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

抗体在各种免疫测定或免疫亲和力纯化系统中被广泛用作关键试剂，这在生物医学分析中至关重要。但是，常规抗体对亲和力和特异性都有一定的局限性，导致这些免疫化学技术的敏感性不足或较低的选择性，在这项研究中，我们试图产生新型的抗体物种，抗极限型抗体（抗体抗体（抗体抗体复合物），并具有抗原抗体复合物和“超级抗体”（具有基因抗体）（具有基因抗体）（具有基因抗体），并具有基因抗体，是基因抗体的。可能使我们能够开发出出色的生物活性小分子（HAPTENS）的分析系统.ANTI-METYPE抗体，这些抗体专门认识到目标触觉和抗Hapten抗体之间的复合物，预计将实现非竞争性免疫测定系统的类固醇和合成药物，并显示出更高的敏感性免疫剂。首先，我们准备了针对岩性酸3-硫酸盐和脱氧胆酸（DCA）的单次chai…更多的N FV片段（SCFV），因为SCFV缺乏免疫球蛋白的恒定区域，因此会诱导此类抗激型抗体。小鼠单克隆抗体对这些胆汁酸的V_H-和V_L基因使用从相应的杂交瘤细胞中提取的RNA放大，然后将其组装到编码5'-v_h-（Gly_4-ser）（Gly_4-ser）_3-v_l-3'的SCFV序列中，并在E. coli中张紧。所得的SCFV蛋白显示出与母小鼠抗体的相关触觉相似的反应性。然后，我们研究了Auti-DCA抗体伞状与相关触觉DCA的亲和力标记，因为这将有效抑制触觉和抗体之间的分离，并增加产生抗激型抗体的机会。我们发现DCA-丙烯酸酯对蛋白质上的原代氨基基团高反应，并且作为标记剂的功能非常好。用亲和力标记的DCA-ANTI-DCA SCFV复合物对小鼠进行免疫接种将为我们提供抗激素抗体，从而实现敏感的非竞争性免疫测定程序。较少的

项目成果

Norihiro Kobayashi: "Production of a monoclonal antibody for sensitive monitoring of deoxycholic acid residues anchored on endogenous proteins"Analytical Sciences. 16. 1133-1138 (2000)

Norihiro Kobayashi：“生产单克隆抗体，用于灵敏监测锚定于内源性蛋白质上的脱氧胆酸残基”分析科学。

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: "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。

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)