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些局限性，从而导致这些免疫化学技术的灵敏度不足或选择性低。在这项研究中，我们试图生成新型的抗体分子，这可能使我们能够开发具有超高灵敏度和特异性的出色分析系统。α型和β型单克隆抗IDiot型抗体，每种抗体的框架区域和抗体抗体的高度敏感性都在敏感性的敏感性中识别出抗体的高度敏感性。很难为触觉建立的系统。选择了ursodoxycholic酸7-n-乙酰葡萄糖酰胺（UDCA 7-NAG）和11-脱氧皮质醇（11-DC），作为α-和β型抗抗体型抗体的联合使用，以及抗Hapten抗体的总使用，以及抗Hapten抗体，以及高度敏感的抗体：70 fg）（11 AMAM）（118 AMAM）。 PG（2.9 FMOL）为11-DC。一项交叉反应研究表明，这两个测定系统都有实际特异性。 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 D110 D1 Mii D1-1 D1) and specificity (cross-reactivity with cortisol, 0.14％，可的松为0.21％），是克隆。在我们的实验室中正在进行中，该SCFV和绿色荧光蛋白之间的融合蛋白的克隆，这将使荧光免疫测定系统具有敏感性。