谷氨酸脱羧酶抗体和蛋白酪氨酸磷酸酶抗体检测的标准化研究

Standardization of assays for autoantibodies to glutamic acid decarboxylase and protein tyrosine phosphatase

目的研究谷氨酸脱羧酶抗体(GADAb)和蛋白酪氨酸磷酸酶2抗体(IA2A)检测的标准化,为合理选择检测方法及准确判定结果提供依据。方法应用国际糖尿病免疫学会(IDS)推荐的WHO单位制,采用放射配体法检测125名健康人的GADAb和IA2A以确定阳性判断标准,通过检测IDS第3次糖尿病自身抗体标准化检测方法评估(DASP2003)工作组提供的100名健康对照及50例新发1型糖尿病患者血清,验证其灵敏度与特异性。并采用新一代的ELISA试剂盒检测41例已知抗体滴度的标本,对比ELISA与标化后放射配体法的一致性。结果GADAb的阳性阈值为18.5U/ml,IA2A为2.7U/ml。DASP2003反馈结果显示,本室GADAb检测的灵敏度82%,特异性98%;IA2A检测的灵敏度64%,特异性100%;受试者工作特征(ROC)曲线分析显示,本室GADAb曲线下面积(AUC)以及统一按95%特异性标化后的灵敏度(AS95)分别为0.946%及86%,IA2A分别为0.824%及68%。在52个回报结果的实验室中综合排名第15位。ELISA试剂盒与标化后的放射配体法检测GADAb的一致率82.9%,Kappa值0.656;检测IA2A的一致率75.6%,Kappa值0.514;结果判定不一致者多集中在阳性边缘水平的标本中。结论经标准化后的GADAb和IA2A放射配体法灵敏度和特异性明显提高;新一代ELISA试剂盒检测GADAb和IA2A可应用于临床,但对于检测结果阴性而临床可疑者,建议采用放射配体检测法核实。

Objective To standardize the radioligand assays for autoantibodies to glutamic acid decarboxylase (GAD-Ab) and protein tyrosine phosphatase (IA-2A).Methods The sera of 125 healthy controls were measured with radioligand assay for GAD-Ab and IA-2A according to a WHO unit system recommended by IDS. That DASP 2003 working group provided the sera of 100 healthy controls and 50 new-onset type 1 diabetic patients were also tested. The sensitivity and specificity of the assay were calculated. A new generation of ELISA kit was then used to measure 41 titer-graded samples in order to test the consistency.Results The positive cut-off value was 18.5 U/ml for GAD-Ab and 2.7 U/ml for IA-2A. The results from DASP 2003 showed that the sensitivity for GAD-Ab was 82 % (41 positive out of 50 type 1 diabetes) and its specificity reached 98 % (2 positive out of 100 healthy controls). The sensitivity for IA-2A was 64 % (32 positive out of 50 type 1 diabetes) and its specificity reached 100 % (no positive out of 100 healthy controls). ROC analysis showed that the area under curve (AUC) and Sensitivity at specificity 95 % (AS95) were 0.946 and 86 % for GAD-Ab respectively, while 0.824 and 68 % for IA-2A. The consistency between ELISA kit and the standardized radioligand assay for GAD-Ab was 82.9 %, with a Kappa value of 0.656. For IA-2A, the consistency and kappa value were 75.6 % and 0.514 respectively. Most of the inconsistent samples belonged to those with borderline positive levels.Conclusion For GAD-Ab and IA-2A, the standardized radioligand assays had high sensitivity and specificity, while the new generation of ELISA kit could be used in clinical practice. The samples from antibody-negative patients by ELISA kits yet suspected to be positive should be confirmed by standardized radioligand assays.