Generation of mutant antibodies with ultrahigh affinity based on a novel and universal selection method and their application to trace analysis

基于新颖且通用的选择方法生成具有超高亲和力的突变抗体及其在痕量分析中的应用

• 批准号: 15390180
• 负责人: KOBAYASHI Norihiro
• 金额: $ 8.58万
• 依托单位: Kobe Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15390180/
• 关键词: antibody; single-chain Fv fragment; phage display; phage antibody; affinity; genetic engineering; cleavable biotin; selection; 新和力

The engineering of hapten-specific antibodies with a much higher affinity than those of conventional antibodies promises hapten immunoassays exhibiting sub-femtomole range sensitivity. From these points of view, we developed a novel and universal method to select phage particles displaying anti-hapten antibody fragments with exceptionally high affinity. 11-Deoxycortisol (11-DC), 17α-hydroxyprogesterone, and estradiol were selected as model target haptens, and were covalently conjugated to biotin via a spacer including a reductively cleavable disulfide bond. These cleavable biotin(CB)-conjugated steroids exhibited desirable biochemical and chemical properties : the conjugates were bound simultaneously with relevant anti-steroid antibody and streptavidin, and the disulfide bond was easily cleaved by the treatment with 50 mM dithiothreitol(DTT). Phage particles displaying high affinity single-chain Fv fragments(scFvs) specific for 11-DC (Ka=1.3×10^<10> M^<-1) were incubated with the CB-conjugated 11-DC and the resulting complexes was captured on immobilized NeutrAvidin. The DTT treatment allowed the recovery of target phages ; this process is fully independent of the dissociation of the antigen-antibody interaction. Five serial rounds of selection enabled the isolation and enrichment of the anti-11-DC-phage from among a 100,000-fold excess of nonspecific phage particles. This method will be universally applicable for selection of extra-high affinity phage antibodies against a wide variety of haptens.

具有比常规抗体高得多的亲和力的半抗原特异性抗体的工程化有望实现表现出亚飞摩尔范围灵敏度的半抗原免疫测定。从这些角度来看，我们开发了一种新的和通用的方法来选择噬菌体颗粒展示抗半抗原抗体片段具有非常高的亲和力。11-选择脱氧皮质醇（11-DC）、17α-羟孕酮和雌二醇作为模型靶半抗原，并通过包括可还原裂解二硫键的间隔区与生物素共价偶联。这些可裂解的生物素（CB）结合的类固醇表现出理想的生化和化学性质：结合物同时与相关的抗类固醇抗体和链霉亲和素，和二硫键很容易被50 mM的二硫苏糖醇（DTT）的处理裂解。将展示对11-DC具有特异性的高亲和力单链Fv片段（scFv）（Ka=1.3×10^<10>M^&lt;-1）的噬菌体颗粒与CB缀合的11-DC孵育，并将所得复合物捕获在固定化的NeutrAvidin上。DTT处理允许靶向抗体的回收;该过程完全独立于抗原-抗体相互作用的解离。五轮连续的选择使得能够从100，000倍过量的非特异性噬菌体颗粒中分离和富集抗11-DC-噬菌体。这种方法将普遍适用于选择针对多种半抗原的超高亲和力噬菌体抗体。

项目成果

Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity

基于独特型-抗独特型的非竞争性酶联免疫吸附测定人尿液中的熊去氧胆酸 7-N-乙酰氨基葡萄糖苷，灵敏度亚飞摩尔范围

• 发表时间: 2003
• 期刊: Journal of Immunological Methods 272
• 作者: Koike K;et al.;Norihiro Kobayashi
• 通讯作者: Norihiro Kobayashi

Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol, Production, characterization and application to a sensitive noncompetitive immunoassay

识别抗 11-脱氧皮质醇高亲和力抗体可变区的单克隆抗独特型抗体，生产、表征及其在敏感非竞争性免疫测定中的应用

• 发表时间: 2003
• 期刊: Journal of Immunological Methods 274
• 作者: Hatakeyama S;et al.;Norihiro Kobayashi
• 通讯作者: Norihiro Kobayashi

薬学生のための臨床化学改定第2版

药学学生临床化学修订版第二版

• 发表时间: 2005
• 作者: 後藤順一;片山善章編
• 通讯作者: 片山善章編

Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody.

• DOI: 10.1021/ac0303620
• 发表时间: 2004-02
• 期刊: Analytical chemistry
• 影响因子: 7.4
• 作者: M. Okuyama;N. Kobayashi;Wakako Takeda;T. Anjo;Y. Matsuki;J. Goto;A. Kambegawa;S. Hori

A nonenzymatic modification of the amino-terminal domain of histone H3 by bile acid acyl adenylate

胆汁酸酰基腺苷酸对组蛋白 H3 氨基末端结构域的非酶修饰

• 发表时间: 2004
• 期刊: Journal of Biological Chemistry 279
• 作者: Yoshida;K.et al.;Nariyasu Mano
• 通讯作者: Nariyasu Mano