Objective: To establish the electrochemiluminescence (ECL) method for insulin autoantibody (IAA) detection in China and preliminarily evaluate its application value. Methods: Proinsulin was labeled with Sulfo-tag and biotin, and then incubated with IAA in serum. The Meso Scale Discovery (MSD) streptavidin plate was used to capture the labeled antigen-antibody complex, which was subsequently detected by MSD electrochemiluminescence machine. IAA index ≥ 0.005 was used as a positive threshold. After optimizing the detection conditions, the correlation and consistency of ECL method and microplate radioimmunoassay were compared. Patients with type 1 diabetes mellitus (T1DM, n=55) and first-degree relatives (n=216) from the Department of Metabolism and Endocrinology of the Second Xiangya Hospital of Central South University from 2016 to 2018, and healthy volunteers (n=413) were selected as subjects, and their serum samples were tested to evaluate the clinical application value of ECL. Results: The optimized conditions included as follows: (1) The optimal concentration of biotin-proinsulin and Sulfo-tag-proinsulin was 800 ng/ml. (2) Serum acidification was achieved by incubating for 45 min. (3) Adding 35 μl diluted antigen, followed by adding 15 μl Tris-HCl to the reaction system, and then incubated with acidified serum overnight to obtain a higher signal-to-noise ratio (S/N). Twenty-two samples were repeatedly detected by ECL method at intervals of 3 months. There was no significant difference in IAA index (P=0.095) and the positive or negative results was completely consistent. The agreement rate between ECL method and microplate radioimmunoassay for IAA was 93.7% (119/127) (Kappa value: 0.858), and the index showed a significant positive correlation (correlation coefficient r=0.749, P<0.001). The area under the curve (AUC) of ECL assay was 0.764 (95%CI: 0.669 to 0.858). The positive rate of IAA in T1DM patients by ECL method was 52.73% (29/55), which was significantly higher than 0.76% (2/263) in healthy controls. The positive rate of IAA in T1DM first-degree relatives group was 0.93% (2/216), and there was no significant difference compared with healthy control group (P=0.854). Conclusion: ECL method of IAA has the features of high sensitivity and specificity, simple operation and no radioactive contamination, indicating its appreciable clinical application value.
目的： 在国内建立胰岛素自身抗体（IAA）的电化学发光检测法，并初步评价其应用价值。 方法： 胰岛素原采用磺基吡啶钌衍生物（Sulfo-tag）和生物素标记后，与血清IAA孵育结合。Meso Scale Discovery（MSD）链霉亲和素电化学发光平板捕获标记的抗原抗体复合物后，采用MSD电化学发光仪进行检测，以IAA指数≥0.005作为阳性阈值。通过优化检测条件后，采用微量平板放射免疫法比较两种方法的相关性和一致性。选取2016至2018年于中南大学湘雅二医院代谢内分泌科门诊或住院的新诊断1型糖尿病（T1DM）患者（55例）及其一级亲属（216名），以及健康志愿者（健康对照组，413名）为研究对象，检测其血清样本，初步评价电化学发光法检测IAA的临床应用价值。 结果： 优化条件包括：（1）生物素标记的胰岛素原和磺基吡啶钌衍生物标记的胰岛素原的最适浓度均为800 ng/ml；（2）酸化血清的方式为水浴30℃静置孵育45 min；（3）在反应体系中先加入35 μl稀释后的抗原，再加入15 μl Tris-HCl缓冲液，然后与酸化血清混匀孵育过夜能获得更高的信噪比（S/N）。电化学发光法间隔3个月重复检测22例样本，IAA指数差异无统计学意义（P=0.095），阴/阳性判定完全一致；与微量平板放射免疫法检测IAA的一致率为93.7%（119/127）（Kappa值为0.858），指数呈正相关（相关系数r=0.749，P<0.001）。电化学发光法受试者工作特征（ROC）曲线下面积（AUC）为0.764（95%CI：0.669~0.858）。电化学发光法检测T1DM患者IAA的阳性率为52.73%（29/55），明显高于健康对照组的0.76%（2/263）（P<0.001）；检测T1DM一级亲属组的阳性率为0.93%（2/216），与健康对照组相比差异无统计学意义（P=0.854）。 结论： 电化学发光法检测IAA有较高的灵敏度和特异度，操作简便无放射污染，具有很好的临床应用价值。.
Keywords: Diabetes mellitus, type 1; Electrochemiluminescence; Insulin autoantibody.