The establishment and application of testing methods of tetraspanin 7 autoantibody in type 1 diabetes

Abstract

Objective: To establish the luciferase immunoprecipitation system assay (LIPS) to test tetraspanin 7 autoantibody (TSPAN7A) and evaluate its value in Chinese type 1 diabetes (T1D) patients. Methods: Renilla luciferase-tagged TSPAN7 plasmids were transfected into 293T cells to obtain Renilla luciferase-tagged TSPAN7 fusion protein. The cell lysate was incubated with sera overnight, followed by addition of protein A-agarose and extensive wash. Finally, the substrate of Renilla luciferase was added and luminescence was detected. Sera from 100 T1D patients [64 males and 36 females,with a mean age of (28±16) years], 119 type 2 diabetes (T2D) patients [78 males and 41 females,with a mean age of (47±12) years] and 98 healthy volunteers [55 males and 43 females,with a mean age of (28±12) years] from the Department of Metabolism and Endocrinology of the Second Xiangya Hospital, Central South University from 2014 to 2017, were tested by LIPS to evaluate the frequency of TSPAN7A. Radioligand binding assay (RLA) was used to test glutamic acid decarboxylase autoantibodies (GADA), protein tyrosine phosphatase-2 autoantibodies (IA-2A) and zinc transporter 8 autoantibodies (ZnT8A). Results: The frequencies of GADA, IA-2A, ZnT8A and TSPAN7A in T1D patients were 72.0%, 40.0%, 29.0% and 25.0%, respectively. After Bonferroni correction, the positivity of TSPAN7A was lower than GADA (P<0.001), but similar with IA-2A (P=0.035) and ZnT8A (P=0.630). The positivity of TSPAN7A in T1D patients was significantly higher than that in T2D (0.84%, P<0.001) and in healthy controls (1.02%, P<0.001). In combination with TSPAN7A, the positivity of islet autoantibodies in T1D patients increased from 82% to 85%. There was no significant difference in clinical characteristics between TSPAN7A-positive T1D and the other three islet autoantibodies-positive patients. Conclusion: This study succeeded in establishing LIPS method to assay TSPAN7A. Moreover, TSPAN7A are valid islet autoantibodies for T1D patients in China.