A novel high-throughput method for accurate, rapid, and economical measurement of multiple Type 1 diabetes autoantibodies
A novel high-throughput method for accurate, rapid, and economical measurement of multiple Type 1 diabetes autoantibodies
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DOI：
10.1016/s0022-1759(00)00259-3
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发表时间：
2000-10-20
影响因子：
2.2
通讯作者：
Hagopian, WA
中科院分区：
文献类型：
--
作者：
Woo, W;LaGasse, JM;Hagopian, WA
预测1型糖尿病的预防性治疗研究需要筛查一般人群，其中85%的新病例发生。即使有基于HLA的预筛选，近20%的儿童将需要多次血清自身抗体检测。高通量。因此，经济和准确的方法是必不可少的。我们已经开发了这样一种放射性结合方法，使用96孔微量滴定板和一种新的免疫复合物捕获方法，通过膜结合蛋白A。每个微量滴定板含有标准阴性对照血清以及低、中和高水平阳性对照血清。所有血清均一式三份进行评价。这很容易为一式三份个体血清和每个96孔板提供质量控制标准。批间变异系数（CV）均小于或等于16%，而批内CV均小于或等于10%。该检测方法被发现是敏感的（检测患者的自身抗体）和特异性（在数千名健康志愿者中的低反应性）。所述形式使用不同的抗原如S-35-met-GAD 65、S-35-met-ICA 512/IA 2、S-35-met-Phogrin、I-125-insulin工作良好，并且可用于在同一孔中同时筛选对GAD 65和ICA 512/IA 2的反应性。诊断准确性与基于微量离心管的蛋白A-琼脂糖GAD 65和IA 2自身抗体放射结合测定法以及基于酸-炭-聚乙二醇（PEG）的竞争性胰岛素自身抗体测定法相比，具有优势。在I-125-胰岛素的情况下，不需要比较不存在与存在冷胰岛素竞争剂的信号。GAD和ICA 512所需的总血清体积仅为6穆尔，IAA仅为15穆尔。该方法的成本低于所有其他常用的格式，应该是有用的人口筛选。(C)2000 Elsevier Science B. V.保留所有权利。
Prediction of Type 1 diabetes for study of preventive therapies requires screening the general population, where 85% of new cases occur. Even with HLA-based prescreening, nearly 20% of all children will need multiple serum autoantibody testings. High-throughput. economical, and accurate methods are therefore essential. We have developed such a radiobinding method, using 96-well microtiter plates and a novel immune complex capture method via membrane-bound Protein A. Each microtiter plate contained a standard negative control serum, and low-, medium-, and high-level positive control sera. All sera were evaluated in triplicate. This readily allowed quality control criteria both for triplicates of individual sera and for each 96-well plate. Inter-assay coefficients of variation (CVs) were all less than or equal to 16%, while intra-assay CVs were all less than or equal to 10%. The assay was found to be sensitive (to detect autoantibodies in patients) and specific (low reactivity in thousands of healthy volunteers). The format worked well using diverse antigens such as S-35-met-GAD65, S-35-met-ICA512/IA2, S-35-met-Phogrin, I-125-insulin, and could be used for simultaneous screening of reactivity to both GAD65 and ICA512/IA2 in the same and well. Diagnostic accuracy compared favorably with microcentrifuge tube-based Protein A-agarose GAD65 and IA2 autoantibody radiobinding assays and with acid-charcoal-polyethylene glycol (PEG) based competitive insulin autoantibody assays. In the case of I-125-insulin, comparing signal in the absence versus presence of cold insulin competitor was not necessary. Total serum volumes required were only 6 mul for GAD and ICA512, and only 15 mul for IAA. The method costs less than all other commonly used formats, and should be useful for population screening. (C) 2000 Elsevier Science B.V. All rights reserved.