Yun Ling1, Pengjun Jiang2, Nan Li3, Qianhua Yan3, Xin Wang4. 1. Department of Laboratory Medicine, The First Affiliated Hospital of Nanjing Medical University, 210029 Nanjing, China. 2. Department of Hematology, Jiangsu Province Hospital of Traditional Chinese Medicine, The Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, 210029 Nanjing, China. 3. Department of Endocrinology, Jiangsu Province Hospital of Traditional Chinese Medicine, The Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, 210029 Nanjing, China. 4. Department of Endocrinology, Jiangsu Province Hospital of Traditional Chinese Medicine, The Affiliated Hospital of Nanjing University of Traditional Chinese Medicine, 210029 Nanjing, China. Electronic address: njmuwangxin@163.com.
Abstract
AIM: Luciferase immunoprecipitation (LIPS) assays show good sensitivity and specificity in testing islet specific autoantibodies for diagnosis of type 1 diabetes (T1D). However, there are currently no LIPS assays available for detecting proinsulin/insulin autoantibody (IAA) previously. We here developed a LIPS assay to measure IAA using nano luciferase (NanoLuc)-proinsulin fusion protein. METHODS: The NanoLuc-proinsulin fusion protein expression plasmid was constructed and transfected to COS1 cells. Expression and binding specificity to IAA of the fusion protein were validated. A LIPS assay using the NL-proinsulin fusion protein was developed and compared to radioimmunoassay (RIPA) in testing sera from 50 healthy controls and 34 T1D patients. RESULTS: The fusion protein was correctly expressed in transfected COS1 cells. Both NANOLUC activity and proinsulin were detected in the medium of transfected COS1 cells. Fusion protein bound to monoclonal anti insulin antibody and sera from T1D patients or NOD mice, these bindings were inhibited by recombinant human insulin. The LIPS assay using the fusion protein showed sensitivity of 47.1% and specificity of 98.0%. Further analysis supported correlation between the IAA indexes of all the T1D samples detected by LIPS assay and radioimmunoassay (RIPA, R2 = 0.6132, p < 0.0001). CONCLUSIONS: The NanoLuc-proinsulin fusion protein based LIPS has the potential to detect IAA for diagnosis of T1D.
AIM: Luciferase immunoprecipitation (LIPS) assays show good sensitivity and specificity in testing islet specific autoantibodies for diagnosis of type 1 diabetes (T1D). However, there are currently no LIPS assays available for detecting proinsulin/insulin autoantibody (IAA) previously. We here developed a LIPS assay to measure IAA using nano luciferase (NanoLuc)-proinsulin fusion protein. METHODS: The NanoLuc-proinsulin fusion protein expression plasmid was constructed and transfected to COS1 cells. Expression and binding specificity to IAA of the fusion protein were validated. A LIPS assay using the NL-proinsulin fusion protein was developed and compared to radioimmunoassay (RIPA) in testing sera from 50 healthy controls and 34 T1D patients. RESULTS: The fusion protein was correctly expressed in transfected COS1 cells. Both NANOLUC activity and proinsulin were detected in the medium of transfected COS1 cells. Fusion protein bound to monoclonal anti insulin antibody and sera from T1D patients or NOD mice, these bindings were inhibited by recombinant humaninsulin. The LIPS assay using the fusion protein showed sensitivity of 47.1% and specificity of 98.0%. Further analysis supported correlation between the IAA indexes of all the T1D samples detected by LIPS assay and radioimmunoassay (RIPA, R2 = 0.6132, p < 0.0001). CONCLUSIONS: The NanoLuc-proinsulin fusion protein based LIPS has the potential to detect IAA for diagnosis of T1D.
Authors: Sarah M Graff; Stephanie R Johnson; Paul J Leo; Prasanna K Dadi; Matthew T Dickerson; Arya Y Nakhe; Aideen M McInerney-Leo; Mhairi Marshall; Karolina E Zaborska; Charles M Schaub; Matthew A Brown; David A Jacobson; Emma L Duncan Journal: JCI Insight Date: 2021-07-08