Single-chain variable fragment technology in forensic toxicological analysis: production of an antibody to fluvoxamine
Single-chain variable fragment technology in forensic toxicological analysis: production of an antibody to fluvoxamine
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单链可变片段技术在法医毒理学分析中的应用：氟伏沙明抗体的生产
作者：
Ako Sasao;Yoshiaki Suwa;T. Aso;Hiroe Kohmatsu;Y. Ohtsu;S. Mishima;K. Yonemitsu;H. Morioka;Y. Nishitani
免疫分析技术由于其简单的程序和快速的结果而被广泛用于法医毒理学和急救医学领域的药物筛选[1，2]。为了建立化合物的免疫测定方法，最费力的步骤是产生对化合物特异的抗体。在这里，我们提出了一种新的重组抗体技术，用于生产单链可变片段（scFv）。scFv是一种小的抗体分子，保留了原始完整免疫球蛋白的高抗原特异性和结合活性[3]。scFv的一个优点是其使用细菌细胞培养物的简单生产，其还通过引入突变提供可变功能。此外，创建展示各种scFv的scFv噬菌体文库可能有助于寻找药物反应性元件。这似乎是一个强大的工具，用于快速创建新的抗药抗体，而无需免疫动物。最近，精神药物中毒的频率在日本有所增加[4]。在这些药物中，氟伏沙明（FLV）是选择性5-羟色胺再摄取抑制剂（SSRI）抗抑郁药物之一，广泛用于治疗抑郁症和强迫症[5]。虽然SSRIs与传统抗抑郁药（如三环类抗抑郁药和单胺氧化酶抑制剂）相比相对安全[6]，但FLV中毒病例的数量随着其处方的增加而增加[7，8]。在这项研究中，我们使用新的重组抗体技术创建了抗FLV scFv。FLV苹果酸盐获自Sigma-Aldrich（St. Louis，MO，USA）。巯基琥珀酰牛血清白蛋白（MS-BSA）是K.藤原，Sojo大学（熊本，日本）。所有其他溶剂和化学品均为分析级，并通过当地供应商购买。将BALB/c小鼠（雌性，4周龄; Kyudo，熊本，日本）饲养在动物资源和发展中心，熊本大学，熊本，日本，并保持在环境控制室（22 ± 2 ℃，50- 70%湿度，0700 - 1900小时光照）中.所有程序均由熊本大学动物实验伦理审查委员会批准。如前所述制备用于诱导抗FLV抗体的免疫原（BSA-FLV），但略有修改[9]。简言之，将溶于1.0 ml 50 mM磷酸盐缓冲液（pH 7.0）中的FLV马来酸盐（4.3 mg;约10 lmol）与N-[c-马来酰亚胺基丁酰氧基]琥珀酰亚胺（GMBS）（0.25 mg;约0.89 lmol）混合，并在室温下搅拌孵育。MS-BSA，估计每个BSA分子含有18个巯基，用3.0 ml 0.1 M磷酸盐缓冲液（pH 7.0）稀释，并立即加入到由Osamu Suzuki编辑的特刊TIAFT 2012中。
Immunoassay techniques are widely used for drug screening in the fields of forensic toxicology and emergency medicine, because of their simple procedures and rapid outcome of results [1, 2]. To create an immunoassay method for a compound, the most laborious step is the production of an antibody that is specific to the compound. Here, we present a new recombinant antibody technology for producing a single-chain variable fragment (scFv). scFv is a small antibody molecule that retains high antigen specificity and binding activity of the original whole immunoglobulin [3]. One advantage of scFv is its simple production using bacterial cell cultures, which also provide variable functionalities by introducing mutations. Furthermore, creating scFv phage libraries, which display various scFvs, might be useful in searching for drug-reactive elements. This seems to be a powerful tool for rapid creation of new antidrug antibodies without immunizing animals. Recently, the frequency of psychotropic drug poisoning has increased in Japan [4]. Among such drugs, fluvoxamine (FLV), one of the selective serotonin reuptake inhibitor (SSRI) antidepressant drugs, is widely used for treatment of depression and obsessive compulsive disorders [5]. Although SSRIs are relatively safe as compared with conventional antidepressant drugs, such as tricyclic antidepressants and monoamine oxidase inhibitors [6], the number of FLV poisoning cases has increased according to the increase in its prescription [7, 8]. In this study, we created an anti-FLV scFv using novel recombinant antibody technology. FLV malate was obtained from Sigma-Aldrich (St. Louis, MO, USA). Mercaptosuccinyl bovine serum albumin (MS-BSA) was a generous gift from Prof. K. Fujiwara, Sojo University (Kumamoto, Japan). All other solvents and chemicals were of analytical grade, and purchased through local suppliers. BALB/c mice (female, 4 weeks old; Kyudo, Kumamoto, Japan) were maintained in the Center for Animal Resources and Development, Kumamoto University, Kumamoto, Japan, and were kept in an environmentally controlled room (22 ± 2 C, 50–70 % humidity, illuminated from 0700 to 1900 hours). All procedures were approved by the Kumamoto University Ethics Review Committee for Animal Experimentation. The immunogen (BSA–FLV) for induction of anti-FLV antibodies was prepared as described previously with a slight modification [9]. Briefly, FLV maleate (4.3 mg; approximately 10 lmol) in 1.0 ml of 50 mM phosphate buffer (pH 7.0) was mixed with N-[c-maleimidobutyryloxy] succinimide (GMBS) (0.25 mg; approximately 0.89 lmol), and incubated at room temperature with stirring. MS-BSA, estimated to contain 18 thiol groups per BSA molecule, was diluted with 3.0 ml of 0.1 M phosphate buffer (pH 7.0), and added immediately to the This article is for the special issue TIAFT2012 edited by Osamu Suzuki.
DOI：
10.1016/s0021-9258(19)50004-7
发表时间：
1992-05
期刊：
The Journal of biological chemistry
影响因子：
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作者：
N. Araki;N. Ueno;B. Chakrabarti;Y. Morino;S. Horiuchi
通讯作者：
N. Araki;N. Ueno;B. Chakrabarti;Y. Morino;S. Horiuchi