DETERMINATION OF ANTIBODY SPECIFICITY USING AFM

使用 AFM 测定抗体特异性

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：本研究将使用原子力量显微镜（AFM）和肽组合文库，以量化和确定直接的抗体特异性。正在研究的抗体（小鼠抗β-内啡肽（克隆3-e7）和小鼠抗Escherichia coli β-半乳糖苷酶（克隆D19-2F3-2））附着在AFM Cantilever尖端和用于探测具有共同连接肽的表面。力/距离曲线是从中产生的，尖端对表面的粘附力为计算。使用AFM评估抗体结合具有优于传统技术涉及酶联免疫吸附测定法（ELISA）将确定强大而弱的结合抗原，而不仅仅是最强的结合抗原。对于抗β-内啡肽抗体，肽 PGGFL和YGGFL附着在玻璃盖板上。初步结果表明抗体的粘附力随着同源而更大肽（YGGFL）比非认知肽（PGGFL）的肽（YGGFL）。其他研究将进行涉及缓冲条件的改变以彻底进行表征抗β-内啡肽抗体的结合。肽组合库将用作抗E进行探测的表面。大肠杆菌 β-半乳糖苷酶衍生化的AFM尖端是确定特异性的一种手段该抗体。库将在硅基板上构造使用新型的喷墨打印机技术。该技术在空间上产生定义的肽组合库。库可以在短时间，针对研究的抗体量身定制，扫描与AFM相对较快，它们是可重复使用的。 AFM的利用探测墨水打印机技术制作的库将提供更快，更可靠和可重现的方法来揭示结合抗体的特征。