College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China.
Biosens Bioelectron. 2011 Oct 15;28(1):91-6. doi: 10.1016/j.bios.2011.07.003. Epub 2011 Jul 13.
The amount of active capture antibodies immobilized per unit square is crucial to developing effective antibody chips, biosensors, immunoassays and other molecular recognition technologies. In this study, we present a novel yet simple method for oriented antibody immobilization at high density based on the formation of an orderly, organized aggregation of immunoglobulin G (IgG) and staphylococcal protein A (SPA). Following the chelation of His-tag with Ni(2+), antibodies were immobilized on a solid surface in a three-dimensional (3D) manner and exposed the analyte receptor sites well, which resulted in a substantial enhancement of the analytical signal with more than 64-fold increase in detection sensitivity. Pull-down assays confirmed that IgG antibody can only bind to Ni(2+) matrix indirectly via a SPA linkage, where the His-tag is responsible for binding Ni(2+) and homologous domains are responsible for binding IgG Fc. The immobilization approach proposed here may be an attractive strategy for the construction of high performance antibody arrays and biosensors as long as the antibody probe is of mammalian IgG.
固定在单位面积上的活性捕获抗体的数量对于开发有效的抗体芯片、生物传感器、免疫分析和其他分子识别技术至关重要。在这项研究中，我们提出了一种新颖而简单的方法，基于免疫球蛋白 G（IgG）和葡萄球菌蛋白 A（SPA）的有序、有组织聚集，实现抗体的高密度定向固定。在 His 标签与 Ni（2+）螯合后，抗体以三维（3D）方式固定在固体表面上，并很好地暴露了分析物受体部位，从而大大增强了分析信号，检测灵敏度提高了 64 倍以上。下拉实验证实，IgG 抗体只能通过 SPA 键间接与 Ni（2+）基质结合，其中 His 标签负责与 Ni（2+）结合，同源结构域负责与 IgG Fc 结合。只要抗体探针是哺乳动物 IgG，这里提出的固定方法可能是构建高性能抗体阵列和生物传感器的有吸引力的策略。
