谷氨酸脱羧酶抗体微量平板放射结合检测法的建立与初步应用  

Micro-plate radiobinding assay of autoantibody to glutamic acid decarboxylase

作　　者：黄干[1] 金河来[1] 王霞[1] 李卉[1] 张松[1] 周智广[1] 

机构地区：[1]中南大学湘雅二医院代谢内分泌研究所、中南大学糖尿病中心,长沙410011

出　　处：《中华核医学杂志》2008年第5期339-343,共5页Chinese Journal of Nuclear Medicine

基　　金：基金项目：国家自然科学基金（30670991）;卫生部优秀青年科技人才专项科研基金（Q9420）;湖南省科学技术厅科技计划（06FJ3155）

摘　　要：目的建立谷氨酸脱羧酶抗体（GAD-Ab）的微量平板放射结合检测（RBA）法并评价其临床价值。方法采用^35S-GAD65抗原与血清在96孔平板中保温24h，而后转入已包被蛋白A的Millipore平板中，平板经洗涤后进行放射性计数，GAD—Ab结果以WHO标准单位（U／ml）表示。检测224名健康人、162例1型糖尿病（T1DM）和210例初诊2型糖尿病（T2DM）患者GAD—Ab浓度，评价其临床应用效果。并分别选择经典放射配体（RLA）法检测GAD—Ab指数呈梯度排列的T1DM患者和健康人共119名，比较RBA法和RLA法检测结果的一致率和相关性。对32名健康人、35例TIDM患者、24例T2DM患者同时采集静脉血和手指血，探索该方法检测手指血GAD—Ab浓度的可行性，同时也与RLA法比较。分别采用直线相关分析、t检验、方差分析、受试者工作特征（ROC）曲线等方法进行统计学处理。结果（1）优化的检测条件包括采用2μl血清与3×10^4计数·min^-1的标记抗原缓慢振荡保温24h，采用4℃PBS缓冲液洗涤沉淀物10次，采用Optiphase Supermix型闪烁液测量放射性计数。（2）该法检测GAD—Ab批内CV为3．8％～10．2％，批间CV5．6％～11．9％；GAD—Ab浓度在40．3～664U／ml范围内线性良好，检测限为3．6U／ml；该方法灵敏度78％，特异性98％，与经典RLA法结果判定一致率97．5％，Kappa值0．95，两者检测值呈显著正相关（r=0．915，P〈0．001）。（3）GAD—Ab在TIDM和T2DM患者阳性率分别为46．9％（76／162）和5．2％（11／210），明显高于健康人的n89％（χ^2值为123．5和10．1，P〈0．001和〈0．01）。（4）该方法检测手指血GAD—Ab与经典RLA法检测静脉血结果判定一致率96．7％，Kappa值0．905，检测结果呈显著正相关（r=0．946，P〈0．001）。结论建立的微量平板RBA法灵敏度、特异性、重复性好，能适用于末梢血GAD—Ab检测，具有较好的临床应用价�Objective Thee purpose of this study was to develop a high-throughput micro-plate radiobinding assay （RBA） of glntamic acid decarboxylase antibody （GAD-Ab） and to evaluate its clinical application. Methods ^35S labeled GAD65 antigen was incubated with sera for 24 h on a 96-well plate, and then transferred to the Millipore plate coated with protein A, which was washed with 4℃ PBS buffer, and then counted by a liquid scintillation counter. The GAD-Ab results were expressed by WHO standard unit （U/ml）. A total of 224 healthy controls, 162 patients with type 1 diabetes mellitus （T1 DM） and 210 patients with newly diagnosed type 2 diabetes （T2DM） were recruited. A total of 119 T1DM and heahhv cases with gradually changing GAD-Ab levels were selected to compare the consistency of micro-plate RBA with conventional radioligand assay （RLA）. Blood samples were obtained from the peripheral vein and finger tip in 32 healthy controls, 35 T1DM and 24 T2DM patients, and tested with micro-plate RBA and then compared with the conventional RLA to investigate the reliability of finger tip sampling. Linear correlation, student＇s t-test, variance analysis and receiver operating characteristic （ROC） curve were performed using SPSS 11.5. Results （ 1 ） The optimized conditions of micro-plate RBA included 2 μl serum incubated with 3 × 10^4 counts/min 35 S-GAD for 24 h under slow vibration, antigen-antibody compounds washed 10 times by 4℃ PBS buffer, and radioactivity counted with Optiphase Supermix scintillation liquid. （2）The intra-batch CV of the micro-plate RBA was 3.8%- 10.2% , and the inter-batch CV was 5.6%- 11.9%. The linearity analysis showed a good correlation when the GAD-Ab in serum samples ranged from 40.3 to 664 U/ml and the detection limit of measurement was 3.6 U/ml. The results from Diabetes Autoantibody Standardization Program （DASP） 2005 showed that the sensitivity and specificity for GAD-Ab were 78% （39 positive among 50 new-onset T1DM） and 98% （2 positive among 100

关 键 词：谷氨酸脱羧酶 抗体 放射免疫测定 糖尿病 胰岛素依赖型 糖尿病 非胰岛素依赖型 

分 类 号：R686[医药卫生—骨科学]