GENERATION OF NOVEL ANTIBODY SPECIES―ANTI-METATYPE ANTIBODIES AND "HYPER-ANTIBODIES" ―AND THEIR APPLICATION TO BIOANALYTICAL SYSTEMS

新抗体种类——抗变型抗体和“超抗体”的产生及其在生物分析系统中的应用

• 批准号: 12557236
• 负责人: GOTO Junichi
• 金额: $ 7.1万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557236/
• 关键词: Antibody engineering; Anti-motatype antibody; Hyper-antibody; Single-chain Fv fragment; Bioanalytical chemistry; Noncompetitive immunoassay; Hapten; Bile acid; 免疫工学; デオキシコール酸; アフィニティーラベル; アシルアデニレート; アシルグルクロニド; ステロイド

Antibodies are widely used as a key reagent in various immunoassays or immunoaffinity purification systems, which are essential in the biomedical analyses. However, conventional antibodies have some limitations on both affinity and specificity, resulting in insufficient sensitivity or low selectivity of these immunochemical techniques, In this study, we attempted to generate novel antibody species, anti-metatype antibodies (the antibodies recognizing antigen-antibody complex) and "hyper-antibodies" (the genetically engineered antibodies having improved affinity and specificity), which may allow us to develop excellent analytical systems for bioactive small molecules (haptens).Anti-metatype antibodies that specifically recognize the complex between a target hapten and an anti-hapten antibody are expected to enable noncompetitive immunoassay systems for steroids and synthetic drugs, showing much higher sensitivity than conventional competitive immunoassays. First, we prepared single-chai … More n Fv fragments (scFvs) against lithocholic acid 3-sulfate and deoxycholic acid (DCA) because scFvs lack the constant regions of immunoglobulins and thus would be advantageous to induce such anti-metatype antibodies. The V_H- and V_L-genes of mouse monoclonal antibodies against these bile acids were amplified by RT-PCR using RNA extracted from the corresponding hybridoma cells, which were then assembled into the scFv sequence encoding 5'-V_H-(Gly_4-Ser)_3-V_L-3' and expressed in E. coli XLOLR strain. The resulting scFv proteins showed similar reactivity against the relevant haptens to those of the parent mouse antibodies.Then we investigated the affinity-labeling of the auti-DCA antibody paratope with a relevant hapten, DCA, because this would be effective to inhibit the dissociation between the haptens and antibodies and increase the chance for generating anti-metatype antibodies. We found DCA-adenylate is highly reactive to primary amino groups on proteins, and works very well as the labeling agent. Immunization of mice with the affinity-labeled DCA-anti-DCA scFv complex would provide us the anti-metatype antibodies that enable sensitive noncompetitive immunoassay procedures. Less

抗体作为关键试剂广泛应用于各种免疫分析或免疫亲和纯化系统中，在生物医学分析中起着至关重要的作用。然而，常规的抗体在亲和力和特异性上都存在一定的局限性，导致这些免疫化学技术的灵敏度不足或选择性低。本研究尝试制备新型抗体种类--抗亚型抗体（识别抗原抗体复合物的抗体）和“超抗体”（具有改进的亲和力和特异性的基因工程抗体），这可能使我们能够开发用于生物活性小分子（半抗原）的优异分析系统。特异性识别靶半抗原和抗半抗原抗体之间的复合物的元型抗体预期能够实现用于类固醇和合成药物的非竞争性免疫测定系统，显示出比常规竞争性免疫测定高得多的灵敏度。首先，我们准备了单柴 ...更多信息 - 抗石胆酸3-硫酸盐和脱氧胆酸（DCA）的scFv片段（scFv），因为scFv缺乏免疫球蛋白的恒定区，因此将有利于诱导这种抗-meta型抗体。从相应的杂交瘤细胞中提取RNA，用RT-PCR方法扩增了抗胆汁酸单克隆抗体的V_H和V_L基因，将其组装成编码5 ′-V_H-（Gly_4-Ser）_3-V_L-3 ′的单链抗体，并在E. coliXLOLR菌株。然后我们研究了用相关的半抗原DCA亲和标记抗DCA抗体互补位，因为这将有效地抑制半抗原和抗体之间的解离，并增加产生抗元型抗体的机会。我们发现DCA-腺苷酸对蛋白质上的伯氨基具有高度反应性，并且作为标记剂非常好。用亲和标记的DCA-抗-DCA scFv复合物免疫小鼠将为我们提供能够进行灵敏的非竞争性免疫测定程序的抗-meta型抗体。少

项目成果

Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)

Norihiro Kobayashi：“基于单克隆抗体的酶联免疫吸附测定人尿液中的甘石胆酸硫酸盐，用于肝功能测试”类固醇。

Jaeick Lee: "Enantioseparation of chiral amino acids as the N(O, S)-ethoxycarbonylated diastereomeric esters by achiral dual-capillary column gas chromatography"Analyst. 126. 2128-2133 (2001)

Jaeick Lee：“通过非手性双毛细管柱气相色谱法将手性氨基酸对映体分离为 N(O,S)-乙氧基羰基化非对映异构酯”，分析师。

Norihiro Kobayashi, Hiroshi Katsumata, Yoshiaki Uto, Junichi Goto, Toshifumi Niwa, Koju Kobayashi, Yoshiaki Mizuuchi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"St

Norihiro Kobayashi、Hiroshi Katsumata、Yoshiaki Uto、Junichi Goto、Toshifumi Niwa、Koju Kobayashi、Yoshiaki Mizuuchi：“基于单克隆抗体的酶联免疫吸附测定人尿中的乙醇胆酸硫酸盐用于肝功能测试”St

Nariyasu Mano, Ayako Nikaido, Takashi Narui, Daisuke Yamasaki, Junichi Goto: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)

Nariyasu Mano、Ayako Nikaido、Takashi Narui、Daisuke Yamasaki、Junichi Goto：“孵育混合物中非对映体氟比洛芬葡萄糖醛酸苷的快速简单定量测定方法”Journal of Chromatography B. 776. 125-131 (2002)

Narihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)

Narihiro Kobayashi：“基于单克隆抗体的酶联免疫吸附测定人尿液中的硫酸乙醇胆酸，用于肝功能测试”类固醇。