Open-Sandwich Enzyme Immunoassay for One-Step Noncompetitive Detection of Corticosteroid 11-Deoxycortisol
Open-Sandwich Enzyme Immunoassay for One-Step Noncompetitive Detection of Corticosteroid 11-Deoxycortisol
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DOI：
10.1021/ac900700a
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发表时间：
2009-10-15
影响因子：
7.4
通讯作者：
Ueda, Hiroshi
中科院分区：
文献类型：
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作者：
Ihara, Masaki;Suzuki, Tatsuya;Ueda, Hiroshi
与相应的竞争性测定相比，非竞争性免疫测定具有提高灵敏度和工作范围的潜力。然而，分子量小于 1000 的单价抗原由于尺寸小而不易进行夹心测定。作为一种可以用抗体克隆进行的非竞争性免疫测定，基于抗体可变区（V-H + V-L）的抗原依赖性稳定性的开放夹心免疫测定（OS-IA）被应用于作为模型目标半抗原的11-脱氧皮质醇（11-DC；M-r 346.5）的定量，11-脱氧皮质醇（11-DC；M-r 346.5）是一种皮质类固醇，用作垂体-肾上腺功能的诊断指标。在微孔板孔中存在样品的情况下，通过一步 OS-IA 检测与固定化 V-L 结合的酶标记 V-H 片段，以飞摩尔检测限测量 11-DC，并且工作范围比相应的竞争测定更宽。此外，发现对类似物的选择性几乎与传统测定相同。还显示了 V-H/V-L 界面处的 V-H 残基诱变对减少背景信号的影响，这意味着 OS-IA 在小分子分析中的更广泛应用。
A noncompetitive immunoassay has the potential for improved sensitivity and working range compared with corresponding competitive assays. However, monovalent antigens with less than 1000 in molecular weight are not susceptible to sandwich assays due to their small size. As a noncompetitive immunoassay that can be performed with a clone of an antibody, an open-sandwich immunoassay (OS-IA) based on the antigen-dependent stabilization of the antibody variable region (V-H + V-L) was applied to the quantification of 11-deoxycortisol (11-DC; M-r 346.5), a corticosteroid serving as a diagnostic index for pituitary-adrenal function, as a model target hapten. By one step OS-IA detection of enzyme-labeled V-H fragment bound to immobilized V-L in the presence of sample in microplate wells, 11-DC was measured with a femtomolar detection limit and the working range was wider than that with corresponding competitive assay. In addition, the selectivity against analogues was found almost identical to that of conventional assays. The effect of the mutagenesis of a V-H residue at the V-H/V-L interface to reduce background signal was also shown, implying the wider application of OS-IA in small molecule analyses.