Kortt A A, Nice E, Gruen L C
CRC for Diagnostic Technologies, 343 Royal Parade, Parkville, 3052, Australia.
Anal Biochem. 1999 Aug 15;273(1):133-41. doi: 10.1006/abio.1999.4183.
The binding of the Fab fragment of monoclonal antibody NC10 to influenza virus N9 neuraminidase, isolated from tern and whale, was measured using an optical biosensor. Both neuraminidases, homotetramers of 190 kDa, were immobilized to avoid multivalent binding, and the binding of the monovalent NC10 Fab to immobilized neuraminidase was analyzed using the 1:1 Langmuir binding model. A contribution of mass transport to the kinetic constants was demonstrated at higher surface densities and low flow rates, and was minimized at low ligand densities and relatively high flow rates (up to 100 microl/min). Application of a global fitting algorithm to a 1:1 binding model incorporating a correction term for mass transport indicated that mass transport was minimized under appropriate experimental conditions; analysis of binding data with a mass transport component, using this model, yielded kinetic constants similar to those obtained with the 1:1 Langmuir binding model applied to binding data where mass transport had been minimized experimentally. The binding constant for binding of NC10 Fab to N9 neuraminidase from tern influenza virus (K(A) = 6.3 +/- 1.3 x 10(7) M(-1)) was about 15-fold higher than that for the NC10 Fab binding to N9 neuraminidase from whale influenza virus (K(A) = 4.3 +/- 0.7 x 10(6) M(-1)). This difference in binding affinity was mainly attributable to a 12-fold faster dissociation rate constant of the whale neuraminidase-NC10 Fab complex and may be due to either (i) the long-range structural effects caused by mutation of two residues distant from the binding epitope or (ii) differences in carbohydrate residues, attached to Asn(200), which form part of the binding epitope on both neuraminidases to which NC10 Fab binds.
使用光学生物传感器测定了从燕鸥和鲸中分离出的单克隆抗体NC10的Fab片段与流感病毒N9神经氨酸酶的结合情况。两种神经氨酸酶均为190 kDa的同四聚体,将其固定以避免多价结合,并使用1:1朗缪尔结合模型分析单价NC10 Fab与固定化神经氨酸酶的结合。在较高表面密度和低流速下证明了传质对动力学常数有贡献,而在低配体密度和相对较高流速(高达100微升/分钟)下传质贡献最小。将全局拟合算法应用于包含传质校正项的1:1结合模型表明,在适当的实验条件下传质最小化;使用该模型对带有传质成分的结合数据进行分析,得到的动力学常数与将1:1朗缪尔结合模型应用于传质已通过实验最小化的结合数据时得到的动力学常数相似。NC10 Fab与来自燕鸥流感病毒的N9神经氨酸酶的结合常数(K(A)=6.3±1.3×10(7) M(-1))比其与来自鲸流感病毒的N9神经氨酸酶的结合常数(K(A)=4.3±0.7×10(6) M(-1))高约15倍。这种结合亲和力的差异主要归因于鲸神经氨酸酶-NC10 Fab复合物的解离速率常数快12倍,可能是由于(i)远离结合表位的两个残基突变引起的远程结构效应,或(ii)附着于Asn(200)的碳水化合物残基的差异,Asn(200)形成了两种神经氨酸酶上NC10 Fab结合的结合表位的一部分。