Biological Physics Group, School of Physics and Astronomy, University of Manchester, Oxford Road, Manchester M13 9PL, UK.
J R Soc Interface. 2012 Oct 7;9(75):2457-67. doi: 10.1098/rsif.2012.0148. Epub 2012 May 2.
The specific recognition between monoclonal antibody (anti-human prostate-specific antigen, anti-hPSA) and its antigen (human prostate-specific antigen, hPSA) has promising applications in prostate cancer diagnostics and other biosensor applications. However, because of steric constraints associated with interfacial packing and molecular orientations, the binding efficiency is often very low. In this study, spectroscopic ellipsometry and neutron reflection have been used to investigate how solution pH, salt concentration and surface chemistry affect antibody adsorption and subsequent antigen binding. The adsorbed amount of antibody was found to vary with pH and the maximum adsorption occurred between pH 5 and 6, close to the isoelectric point of the antibody. By contrast, the highest antigen binding efficiency occurred close to the neutral pH. Increasing the ionic strength reduced antibody adsorbed amount at the silica-water interface but had little effect on antigen binding. Further studies of antibody adsorption on hydrophobic C8 (octyltrimethoxysilane) surface and chemical attachment of antibody on (3-mercaptopropyl)trimethoxysilane/4-maleimidobutyric acid N-hydroxysuccinimide ester-modified surface have also been undertaken. It was found that on all surfaces studied, the antibody predominantly adopted the 'flat on' orientation, and antigen-binding capabilities were comparable. The results indicate that antibody immobilization via appropriate physical adsorption can replace elaborate interfacial molecular engineering involving complex covalent attachments.
单克隆抗体（抗人前列腺特异性抗原，抗-hPSA）与其抗原（人前列腺特异性抗原，hPSA）之间的特异性识别在前列腺癌诊断和其他生物传感器应用中具有广阔的应用前景。然而，由于界面组装和分子取向的空间限制，结合效率通常非常低。在这项研究中，光谱椭圆术和中子反射被用来研究溶液 pH 值、盐浓度和表面化学如何影响抗体的吸附以及随后的抗原结合。发现抗体的吸附量随 pH 值而变化，最大吸附发生在 pH5 到 6 之间，接近抗体的等电点。相比之下，抗原结合效率最高发生在接近中性 pH 值的地方。增加离子强度会减少硅烷-水界面处的抗体吸附量，但对抗原结合几乎没有影响。还进一步研究了抗体在疏水性 C8（辛基三甲氧基硅烷）表面上的吸附以及抗体在（3-巯丙基）三甲氧基硅烷/4-马来酰亚胺丁酸 N-羟基琥珀酰亚胺酯修饰表面上的化学附着。结果表明，在所有研究的表面上，抗体主要采用“平躺”取向，并且具有相当的抗原结合能力。结果表明，通过适当的物理吸附固定抗体可以替代涉及复杂共价键合的复杂界面分子工程。
