van den Elsen J M, van Pomeren E, Poolman J T, Wilting J, Herron J N, Crommelin D J
Department of Pharmaceutics, Faculty of Pharmacy, Utrecht Institute of Pharmaceutical Sciences, Utrecht University, The Netherlands.
Anal Biochem. 1997 May 1;247(2):382-8. doi: 10.1006/abio.1997.2064.
This paper describes a method for determining the affinity constant (Ka) of the binding between an antibody Fab fragment and a membrane-embedded protein epitope under equilibrium conditions. Monoclonal antibody MN12H2, directed against outer membrane protein PorA of Neisseria meningitidis, is used in a competitive fluorescence polarization assay with a cyclic peptide-fluorescein conjugate as a tracer antigen. Displacement experiments with PorA-containing and PorA-deficient meningococcal outer membrane vesicles revealed highly specific binding of MN12H2 Fab to the membrane-embedded PorA P1.16 epitope with Ka of 1.5 x 10(8) M-1.
本文描述了一种在平衡条件下测定抗体Fab片段与膜嵌入蛋白表位之间结合亲和力常数（Ka）的方法。针对脑膜炎奈瑟菌外膜蛋白PorA的单克隆抗体MN12H2，用于以环肽 - 荧光素偶联物作为示踪抗原的竞争性荧光偏振测定。用含PorA和不含PorA的脑膜炎球菌外膜囊泡进行的置换实验表明，MN12H2 Fab与膜嵌入的PorA P1.16表位具有高度特异性结合，Ka为1.5×10（8）M-1。
