Ankelo Matti, Westerlund Annette, Blomberg Kaj, Knip Mikael, Ilonen Jorma, Hinkkanen Ari E
JDRF Center for Prevention of Type 1 Diabetes in Finland.
Clin Chem. 2007 Mar;53(3):472-9. doi: 10.1373/clinchem.2005.064568. Epub 2007 Jan 26.
Autoantibodies to glutamic acid decarboxylase (GADAs), specifically the 65-kDa isoform GAD65, and autoantibodies to the protein tyrosine phosphatase-like molecule IA-2 (IA-2As) predict development of diabetes. Our aim was to develop a time-resolved immunofluorometric (TR-IFMA) dual-label assay method for the simultaneous detection of these autoantibodies and to evaluate the diagnostic sensitivity of the method compared with single-label TR-IFMA and fluid-phase radiobinding assay (RBA) in screening children with type 1 diabetes.
We incubated combined biotinylated GAD65 and IA-2 proteins, glutathione S-transferase (GST)-IA-2, europium-labeled GAD65, terbium-labeled anti-GST antibody, and serum sample or calibrator and transferred aliquots to a streptavidin-coated 96-well microtiter plate for a second incubation. After washing, we added Delfia Enhancement solution to each well and measured the fluorescence of Eu. We developed the Tb fluorescence signal by use of the Delfia Enhancer solution and measured it. We analyzed serum samples from a cohort of 100 children with newly diagnosed type 1 diabetes.
The correlation coefficients between the autoantibody concentrations measured by dual- and single-label TR-IFMA assays were 0.962 for GADA and 0.874 for IA-2A. Among 100 children with newly diagnosed diabetes, 65 of them were GADA positive in the dual-label assay, 64 in the single-label assay, and 66 in the RBA GADA assay. Seventy-four of the children tested positive for IA-2A in both TR-IFMA assay types, and 79 in the RBA IA-2A assay.
The novel dual-label immunofluorometric assay performed comparably to the separate, single-label GADA and IA-2A assays in screening for beta-cell autoimmunity in children with newly diagnosed type 1 diabetes.
谷氨酸脱羧酶（GADA）自身抗体，特别是65 kDa亚型GAD65，以及蛋白酪氨酸磷酸酶样分子IA - 2自身抗体（IA - 2A）可预测糖尿病的发生。我们的目的是开发一种时间分辨免疫荧光分析（TR - IFMA）双标记检测方法，用于同时检测这些自身抗体，并在筛查1型糖尿病儿童时，与单标记TR - IFMA和液相放射结合分析（RBA）相比，评估该方法的诊断敏感性。
我们将生物素化的GAD65和IA - 2蛋白、谷胱甘肽S - 转移酶（GST） - IA - 2、铕标记的GAD65、铽标记的抗GST抗体与血清样本或校准品一起孵育，然后将等分试样转移至链霉亲和素包被的96孔微量滴定板中进行第二次孵育。洗涤后，向每个孔中加入Delfia增强溶液并测量铕的荧光。我们使用Delfia增强剂溶液使铽荧光信号显色并进行测量。我们分析了100名新诊断的1型糖尿病儿童队列的血清样本。
双标记和单标记TR - IFMA检测所测自身抗体浓度之间的相关系数，GADA为0.962，IA - 2A为0.874。在100名新诊断的糖尿病儿童中，双标记检测中有65名GADA呈阳性，单标记检测中有64名，RBA GADA检测中有66名。在两种TR - IFMA检测类型中，74名儿童的IA - 2A检测呈阳性，RBA IA - 2A检测中有79名。
在筛查新诊断的1型糖尿病儿童的β细胞自身免疫方面，新型双标记免疫荧光检测与单独的单标记GADA和IA - 2A检测表现相当。
