Stigter E C A, de Jong G J, van Bennekom W P
Department of Biomedical Analysis, Faculty of Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Biosens Bioelectron. 2005 Sep 15;21(3):474-82. doi: 10.1016/j.bios.2004.11.008. Epub 2004 Dec 19.
A study was initiated to investigate the use of surface plasmon resonance (SPR) for the detection in plasma of a high pI model protein, recombinant human interferon-gamma (IFN-gamma). Initially a number of self-assembled monolayers (SAMs) and hydrogel-derivatised SAM-coatings were characterised for the adsorptive and desorptive properties of plasma components. Next a monoclonal anti-IFN-gamma antibody, MD-2, was covalently attached to dextran-modified mercaptoundecanoic acid surfaces that performed best. On coatings consisting of carboxyl-modified dextran (CMD) a difference in interaction behaviour was observed when IFN-gamma was injected in either buffer or diluted plasma. During the injection of IFN-gamma in buffer, an acceleration of the interaction process was observed and the signal continued to increase after the injection plug had passed. Upon injection of diluted plasma spiked with IFN-gamma, the response increased without acceleration of the binding process. After the injection was finished, some of the bound material desorbed as expected, resulting in a signal decrease. On non-charged dextrans, the interaction between the antibody-modified surface and IFN-gamma in either plasma or buffer was similar. During sample injection the response increased with a binding rate depending on the concentration of IFN-gamma present in solution. When the injection was finished, some of the bound material was washed away from the surface and only a minor contribution of non-specific adsorbed plasma components was noticeable. From the coatings tested, the non-modified dextran-coated SPR sensor disks prove to be best suited for the detection of IFN-gamma in complex matrices like plasma. The interaction of IFN-gamma in both diluted plasma and buffer is comparable and concentrations of IFN-gamma of 250 ng ml-1 and higher can be detected in both buffer and 100x-diluted plasma. The non-specific adsorption of plasma components is low, whereas the specific IFN-gamma response is relatively high.
开展了一项研究，以调查表面等离子体共振（SPR）用于检测血浆中高pI模型蛋白重组人干扰素-γ（IFN-γ）的情况。最初，对一些自组装单分子层（SAMs）和水凝胶衍生的SAM涂层进行了表征，以研究血浆成分的吸附和解吸特性。接下来，将单克隆抗IFN-γ抗体MD-2共价连接到性能最佳的葡聚糖修饰的巯基十一烷酸表面。在由羧基修饰的葡聚糖（CMD）组成的涂层上，当在缓冲液或稀释血浆中注入IFN-γ时，观察到相互作用行为存在差异。在缓冲液中注入IFN-γ期间，观察到相互作用过程加速，并且在注入塞通过后信号继续增加。在注入加有IFN-γ的稀释血浆时，响应增加，但结合过程没有加速。注射完成后，一些结合的物质如预期那样解吸，导致信号下降。在不带电荷的葡聚糖上，抗体修饰表面与血浆或缓冲液中的IFN-γ之间的相互作用相似。在样品注入期间，响应以取决于溶液中IFN-γ浓度的结合速率增加。注射完成后，一些结合的物质从表面被冲洗掉，只有少量非特异性吸附的血浆成分是明显的。在所测试的涂层中，未修饰的葡聚糖包被的SPR传感器盘被证明最适合于检测血浆等复杂基质中的IFN-γ。IFN-γ在稀释血浆和缓冲液中的相互作用相当，在缓冲液和100倍稀释血浆中均可检测到浓度为250 ng ml-1及更高的IFN-γ。血浆成分的非特异性吸附较低，而特异性IFN-γ响应相对较高。
