胰岛素自身抗体的改良放射免疫分析法

目的: 建立胰岛素自身抗体（IAA）的改良放射免疫分析法，并探讨IAA对1型糖尿病（T1DM）的诊断价值。方法:125I-胰岛素抗原与血清在EP管内混匀，旋转孵育，用蛋白A-琼脂糖沉淀抗原抗体复合物。沉淀物经TBST缓冲液洗涤后，置于液体闪烁计数仪上计数，结果以IAA指数表示。通过与国际标准化实验室及国内商品化IAA放射免疫试剂盒检测结果比较，并检测120例健康对照和32例初发T1DM患者IAA水平，评价其一致性、灵敏度和特异性，以及临床应用价值。结果: ①该方法的批内变异系数（CV）为5.8%~8.3%，批间CV 为7.0%~11.0%。41例标本的IAA指数与国际标准化实验室呈显著正相关（r=0.783，P＜0.01）,结果判断一致率为100%。82例IAA指数呈梯度排列的标本检测结果与国内放射免疫试剂盒一致率为89%，9例不符合的标本均为该方法检测为低滴度阳性（指数在0.05~1.00）而试剂盒检测阴性；②IAA指数的阳性阈值为0.051（为120例健康人的99.5%百分位点上限），T1DM 患者IAA检出率为18.8%(6/32)，高于正常对照的0.8%（ P<0.001）。其中15岁以下病程≤半年的T1DM患者IAA检出率为28.6%（4/14），15岁以上IAA阳性率为11.1%（2/18）。结论: 建立的IAA改良放射免疫分析法敏感性与特异性好，适于临床应用，尤其是低滴度IAA阳性者。

Improved radioligand assay of insulin autoantibody

OBJECTIVE: To establish an improved radioligand assay of insulin autoantibody (IAA) and to investigate the diagnostic role of IAA in type 1 diabetes (T1DM). METHODS: 125I-insulin antigens were mixed with sera samples in the Eppendorf tubes. The immunocomplexes were precipitated with protein A-agarose, then washed with TBST buffer and counted the cpm value using liquid scintillation counter. The assay results were expressed by IAA index and compared with those of an international standardized laboratory and a domestic anti-insulin antibody kit. Sera of 32 recent-onset type 1 diabetic patients and 120 healthy controls were screened for IAA and the consistency, sensitivity, specificity and the diagnostic value of IAA were evaluated. RESULTS: (1) The intra-assay coefficient of variation (CV) was 5.8% - 8.3%, and the inter-assay CV 7.0% - 11.0%. The IAA indices of 41 samples tested in our laboratory and in an international standardized laboratory were strongly positively related (r = 0. 783, P < 0. 01) (100.0% concordance). Arrayed according to their antibody titers, the IAA indices of 82 samples measured in our laboratory and in a domestic anti-insulin antibody kit were also strongly positively related (r = 0.982, P < 0.0001) (89.0% concordance, Kappa value 0.813), but 9 samples were positive with low titer (index 0.05 - 1.00) by the radioligand assay or negative by the kit. 2) The IAA index cut-off point was determined according to the upper limit of 99.5% percentile of 120 healthy controls. The index of 0.051 or higher was defined as positive. The IAA positivity prevalence was 18.8% (6/32) in type I diabetes and 0.8% (1/120) in the healthy controls (P < 0.001). The positivity frequency of IAA was 28.6% (4/14) in patients younger than 15 years of age and the duration less than half a year, while 11.1% (2/ 18) in those older than 15. CONCLUSION: The improved radioligand assay for IAA has a high sensitivity and specificity and can be used in clinical practice, especially for the low-titer positive subjects.