Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor - A comparison of immobilisation strategies
Kinetic analysis of the interaction between the monoclonal antibody A33 and its colonic epithelial antigen by the use of an optical biosensor - A comparison of immobilisation strategies
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DOI：
10.1016/s0021-9673(97)00087-3
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发表时间：
1997-07-25
影响因子：
4.1
通讯作者：
Nice, EC
中科院分区：
文献类型：
--
作者：
Catimel, B;Nerrie, M;Nice, EC
人源化A33单克隆抗体及其相应的F（ab）（2）'或Fab'片段与结肠上皮A33抗原的相互作用，通过微制备HPLC从结肠癌细胞系LIM 1215的膜提取物中纯化，用BIAcore 2000生物传感器使用表面等离子体共振检测进行了研究。使用替代的固定化化学来控制固定化抗体和Fab'片段在生物传感器表面上的表面取向。与常规N-羟基琥珀酰亚胺（NHS）/N-乙基-N '-二甲基氨基丙基碳二亚胺（EDC）化学相比，这导致显著更高的摩尔结合活性。这种信号的增加导致检测灵敏度的同时增加，这有助于分析低水平的A33抗原。测定了用不同固定化化学获得的表观缔合速率（k（a））和解离速率（k（d））常数。这些分析表明，获得的免疫球蛋白的动力学常数没有显着影响的方法的固定。使用NHS/EDC化学固定的F（ab）（2）'和Fab'片段显示显著较低的表观亲和力。相比之下，使用Fab'片段的硫醇偶联化学得到了观察到的K-A的五倍增加，导致与用完整IgG分子观察到的亲和力相似的亲和力。(C)1997年Elsevier Science B.V.
The interaction between the humanised A33 monoclonal antibody and the corresponding F(ab)(2)' or Fab' fragments with the colonic epithelial A33 antigen, purified by micropreparative HPLC from membrane extracts of the colonic carcinoma cell line LIM 1215, has been studied with the BIAcore 2000 biosensor using surface plasmon resonance detection. The surface orientation of immobilised antibody and the Fab' fragment onto the biosensor surface was controlled using alternative immobilisation chemistries. This resulted in significantly higher molar binding activities compared with the conventional N-hydroxysuccinimide (NHS)/N-ethyl-N'-dimethylaminopropylcarbodiimide (EDC) chemistry. This increase in signal resulted in a concomitant increase in sensitivity of detection, which facilitates analysis of low levels of A33 antigen. The apparent association rate (k(a)) and dissociation rate (k(d)) constants obtained with the different immobilisation chemistries were determined. These analyses showed that the kinetic constants obtained for the Ige were not significantly affected by the method of immobilisation. F(ab)(2)' and Fab' fragments immobilised using NHS/EDC chemistry showed significantly lower apparent affinity. By contrast the use of the thiol coupling chemistry with the Fab' fragment gave a five fold increase in observed K-A, resulting in a similar affinity to that observed with the intact IgG molecule. (C) 1997 Elsevier Science B.V.