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.
Highlights
Zinc is an essential element for all cells and plays an important role in insulin secretion
Our study demonstrated that Zinc transporter 8 (ZnT8) as a novel autoantigen containing many immunodominant epitopes, which were recognized in type 1 diabetes patients (Xu et al, 2015)
From our phage display library, we can screen all antibodies of type 1 diabetes (T1D), which is valuable for further studies of antibodies in T1D
Summary
Zinc is an essential element for all cells and plays an important role in insulin secretion. The proteins of ZnT family play a role as zinc ion diffusion facilitators, which allow diffusion of zinc ion across biological membrane. Among ZnT family members, Chimienti et al first showed that ZnT8, encoded by the gene SLC30A8, was the most expressed transporter in hu-. The overexpression of ZnT8 in islet cells stimulates zinc accumulation and enhances glucose-stimulated insulin secretion compared with control cells (Chimienti et al, 2006). These findings showed that ZnT8 is essential for zinc accumulation and the storage, secretion, and the action of insulin
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