电化学发光法检测胰岛β细胞自身抗体的初步临床应用

The preliminary clinical application of electrochemiluminescence assays for islet β cell autoantibodies

目的建立电化学发光法(ECL)用以检测胰岛自身抗体,设定阳性标准并探讨其在1型糖尿病(T1DM)的预测及诊断方面的意义。方法选取2016至2018年南京医科大学附属逸夫医院内分泌科就诊的糖尿病患者(360例)及来自南京医科大学的健康志愿者(124名)为研究对象,采用ECL法检测180例T1DM患者、180例2型糖尿病(T2DM)患者及124名健康志愿者血清中谷氨酸脱羧酶抗体(GADA65)、胰岛素自身抗体(IAA)、蛋白酪氨酸磷酸酶抗体(IA-2A)。结果用抗体指数来表示,并取健康志愿者各抗体指数的99%百分位数作为该抗体的阳性临界值。采用卡方检验分析ECL方法检测的T1DM及T2DM两组患者抗体阳性率。结果ECL法检测GADA65、IAA和IA-2A的阳性临界值分别为0.047、0.009和0.002 9。GADA65、IAA和IA-2A批内变异系数分别为1.88%~8.30%、1.39%~3.40%、1.23%~3.17%,批间变异系数分别为3.9%~7.3%、2.7%~9.8%、2.2%~7.4%,结果判断重复性为99.8%。用该方法检测TIDM患者GADA65、IAA、IA-2A的阳性率分别为52%(94/180)、45%(81/180)、21%(39/180),T2DM患者阳性率分别为GADA65 2.2%(4/180)、IAA 2.2%(4/180)、IA-2A 2.2%(4/180),T1DM患者及T2DM患者GADA65、IAA和IA-2A阳性率差异均有统计学意义(χ2=113.57、32.35、91.31,均P<0.001)。比较放射免疫配体法及ECL两种方法检测GADA65、IA-2A阳性率差异无统计学意义(均P>0.05)。结论ECL法检测胰岛自身抗体灵敏度高,特异性强,可重复性好,可用于T1DM的预测及诊断。

Objective To establish a new standard for measurement islet autoantibodies by electrochemiluminescence (ECL) assay and to discuss its applications for type 1 diabetes mellitus (T1DM) prediction and diagnosis. Methods ECL assay was used to measure serum levels of glutamic acid decarboxylase antibody (GADA65), insulin autoantibody (IAA) and protein tyrosine phosphatase antibody (IA-2A) in 124 healthy volunteers, 180 T1DM patients, and 180 type 2 diabetes mellitus (T2DM) patients. Patients admitted in Sir Run Run Hospital, Nanjing Medical University from December 2016 to June 2018 with diagnosis of T1DM or T2DM were enrolled in the study. Healthy volunteers were recruited from students in Nanjing Medical University. The serum antibody level was represented as antibody index. The positive cut-off limit for each antibody was defined as 99% of the indicated antibody index from healthy volunteers. The chi-square test was used to analyze the positivity of GADA65, IAA, IA-2A of T1DM and T2DM patients detected by ECL method. Results The cut-off points for GADA65, IAA and IA-2A were 0.047, 0.009 and 0.0029 respectively by ECL methods. The intra-assay coefficients of variation (CV) for GADA65, IAA and IA-2A were 1.88%-8.30%, 1.39%-3.40%, 1.23%-3.17% respectively, and the inter-assay CV were 3.9%-7.3%, 2.7%-9.8%, 2.2%-7.4% respectively. The repeatability of the result was 99.8%. The positivity of GADA65, IAA,IA-2A were 52%(94/180), 45%(81/180), 21%(39/180) respectively in T1DM patients and were 2.2%(4/180), 2.2%(4/180), 2.2%(4/180) respectively in T2DM patients. There were statistic significances in positivity between T1DM and T2DM for all three antibodies (χ2=113.57,32.35,91.31, all P<0.001). The positive ratio of GADA65 and IA-2A detected by ECL or radio-binding assay shows no difference. Conclusion The ECL assay has high sensitivity, specificity and repeatability and could be used for the prediction and diagnosis of T1DM.