College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China.
College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan Province, China.
Biochem Biophys Res Commun. 2014 Jul 18;450(1):429-32. doi: 10.1016/j.bbrc.2014.05.135. Epub 2014 Jun 5.
Polystyrene is a common substrate material for protein adsorption in biosensors and bioassays. Here, we present a new method for multilayered, site-directed immobilization of antibody on polystyrene surface through the linkage of a genetically engineered ligand and the assembly of staphylococcal protein A (SPA) with immunoglobulin G (IgG). In this method, antibodies were stacked on polystyrene surface layer by layer in a potential three-dimensional way and exposed the analyte-binding sites well. Enzyme-linked immunosorbent assay (ELISA) revealed that the new method showed a 32-fold higher detection sensitivity compared with the conventional one. Pull-down assay and Western blot analysis further confirmed that it is different from the ones of monolayer adsorption according to the comparison of adsorption capacity. The differentiated introduction of functional ligands, which is the key of this method, might offer a unique idea as a way to interfere with the dynamic behavior of a protein complex during the process of adsorption.
聚苯乙烯是生物传感器和生物测定中蛋白质吸附的常见基质材料。在这里，我们通过基因工程配体的连接和葡萄球菌蛋白 A（SPA）与免疫球蛋白 G（IgG）的组装，提出了一种在聚苯乙烯表面进行多层、定点固定抗体的新方法。在这种方法中，抗体通过静电吸附逐层在聚苯乙烯表面上组装，很好地暴露了分析物结合位点。酶联免疫吸附测定（ELISA）显示，与传统方法相比，新方法的检测灵敏度提高了 32 倍。下拉实验和 Western blot 分析进一步证实，根据吸附容量的比较，它与单层吸附不同。功能配体的差异引入是该方法的关键，这可能为干扰吸附过程中蛋白质复合物的动态行为提供一种独特的思路。
