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.
目的： 建立四跨膜蛋白7自身抗体（TSPAN7A）的荧光素酶免疫共沉淀检测技术（LIPS），并评估TSPAN7A在中国1型糖尿病（T1D）人群中的应用价值。 方法： 将插入人全长四跨膜蛋白7 cDNA的海肾荧光素酶重组质粒转染293T细胞，获得含四跨膜蛋白7与海肾荧光素酶融合蛋白的细胞裂解液。待测血清与该细胞裂解液孵育过夜后，用蛋白A-琼脂糖沉淀免疫复合物，洗涤，加入海肾荧光素酶底物并检测荧光读数，以199名健康对照的第99百分位数作为阳性阈值。采用LIPS法检测2014至2017年中南大学湘雅二医院代谢内分泌科就诊的100例T1D患者［男64例，女36例，年龄（28±16）岁］、119例2型糖尿病（T2D）患者［男78例，女41例，年龄（47±12）岁］以及98名健康对照［男55名，女43名，年龄（28±12）岁］血清中TSPAN7A水平。同时采用放射配体法（RLA）检测谷氨酸脱羧酶自身抗体（GADA）、蛋白酪氨酸磷酸酶自身抗体（IA-2A）、锌转运体8自身抗体（ZnT8A）水平，初步评估TSPAN7A的临床应用价值。 结果： 100例T1D患者中GADA、IA-2A、ZnT8A、TSPAN7A的阳性率分别为72.0%、40.0%、29.0%、25.0%，经Bonferroni法校正后，TSPAN7A阳性率低于GADA（P<0.001），而与IA-2A（P=0.035）和ZnT8A比较差异无统计学意义（P=0.630）。T1D患者TSPAN7A阳性率高于T2D的0.84%（1/119；25.0%比0.84%，P<0.001）和健康对照的1.02%（1/98；25.0%比1.02%，P<0.001）。在其他抗体基础上联合检测TSPAN7A可使T1D抗体阳性检出率由82.0%提高至85.0%。TSPAN7A阳性T1D患者与其他3种胰岛自身抗体阳性者临床特征差异均无统计学意义（均P>0.05）。 结论： 本研究成功建立了TSPAN7A的荧光素酶免疫共沉淀检测技术，TSPAN7A是中国T1D人群的一种新型胰岛抗体。.