Detection of autoantibodies to protein tyrosine phosphatase-like protein IA-2 with a novel time-resolved fluorimetric assay
Detection of autoantibodies to protein tyrosine phosphatase-like protein IA-2 with a novel time-resolved fluorimetric assay
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DOI：
10.1373/49.6.916
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发表时间：
2003-06-01
影响因子：
9.3
通讯作者：
Hinkkanen, AE
中科院分区：
文献类型：
--
作者：
Westerlund-Karlsson, A;Suonpää, K;Hinkkanen, AE
背景：抗胰腺谷氨酸脱羧酶(GAD65；65 kDa谷氨酸脱羧酶)、蛋白酪氨酸磷酸酶样蛋白IA-2和胰岛素的循环自身抗体可作为1型糖尿病的预测标志物。我们基于时间分辨荧光法建立了一种检测血清中IA-2自身抗体(IA-2As)的新方法，该方法比放射结合分析(RBAS)和ELISAS等方法具有许多优点。方法：IA-2(IA-2IC)的胞内部分通过与血清样品和谷胱甘肽S转移酶-IA-2IC融合蛋白的同时孵育而被生物素化并结合到包被链霉亲和素的96孔板上。用铕标记的抗GST抗体检测血清中自身抗体捕获的GST-IA-2IC，并在时间分辨荧光仪上测量信号。对100例新诊断的1型糖尿病患者和100例正常对照的血清样本进行分析。结果：在100份1型糖尿病患者的血清样本中，时间分辨荧光法检测出74份含有IA-2A的血清，而澳洲央行检测到80份IA-2A阳性的样本。在澳大利亚央行呈阳性但未被时间分辨荧光分析检测到的6个样本中，有5个在澳大利亚央行仅呈弱阳性。结论：时间分辨荧光法检测IA-2As是一种简便的、非放射性的分析方法，其特异性和灵敏度可与RBA法相媲美。与传统的澳洲央行相比，这种分析可以大幅缩短执行时间。(C)2003年美国临床化学协会。
Background: Circulating autoantibodies to pancreatic glutamic acid decarboxylase (GAD65; the 65-kDa isoform of glutamic acid decarboxylase), protein tyrosine phosphatase-like protein IA-2, and insulin can be used as predictive markers of type 1 diabetes. We developed a novel assay for the detection of IA-2 autoantibodies (IA-2As) in serum based on time-resolved fluorimetry, hypothesizing that this kind of assay could provide several advantages over methods described to date, including radiobinding assays (RBAs) and ELISAs.Methods: The intracellular part of IA-2 (IA-2ic) was biotinylated and bound to streptavidin-coated 96-well plates by simultaneous incubation with serum samples and glutathione S-transferase (GST)-IA-2ic fusion protein. GST-IA-2ic captured by autoantibodies in the serum was detected with europium-labeled anti-GST antibody, and the signal was measured in a time-resolved fluorimeter. A serum sample panel from 100 patients with newly diagnosed type 1 diabetes and 100 unaffected controls was analyzed with the new assay and a conventional RBA.Results: Among the 100 serum samples from patients with type 1 diabetes, the time-resolved fluorimetric assay identified 74 IA-2A-containing sera, whereas the RBA detected 80 IA-2A-positive samples. Five of the six samples positive in the RBA but not detected by the time-resolved fluorimetric assay were only weakly positive in the RBA. The performance time of the time-resolved fluorimetric assay was 2.5 h compared with 10-12 h required by the RBA.Conclusions: The time-resolved fluorimetric assay provides a simple, nonradioactive analysis method for the detection of IA-2As with a specificity and a sensitivity comparable to the RBA method. This assay allows substantial reduction in performance time compared with the conventional RBA. (C) 2003 American Association for Clinical Chemistry.