DETERMINATION OF ANTIBODY SPECIFICITY USING AFM

描述(申请人提供)：这项研究将使用原子力 显微镜(AFM)和多肽组合文库的定量和测定 直接反映抗体的特异性。正在调查的抗体 (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D19-2F3-2)连接到AFM悬臂尖端，并 用于探测带有共价结合多肽的表面。力/距离曲线 由此产生尖端与表面的粘附力 计算出来的。使用原子力显微镜评估抗体结合具有以下优点 常规技术涉及酶联免疫吸附试验(ELISA)在 强结合抗原和弱结合抗原将被识别，而不仅仅是 最强的结合抗原。对于抗β-内啡肽抗体的多肽 PGGFL和YGGFL固定在玻璃盖滑面上。初步结果 说明同源抗体对抗体的粘附力较大。 多肽(YGGFL)比非同源多肽(PGGFL)少。其他研究 将执行涉及更改缓冲条件的操作以彻底 鉴定抗β-内啡肽抗体的结合力。多肽组合 文库将作为与抗E.大肠埃希氏菌 β-半乳糖苷酶衍生化AFM针尖作为确定特异性的手段 这种抗体。这些文库将建在硅衬底上 使用一种新的喷墨打印机技术。这项技术在空间上产生 已定义的多肽组合文库。这些库可以在一个 短时间，为正在研究的抗体量身定做，扫描 相对较快的原子力显微镜，他们是可重复使用的。原子力显微镜的应用 探针库由喷墨打印机技术制作而成，将为 用于解开绑定的更快、更可靠和可重现的方法 抗体的特征。

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.