Nonspecific amine immobilization of ligand can be a potential source of error in BIAcore binding experiments and may reduce binding affinities
Nonspecific amine immobilization of ligand can be a potential source of error in BIAcore binding experiments and may reduce binding affinities
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DOI：
10.1006/abio.1997.2333
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发表时间：
1997-11-01
影响因子：
2.9
通讯作者：
Hudson, PJ
中科院分区：
文献类型：
--
作者：
Kortt, AA;Oddie, GW;Hudson, PJ
单价形式的NC 41（一种抗病毒神经氨酸酶抗体）和抗独特型抗体11- 1G 10的相互作用已被用作BIAcore分析的模型系统，以证明非特异性胺偶联程序导致的潜在问题。为了避免由于抗体二价引起的并发症，使用单价Fab片段和单体重组scFv。当通过胺偶联固定时，发现11- 1G 10抗独特型片段与通过C-末端硫醇残基使用定点固定和从溶液平衡测量获得的结果相比对NC 41具有人为降低的亲和力。另一方面，当通过游离胺基非特异性固定时，NC 41抗体片段能够保持其11- 1G 10结合亲和力。这些数据，结合两种抗体的已知序列，表明通过靠近11- 1G 10 V-H结构域的CDR 2区域或在其内部的一个或多个赖氨酸残基的非特异性固定是与NC 41相互作用强度降低的原因。这些结果强调需要使用特定位点的固定策略时，需要准确的动力学测量。(C)北京：科学出版社.
The interaction of monovalent forms of NC41, an anti-viral neuraminidase antibody, and the antiidiotype antibody 11-1G10 has been used as a model system for BIAcore analysis to demonstrate the potential problems resulting from the nonspecific amine coupling procedure. To avoid complications due to antibody bivalency, monovalent Fab fragments and monomeric recombinant scFvs were used. When immobilized by amine coupling, the 11-1G10 anti-idiotype fragments were found to have an artificially reduced affinity for NC41 compared to the results obtained using site-directed immobilization via C-terminal thiol residue and from solution equilibrium measurements. The NC41 antibody fragments, on the other hand, were able to retain their 11-1G10 binding affinity when immobilized nonspecifically through free amine groups. These data, in combination with the known sequences of the two antibodies, suggested that nonspecific immobilization through one or more lysine residues close to or within the CDR2 region of the 11-1G10 V-H domain was responsible for the reduced strength of the interaction with NC41. These results emphasize the need to use site-specific immobilization strategies when accurate kinetic measurements are required. (C) 1997 Academic Press.