Liu B, Marks J D
Department of Anesthesia, Department of Pharmaceutical Chemistry, University of California, San Francisco, Room 3C-38, USA.
Anal Biochem. 2000 Nov 1;286(1):119-28. doi: 10.1006/abio.2000.4788.
Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.
二维凝胶电泳是一种强大的工具，可用于鉴定在定性或定量上存在不同疾病状态的患者之间存在差异的蛋白质。如果能够获得可用于检测和定量不同蛋白质的时空模式以及细胞和组织定位的抗体，将极大地促进对这些蛋白质差异的进一步表征。为了产生这样的抗体，人们开发了一些方法，这些方法能够成功地从噬菌体展示文库中筛选出针对从SDS-PAGE凝胶印迹到硝酸纤维素膜上的抗原的单克隆噬菌体抗体。首先，已确定硝酸纤维素膜和聚偏二氟乙烯膜产生的背景噬菌体结合水平明显低于所研究的其他两种膜。其次，已确定用鱼明胶封闭并在0.5M NaCl存在下进行结合可将非特异性结合降低10000倍，并在抗原浓度低至1 ng/mm²时产生大于500倍的富集率。当将优化条件应用于噬菌体抗体文库时，针对从SDS-PAGE凝胶印迹到硝酸纤维素膜上的蛋白质ErbB2和牛血清白蛋白产生了单克隆噬菌体抗体库。根据文库中每个噬菌体展示的是单拷贝还是多拷贝抗体，仅用10到1 ng的抗原就获得了抗体。这些抗体在ELISA和蛋白质印迹中均可用作试剂。
