Wang Lu, Zhou Lele, Ma Ning, Wan Yizhen, Zhang Yu, Xu Bin, Qian Weiping
State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Center of Clinical Laboratory Science, The Affiliated Cancer Hospital of Nanjing Medical University & Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research, Nanjing 210009, China.
Int J Biol Macromol. 2023 Apr 30;235:123872. doi: 10.1016/j.ijbiomac.2023.123872. Epub 2023 Mar 5.
Antigen binding fragments (Fabs) employed in research are typically generated by the papain digestion of monoclonal antibodies. However, the interaction between papain and antibodies at the interface remains unclear. Herein, we developed ordered porous layer interferometry for the label-free monitoring of the interaction between the antibody and papain at liquid-solid interfaces. Human immunoglobulin G (hIgG) was used as the model antibody, and different strategies were employed to immobilize it on the surface of silica colloidal crystal (SCC) films which are optical interferometric substrates. It was observed that different immobilization strategies induced different changes in the optical thickness (OT) of SCCs. The order of rate of the changes of OT from largest to smallest was IgG immobilized by protein A orientation, glutaraldehyde coupling, and physical adsorption. This phenomenon can be explained by the varied orientations of the antibodies created at the interface by the different modification procedures. The Fab-up orientation maximized the exposure of the hinge region sulfhydryl group and easily underwent conformational transitions because hIgG was immobilized by protein A. This process stimulates papain to produce the highest degree of activity, resulting in the greatest decrease in OT. This study provides insights into the catalysis of papain on antibodies.
研究中使用的抗原结合片段（Fabs）通常通过木瓜蛋白酶消化单克隆抗体产生。然而，木瓜蛋白酶与抗体在界面处的相互作用仍不清楚。在此，我们开发了有序多孔层干涉术，用于无标记监测抗体与木瓜蛋白酶在液固界面的相互作用。人免疫球蛋白G（hIgG）用作模型抗体，并采用不同策略将其固定在作为光学干涉基底的二氧化硅胶体晶体（SCC）薄膜表面。观察到不同的固定策略会引起SCCs光学厚度（OT）的不同变化。OT变化速率从大到小的顺序为通过蛋白A定向、戊二醛偶联和物理吸附固定的IgG。这种现象可以通过不同修饰程序在界面处产生的抗体不同取向来解释。Fab向上取向使铰链区巯基的暴露最大化，并且由于hIgG通过蛋白A固定，容易发生构象转变。这个过程刺激木瓜蛋白酶产生最高程度的活性，导致OT的最大下降。本研究为木瓜蛋白酶对抗体的催化作用提供了见解。
