DETERMINATION OF ANTIBODY SPECIFICITY USING AFM

使用 AFM 测定抗体特异性

• 批准号: 6317096
• 负责人: CHARLES W SOKOLIK
• 金额: $ 10.65万
• 依托单位: DENISON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6317096
• 关键词: acidity /alkalinity; adhesions; antibody specificity; atomic force microscopy; beta galactosidase; bioengineering /biomedical engineering; bioimaging /biomedical imaging; biophysics; combinatorial chemistry; endorphins; high throughput technology; intermolecular interaction; ionic strengths; molecular site; monoclonal antibody; peptide chemical synthesis; peptide library; protein sequence; protein structure function; statistics /biometry; structural biology; surface property; synthetic antigens; technology /technique development; temperature

Description (provided by applicant): This research will use atomic force microscopy (AFM) and peptide combinatorial libraries to quantify and determine directly the specificity of an antibody. The antibody under investigation (mouse anti-beta-endorphin (clone 3-E7) and mouse anti-Escherichia coli beta-galactosidase (clone D19-2F3-2)) is attached to the AFM cantilever tip and used to probe surfaces with covalently attached peptides. Force/distance curves are generated from which the adhesion force of the tip to the surface is calculated. The use of AFM to assess antibody binding has the advantage over conventional techniques involving enzyme-linked immunosorbant assays (ELISA) in that strong and weak binding antigens will be identified rather than just the strongest binding antigen. For the anti-beta-endorphin antibody the peptides PGGFL and YGGFL are attached to a glass cover slip surface. Preliminary results indicate that the adhesion force for the antibody is greater with the cognate peptide (YGGFL) than for the non-cognate peptide (PGGFL). Additional studies will be performed involving alterations to buffer conditions to thoroughly characterize the binding of anti-beta-endorphin antibody. Peptide combinatorial libraries will be used as the surfaces for probing with the anti-E. coli beta-galactosidase derivatized AFM tip as a means to determine the specificity of this antibody. The libraries will be constructed on a silicon substrate using a novel ink-jet printer technology. This technology produces spatially defined peptide combinatorial libraries. The libraries can be synthesized in a short period of time, tailored for the antibody being studied, scanned relatively quickly with AFM, and they are reusable. The utilization of the AFM to probe libraries made by the ink-jet printer technology will provide for a faster, more reliable and reproducible methodology for uncovering the binding characteristics of an antibody.

描述（申请人提供）：本研究将使用原子力 显微镜（AFM）和肽组合库，以定量和确定 抗体的特异性。正在研究的抗体 （小鼠抗β-内啡肽（克隆3-E7）和小鼠抗大肠杆菌 β-半乳糖苷酶（克隆D19- 2F 3 -2））连接到AFM悬臂尖端， 用于探测具有共价连接的肽的表面。力/距离曲线 由此产生尖端对表面的粘附力， 计算了使用AFM评估抗体结合具有优于 涉及酶联免疫吸附测定（ELISA）的常规技术 强结合抗原和弱结合抗原将被识别，而不仅仅是 最强结合抗原。对于抗β-内啡肽抗体， PGGFL和YGGFL附着在玻璃盖玻片表面上。初步结果 表明抗体与同源物的粘附力更大 与非同源肽（PGGFL）相比，非同源肽（YGGFL）具有更高的同源性。其他研究 将进行涉及改变缓冲条件，以彻底 表征抗β-内啡肽抗体的结合。肽组合 文库将被用作用抗E.杆菌 β-半乳糖苷酶衍生的AFM针尖作为确定特异性的手段 这个抗体。这些图书馆将建在硅基片上 使用一种新的喷墨打印机技术。这项技术在空间上产生 定义的肽组合文库。所述文库可以在一个或多个聚合物中合成。 短时间内，为正在研究的抗体量身定制，扫描 用原子力显微镜测量相对快，而且它们是可重复使用的。原子力显微镜的应用 探测由喷墨打印机技术制作的图书馆将提供一个 更快、更可靠和可重复的方法， 抗体的特性。

项目成果

A simple and sensitive assay for measuring very small volumes of microprinted solutions.

• DOI: 10.4137/aci.s7827
• 发表时间: 2011
• 期刊: Analytical chemistry insights
• 作者: Sokolik CW;Walker AS;Nishioka GM
• 通讯作者: Nishioka GM