Ch'ng Angela Chiew Wen, Hamidon Nurul Hamizah Binti, Konthur Zoltán, Lim Theam Soon
Analytical Biochemistry Research Centre, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800, Minden, Penang, Malaysia.
Max Planck Institute of Colloids and Interfaces, Mühlenberg 1, 14476, Potsdam, Germany.
Methods Mol Biol. 2018;1701:301-319. doi: 10.1007/978-1-4939-7447-4_16.
The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface. Magnetic nanoparticles allow for improved antigen binding due to their large surface area. The Kingfisher Flex magnetic particle processing system was originally designed to aid in RNA, DNA, and protein extraction using magnetic beads. However, the system can be programmed for antibody phage display panning. The automation allows for a reduction in human error and improves reproducibility in between selections with the preprogrammed movements. The system requires minimum human intervention to operate; however, human intervention is needed for post-panning steps like phage rescue. In addition, polyclonal and monoclonal ELISA can be performed using the semi-automated platform to evaluate the selected antibody clones. This chapter will summarize the suggested protocol from the panning stage till the monoclonal ELISA evaluation. Other than this, important notes on the possible optimization and troubleshooting are also included at the end of this chapter.
重组人抗体的应用主要在诊断和治疗领域迅速增长。为了鉴定针对特定抗原的抗体，使用不同的展示技术进行淘选筛选。由于噬菌体展示技术在生物淘选实验中的稳健性和效率，它仍然是首选平台。使用聚苯乙烯塑料、磁珠和硝酸纤维素作为固定固相表面，既有手动的也有半自动的淘选筛选。磁性纳米颗粒因其大表面积而具有更好的抗原结合能力。翠鸟Flex磁性颗粒处理系统最初设计用于使用磁珠辅助RNA、DNA和蛋白质提取。然而，该系统可被编程用于抗体噬菌体展示淘选。自动化减少了人为误差，并通过预编程的动作提高了筛选之间的可重复性。该系统操作所需的人工干预最少；然而，在淘选后步骤如噬菌体拯救中需要人工干预。此外，可使用半自动平台进行多克隆和单克隆ELISA来评估所选抗体克隆。本章将总结从淘选阶段到单克隆ELISA评估的建议方案。除此之外，本章末尾还包括关于可能的优化和故障排除的重要注意事项。
