Biological Physics Lab, School of Physics and Astronomy, University of Manchester, Schuster Building, Oxford Road, Manchester M13 9PL, United Kingdom.
Langmuir. 2011 Jun 21;27(12):7654-62. doi: 10.1021/la201245q. Epub 2011 May 25.
Antibody orientation and its antigen binding efficiency at interface are of particular interest in many immunoassays and biosensor applications. In this paper, spectroscopic ellipsometry (SE), neutron reflection (NR), and dual polarization interferometry (DPI) have been used to investigate interfacial assembly of the antibody [mouse monoclonal anti-human prostate-specific antigen (anti-hPSA)] at the silicon oxide/water interface and subsequent antigen binding. It was found that the mass density of antibody adsorbed at the interface increased with solution concentration and adsorption time while the antigen binding efficiency showed a steady decline with increasing antibody amount at the interface over the concentration range studied. The amount of antigen bound to the interfacial immobilized antibody reached a maximum when the surface-adsorbed amount of antibody was around 1.5 mg/m(2). This phenomenon is well interpreted by the interfacial structural packing or crowding. NR revealed that the Y-shaped antibody laid flat on the interface at low surface mass density with a thickness around 40 Å, equivalent to the short axial length of the antibody molecule. The loose packing of the antibody within this range resulted in better antigen binding efficiency, while the subsequent increase of surface-adsorbed amount led to the crowding or overlapping of antibody fragments, hence reducing the antigen binding due to the steric hindrance. In situ studies of antigen binding by both NR and DPI demonstrated that the antigen inserted into the antibody layer rather than forming an additional layer on the top. Stability assaying revealed that the antibody immobilized at the silica surface remained stable and active over the monitoring period of 4 months. These results are useful in forming a general understanding of antibody interfacial behavior and particularly relevant to the control of their activity and stability in biosensor development.
抗体在界面上的取向及其抗原结合效率在许多免疫分析和生物传感器应用中尤为重要。在本文中，光谱椭圆术（SE）、中子反射（NR）和双偏振干涉测量（DPI）被用于研究抗体[鼠单克隆抗人前列腺特异性抗原（抗-hPSA）]在氧化硅/水界面上的界面组装以及随后的抗原结合。结果发现，吸附在界面上的抗体的质量密度随着溶液浓度和吸附时间的增加而增加，而抗原结合效率随着界面上抗体量的增加而稳定下降，研究浓度范围内。当界面上吸附的抗体量约为 1.5mg/m2 时，与界面固定抗体结合的抗原量达到最大值。这种现象可以通过界面结构的堆积或拥挤得到很好的解释。NR 表明，Y 形抗体在低表面质量密度下在界面上平躺，厚度约为 40Å，相当于抗体分子的短轴长度。在这个范围内，抗体的松散堆积导致更好的抗原结合效率，而随后表面吸附量的增加导致抗体片段的拥挤或重叠，从而由于空间位阻而降低抗原结合。NR 和 DPI 对抗原结合的原位研究表明，抗原插入到抗体层中，而不是在顶部形成额外的层。稳定性测定表明，固定在硅表面的抗体在 4 个月的监测期内保持稳定和活性。这些结果有助于形成对抗体界面行为的一般理解，特别是对控制其在生物传感器开发中的活性和稳定性具有重要意义。
