蛋白酪氨酸磷酸酶自身抗体放射配体检测法的建立与临床应用

The radioligand assay of protein-tyrosine-phosphatase autoantibody and clinical implications

目的 建立蛋白酪氨酸磷酸酶自身抗体 (IA 2A)的放射配体测定法。 方法 经试管内转录与翻译 ,获得3 5 S标记的重组人IA 2抗原。3 5 S IA 2与血清在自制旋转孵育器上混匀过夜 ,用蛋白A 琼脂糖沉淀抗原抗体复合物。沉淀复合物经TBST缓冲液洗涤后 ,置液体闪烁计数仪计数 ,结果以IA 2A指数表示。并检测国人 111例 1型和 113例 2型糖尿病患者 ,以及 12 5名健康志愿者的IA 2A水平 ,评价其敏感性、特异性及临床应用价值。 结果 该方法的批内变异系数 (CV) 3 .2 %～ 9 .7% ,批间CV 5.6%～ 11.7% ;DASP 2 0 0 3回报结果显示其敏感性 64% ,特异性 10 0 % ;该方法检测 45份标本的IA 2A指数与国际标准化实验室结果呈显著正相关 (r =0 .690 ,P <0 .0 1) ,且阴、阳性判断完全一致 ;IA 2A指数阳性阈值 0 .0 0 65(为 12 5名健康者的 99.5%百分位点上限 ) ;该方法检测国人 1型糖尿病患者阳性率 2 3 .4% (2 6/ 111) ,其中病程 <1年的阳性率 3 3 .3 % (17/ 51) ,病程≥ 1年的阳性率 15.0 % (9/ 60 ) ,差异有显著意义 (χ2 =5.17,P <0 .0 5)。 2型糖尿病患者阳性率 1.8% (2 /113 ) ,显著低于 1型糖尿病患者 (χ2 =2 4.0 ,P <0 .0 0 1)。 结论 建立的IA 2A放射配体检测法灵敏、特异性和重复性好 。

ObjectiveTo establish a radioligand assay for protein tyrosine phosphatase autoantibody (IA-2A).Methods 35 S labeling recombinant human IA-2 antigen wa s obtained by in vitro transcription and translation, and then mixed with serum samples in self-made rotating incubator overnight. The IA-2 immunocomplex was precipitated with protein A-agarose, then washed with TBST buffer and counted i n CPM using liquid scintillation counter The results were expressed by IA-2A index. The sensitivity and specificity of the assay were evaluated in the 3rd Di abetes Autoantibody Standardization Program (DASP) 2003 sponsored by the Immuno logy of Diabetes Society (IDS). Also, IA-2A was screened in Chinese diabetic patients (type 1, n=111; type 2, n=113) and healthy controls(n=125 ).ResultsThe intra-assay coefficient of variation (CV ) was 3.2%～9.7%, inter-assay CV 5.6%～11.7%. The IA-2A indices of 45 sa mp les were tested in our laboratory and an international standardized laboratory, which were totally consistent. The IA-2A index cut point was determined accord ing to the upper limit of 99.5 percentile of 125 healthy controls. The index of 0.0065 or higher was defined as positive. The IA-2A positive prevalence was 23 .4 (26/111) in type 1 diabetes and 1.8 (2/113) in type 2 diabetes (χ 2=24.0 ,P<0.001). The IA-2A positive percentage in type 1 diabetic patients was higher in the course of less than 1 year than over 1 year (33.3% vs 15.0%, P <0.05).ConclusionThe established IA-2A radioligand assay h as a high sensitivity, specificity and reproductivity, and it could be used wid ely in clinical practice.