Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface
Development of a one-step ELISA method using an affinity peptide tag specific to a hydrophilic polystyrene surface
复制标题
DOI：
10.1016/j.jbiotec.2006.07.011
复制
发表时间：
2007-01-01
影响因子：
4.1
通讯作者：
Nakanishi, Kazuhiro
中科院分区：
文献类型：
--
作者：
Kumada, Yoichi;Katoh, Shigeo;Nakanishi, Kazuhiro
将与亲和肽标签PS 19（RAFIASRRIKRP）遗传融合的谷胱甘肽S-转移酶优先固定在亲水性PS（phi-PS）板上，而不受共存蛋白质分子的干扰，所述亲和肽标签PS 19对亲水性聚苯乙烯（PS）表面具有特异性亲和力。此外，与肽KPS 19 R10化学缀合的兔IgG显示出比与PS 19肽缀合的兔IgG更高的对phi-PS板的固定化亲和力，其中PS 19中的（10）Lys被Arg取代，并且在N-末端添加一个Lys残基作为戊二醛的偶联位点。基于这些发现，使用phi-PS板和肽标签连接的配体蛋白允许实现一步或两步酶联免疫吸附测定（ELISA），与使用疏水PS（pho-PS）板的常规ELISA方法相比，操作时间大幅减少，同时保持高灵敏度。与常规ELISA方法相比，一步ELISA法在夹心模式下检测牛胰岛素时，由于减少了洗涤和孵育步骤，灵敏度得到了较大程度的提高。本文提出的方法将是用于各种ELISA技术的通用方法，例如使用与PS特异性亲和肽遗传融合或化学缀合的抗原、抗体和链霉亲和素作为配体蛋白的夹心和竞争性ELISA。(c)2006 Elsevier B. V.保留所有权利。
Glutathione S-transferase genetically fused with an affinity peptide tag, PS 19 (RAFIASRRIKRP) having a specific affinity for a hydrophilic polystyrene (PS) surface, was preferentially immobilized on a hydrophilic PS (phi-PS) plate without suffering from interference by coexisting protein molecules. Furthermore, rabbit IgG chemically conjugated with a peptide, KPS 19R10, in which (10)Lys in PS 19 was replaced with Arg and one Lys residue was added at the N-terminus as a coupling site for glutaraldehyde, showed a higher immobilization affinity to the phi-PS plate than that conjugated with the PS 19 peptide. On the basis of these findings, the use of a phi-PS plate and peptide tag-linked ligand proteins permitted a one-step or two-step enzyme-linked immumosorbent assay (ELISA) to be achieved, resulting in a substantial reduction in operational time compared with the conventional ELISA method using a hydrophobic PS (pho-PS) plate, while maintaining a high sensitivity. Furthermore, the sensitivity was increased to a greater extent compared to the conventional ELISA meihod when the one-step ELISA was applied to the detection of bovine insulin in a sandwich mode, due to the reduced number of washing and incubation steps. The method proposed here would be a versatile method for use in various ELISA techniques such as sandwich and competitive ELISAs using an antigen, an antibody and streptavidin that are genetically fused or chemically conjugated with the PS-specific affinity peptide as the ligand protein. (c) 2006 Elsevier B.V. All rights reserved.