Effect of surface packing density of interfacially adsorbed monoclonal antibody on the binding of hormonal antigen human chorionic gonadotrophin
Effect of surface packing density of interfacially adsorbed monoclonal antibody on the binding of hormonal antigen human chorionic gonadotrophin
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DOI：
10.1021/jp0538161
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发表时间：
2006-02-02
影响因子：
3.3
通讯作者：
Williams, DE
中科院分区：
文献类型：
--
作者：
Xu, H;Lu, JR;Williams, DE
界面吸附的小鼠单克隆抗体（类型IgG 1，抗β-hCG）在亲水性氧化硅/水界面已被研究，通过椭圆偏振光谱和中子反射，然后通过评估结合的激素抗原，人绒毛膜促性腺激素（hCG），吸附的抗体分子。对于抗体，吸附量在等电点pH 6附近达到最大值;这种pH依赖性模式可以通过增加盐浓度来改变，对于其他蛋白质也观察到这种趋势。中子反射揭示了从吸附的抗体形成40埃均匀层，表明平贴取向。随后的hCG结合表明，在界面处结合抗体的hCG的摩尔比高达0.7，在低表面覆盖度的抗体，并随着表面抗体浓度的增加而降低。结果表明，随着表面上抗体分子堆积密度的增加，hCG进入的空间位阻程度增加。与先前发表的晶体结构研究的比较表明可变区的扭曲允许抗原进入。hCG的结合也被发现是pH依赖性的，其最大值在IP附近，如果溶液的离子强度低（20 mM）。然而，如果离子强度增加到200 mM，则hCG结合受到抗原和表面之间的空间位阻和静电相互作用的组合的影响。这些结果与提高生物技术的性能高度相关，例如基于抗体固定的生育力测试垫和生物传感器。
Interfacial adsorption of a mouse monoclonal antibody (type IgG1, anti-beta-hCG) at the hydrophilic silicon oxide/water interface has been studied by spectroscopic ellipsometry and neutron reflection, followed by assessment of binding of a hormonal antigen, human chorionic gonadotrophin (hCG), onto the adsorbed antibody molecules. The amount of adsorption reached a maximum around the isoelectric pH JP) of 6 for the antibody; this pH-dependent pattern could be altered by increasing salt concentration, a trend also observed for other proteins. Neutron reflection revealed the formation of a 40 angstrom uniform layer from the adsorbed antibody, indicating a flat-on orientation. The subsequent hCG binding showed that the molar ratio of hCG bound to antibody at the interface was as high as 0.7 at low surface coverage of antibody and decreased with increasing surface antibody concentration. The results point to an increasing extent of steric hindrance to hCG access with increasing packing density of antibody molecules on the surface. Comparison with previously published crystal Structure studies suggests twisting of the variable region to allow access of the antigen. The binding of hCG was also found to be pH-dependent with its maximum around the IP, if the ionic strength of the solution was low (20 mM). However, if the ionic strength was increased to 200 mM, then hCG binding was influenced by a combination of steric hindrance and electrostatic interaction between the antigen and the surface. These results are highly relevant to the improvement of the performance of biotechnologies such as fertility test pads and biosensors based on antibody immobilization.