu
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China.
Tao
T
Yang
Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, 210029, China. zhangmei@njmu.edu.cn.
Mei
M
Zhang
D001435:Bacteriophages / Q000235:genetics*; D027682:Cation Transport Proteins / Q000032:analysis* / Q000276:immunology; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*; D000075703:Zinc Transporter 8
D027682:Cation Transport Proteins; D011994:Recombinant Proteins; C492522:SLC30A8 protein, human; D057127:Single-Chain Antibodies; D000075703:Zinc Transporter 8
Zinc transporter 8 (ZnT8); phage display; single-chain variable fragment (scFv); type 1 diabetes (T1D)