A novel electrochemiluminescence method for detecting insulin autoantibody

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 mul diluted antigen, followed by adding 15 mul 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.