QUANTITATIVE-DETERMINATION OF SURFACE CONCENTRATION OF PROTEIN WITH SURFACE-PLASMON RESONANCE USING RADIOLABELED PROTEINS
QUANTITATIVE-DETERMINATION OF SURFACE CONCENTRATION OF PROTEIN WITH SURFACE-PLASMON RESONANCE USING RADIOLABELED PROTEINS
复制标题
DOI：
10.1016/0021-9797(91)90284-f
复制
发表时间：
1991-05-01
影响因子：
9.9
通讯作者：
URBANICZKY, C
中科院分区：
文献类型：
--
作者：
STENBERG, E;PERSSON, B;URBANICZKY, C
描述了一种将蛋白质表面绝对浓度与表面等离子体共振（SPR）响应相关联的方法。采用不同的光学技术确定了传感器芯片上各层的厚度和光学常数。在流动注射系统中，稳态SPR响应与表面闪烁计数器吸附的放射性标记蛋白的绝对数量相关。所使用的蛋白，14c标记的人转铁蛋白和乳糜蛋白酶原A，以及35s标记的体内单克隆抗体，通过静电相互作用吸附在传感器芯片上的羧甲基化葡聚糖水凝胶上。对于这些蛋白质，表面浓度从2到50 ng mm−2与SPR响应呈线性对应，特异性响应在0.10±0.01°(ng mm−2)−1范围内，与蛋白质大小无关。该SPR仪器估计蛋白质的最低可检测表面浓度为50 pg mm−2。已经建立了光学模型来描述SPR响应如何依赖于表面水凝胶体积内吸附蛋白质的分布。利用薄膜光学程序计算了不同模型下的理论SPR响应。与实验数据比较表明，该蛋白分布在约100 nm厚的葡聚糖水凝胶层内。
A methodology to correlate the absolute surface concentration of protein to the surface plasmon resonance (SPR) response is described. The thickness and the optical constants for each layer on the sensor chip used were determined with different optical techniques. In a flow injection system, the steady-state SPR response was correlated to the absolute amount of radiolabeled protein adsorbed by using a surface scintillation counter. The proteins used,14C-labeled human transferrin and chymotrypsinogen A, as well asin vivo35S-labeled monoclonal antibodies, were adsorbed via electrostatic interaction to a carboxymethylated dextran hydrogel on the sensor chip. For these proteins, surface concentrations from 2 to 50 ng mm−2correspond linearly to the SPR response, with specific response in the range 0.10 ± 0.01° (ng mm−2)−1, independent of protein size. The minimum detectable surface concentration of protein is estimated to be 50 pg mm−2with this SPR instrument. Optical models have been developed to describe how the SPR response depends on the distribution of the adsorbed protein within the hydrogel volume at the surface. With a thin-film optical program, the theoretical SPR responses for the different models were calculated. Comparison with experimental data shows that the protein is distributed within an approximately 100-nm-thick dextran hydrogel layer.