Development of immunoassay for fluvoxamine detection using recombinant single-chain variable fragment antibody
Development of immunoassay for fluvoxamine detection using recombinant single-chain variable fragment antibody
复制标题
开发使用重组单链可变片段抗体检测氟伏沙明的免疫分析方法
DOI：
10.1007/s11419-017-0358-9
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发表时间：
2017
影响因子：
2.2
通讯作者：
Yoko Nishitani
中科院分区：
文献类型：
--
作者：
Ako Sasao;Michiyo Takaki;Yuki Ohtsu;Satoko Mishima;Kosei Yonemitsu;Hiroshi Morioka;Yoko Nishitani
在法医毒理学中，免疫分析法因其检测过程简单、结果快速而得到广泛应用。然而，商业免疫测定产品仅可用于有限数量的药物。制备抗药抗体是建立免疫分析系统的关键，但耗时。本研究以抗氟伏沙明（fluvoxamine，FLV）的单链抗体（single-chain variable fragment，scFv）为探针，建立了一种用于药物筛选的间接竞争酶联免疫吸附试验（icELISA）检测体系。为了阐明结构域顺序对单链抗体结合活性的影响，我们制备了两种具有不同结构域顺序的单链抗体（HL，VH-接头-VL和LH，VL-接头-VH），并检测了它们对FLV的动力学参数。scFv显示出足够的FLV结合活性（KD= 3.8和7.6 nM），并且HL scFv比LH scFv稍微更有利于FLV结合。所建立的检测限为10-200 ng/mL，在100 μM以下无交叉反应，但对氯丙嗪和丙咪嗪无交叉反应。我们还定量了法医尸检病例中血浆中的FLV浓度，结果表明，该方法可以有效地应用于FLV的定量，而无需提取步骤。尽管针对小分子药物的重组抗体在免疫分析中尚未得到广泛应用，但由于其自身的优势，我们可以预测，它们在不久的将来可能成为筛选药物的有力工具。
In forensic toxicology, immunoassays for drug screening are widely used because of the simple test procedures and instantaneous outcome of results. However, commercial immunoassay products are available for only a limited number of drugs. Preparation of antidrug antibodies is a crucial, but time-consuming in creating an immunoassay system. In this study, we focused on the application of a single-chain variable fragment (scFv) antibody for drug screening and developed a fluvoxamine (FLV) detection system for indirect competitive enzyme-linked immunosorbent assay (icELISA) using the scFv against FLV. To clarify the influence of domain order on the scFv binding activities, we prepared two kinds of scFv with different domain orders (HL, VH-linker-VL, and LH, VL-linker-VH) and examined their kinetic parameters against FLV. The scFvs showed sufficient FLV binding activities (KD= 3.8 and 7.6 nM), and the HL scFv was slightly more favorable for FLV binding than the LH scFv. The developed icELISA using the HL scFv could detect FLV in the range of 10–200 ng/mL, and the scFv has no cross-reactivity below 100 μM except for chlorpromazine and imipramine. We also quantified the plasma FLV concentrations in forensic autopsy cases, and the results showed that this method could be applied effectively for FLV quantification without the need for extraction steps. Although recombinant antibodies against small molecule drugs for immunoassays have not yet been commonly used, we can predict that they could be a powerful tool to screen drugs in the near future, because of their advantages.