Woo W, LaGasse J M, Zhou Z, Patel R, Palmer J P, Campus H, Hagopian W A
Department of Medicine, University of Washington, Seattle, WA 98195, USA.
J Immunol Methods. 2000 Oct 20;244(1-2):91-103. doi: 10.1016/s0022-1759(00)00259-3.
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 </=16%, while intra-assay CVs were all </=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 35S-met-GAD65, 35S-met-ICA512/IA2, 35S-met-Phogrin, and 125I-insulin, and could be used for simultaneous screening of reactivity to both GAD65 and ICA512/IA2 in the same 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 125I-insulin, comparing signal in the absence versus presence of cold insulin competitor was not necessary. Total serum volumes required were only 6 microl for GAD and ICA512, and only 15 microl for IAA. The method costs less than all other commonly used formats, and should be useful for population screening.
为了研究预防性治疗方法而对1型糖尿病进行预测，需要对普通人群进行筛查，因为85%的新发病例都出现在这一群体中。即使采用基于HLA的预筛查，所有儿童中仍有近20%需要进行多次血清自身抗体检测。因此，高通量、经济且准确的方法至关重要。我们开发了这样一种放射结合方法，使用96孔微量滴定板和一种通过膜结合蛋白A的新型免疫复合物捕获方法。每个微量滴定板都包含一个标准阴性对照血清，以及低、中、高浓度的阳性对照血清。所有血清均进行一式三份评估。这很容易为单个血清的三份重复样本以及每个96孔板建立质量控制标准。批间变异系数（CVs）均≤16%，而批内CVs均≤10%。该检测方法被发现具有敏感性（能检测患者中的自身抗体）和特异性（在数千名健康志愿者中反应性较低）。该方法使用多种抗原（如35S-甲硫氨酸-GAD65、35S-甲硫氨酸-ICA512/IA2、35S-甲硫氨酸-Phogrin和125I-胰岛素）效果良好，并且可用于在同一孔中同时筛查对GAD65和ICA512/IA2的反应性。与基于微量离心管的蛋白A-琼脂糖GAD65和IA2自身抗体放射结合检测以及基于酸-活性炭-聚乙二醇（PEG）的竞争性胰岛素自身抗体检测相比，该方法的诊断准确性更优。对于125I-胰岛素，无需比较有无冷胰岛素竞争者时的信号强度差异。检测GAD和ICA512所需的血清总体积仅为6微升，检测IAA仅需15微升。该方法的成本低于所有其他常用方法，应可用于人群筛查。
