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STUDIES ON GENERATION OF NOVEL ANTIBODY MOLECULES FOR MOLECULAR RECOGNITION

用于分子识别的新型抗体分子的生成研究

基本信息

批准号：
09557186
负责人：
GOTO Junichi
金额：
$ 8.32万
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：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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 attempt to generate novel antibody molecules, which may allow us to develop excellent analytical systems with ultrahigh sensitivity and specificity.α-type and β-type monoclonal anti-idiotype antibodies, each recognizing the framework region and paratope of an anti-hapten antibody have been produced by the B-cell hybridoma technique, for developing a highly sensitive noncompetitive immunoassay system which has been difficult to establish for haptens. Ursodeoxycholic acid 7-N-acetylglucosaminide (UDCA 7-NAG) and 11-deoxycortisol (11-DC) were selected as the model hapten. The combined use of the α- and β-type anti-idiotype antibodies together with the anti-hapten antibody provided a highly sensitive assay system : the detection limit was 70 fg (118 amol) for UDCA 7-NAG and 1 pg (2.9 fmol) for 11-DC. A cross-reaction study revealed that both assay systems have practical specificity. The UDCA 7-NAG noncompetitive assay system has already been well validated to be available for direct measurement (without any pretreatment) of human urine levels of this metabolite.On the other hand, a single-chain Fv fragment (scFv) against 11-DC, which shows very high affinity (Ka 1.3 x 10ィイD110ィエD1 MィイD1-1ィエD1) and specificity (cross-reactivity with cortisol, 0.14%, cortisone 0.21%), has been clones. Cloning of a fusion protein between this scFv and green fluorescent protein, which would enable a sensitive fluoroimmunoassay system, is now ongoing in our laboratory.

抗体作为一种关键试剂广泛应用于各种免疫测定或免疫亲和纯化系统中，在生物医学分析中具有重要作用。然而，传统的抗体在亲和力和特异性上都有一定的局限性，导致这些免疫化学技术的灵敏度不足或选择性较低。在这项研究中，我们试图产生新的抗体分子，这可能使我们能够开发具有超高灵敏度和特异性的优秀分析系统。利用b细胞杂交瘤技术制备了α型和β型单克隆抗独特型抗体，它们分别识别半抗原抗体的框架区和副表面，用于建立半抗原的高灵敏度非竞争性免疫分析系统。选择熊去氧胆酸7- n -乙酰氨基葡萄糖（UDCA - 7-NAG）和11-脱氧皮质醇（11-DC）作为模型半抗原。α-型和β型抗独特型抗体与抗半抗原抗体联合使用提供了一个高度敏感的检测系统：UDCA 7-NAG的检测限为70 fg (118 amol)， 11-DC的检测限为1 pg （2.9 fmol）。交叉反应研究表明，这两种检测系统都具有实际特异性。UDCA 7-NAG非竞争性分析系统已被充分验证可用于直接测量（无需任何预处理）人类尿液中该代谢物的水平。另一方面,一个单链阵线对11-DC片段(scFv),它显示了很高的亲和力(Ka 1.3 x 10ィイD110ィエD1 MィイD1-1ィエD1)和特异性(大皮质醇,0.14%,可的松0.21%),已克隆。该scFv和绿色荧光蛋白之间的融合蛋白的克隆，将使一个敏感的荧光免疫分析系统，目前正在我们的实验室进行。