Vashist Sandeep Kumar, Dixit Chandra Kumar, MacCraith Brian D, O'Kennedy Richard
Centre for Bioanalytical Sciences (CBAS), National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland.
Analyst. 2011 Nov 7;136(21):4431-6. doi: 10.1039/c1an15325k. Epub 2011 Sep 8.
Antibody immobilization strategies (random, covalent, orientated and combinations of each) were examined to determine their performance in a surface plasmon resonance-based immunoassay using human fetuin A (HFA) as the model antigen system. The random antibody immobilization strategy selected was based on passive adsorption of anti-HFA antibody on 3-aminopropyltriethoxysilane (APTES)-functionalized gold (Au) chips. The covalent strategy employed covalent crosslinking of anti-HFA antibody on APTES-functionalized chips using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) and sulfo-N-hydroxysuccinimide (SNHS). The orientation strategy used passive adsorption of protein A (PrA) on Au chips, with subsequent binding of the anti-HFA antibody in an orientated fashion via its fragment crystallisable (Fc) region. In the covalent-orientated strategy, PrA was first bound covalently, to the surface, which in turn, then binds the anti-HFA antibody in an orientated manner. Finally, in the most widely used strategy, covalent binding of anti-HFA antibody to carboxymethyldextran (CM5-dextran) was employed. This immobilization strategy gave the highest anti-HFA antibody immobilization density, whereas the highest HFA response was obtained with the covalent-orientated immobilization strategy. Therefore, the covalent-orientated strategy was the best for SPR-based HFA immunoassay and can detect 0.6-20.0 ng/mL of HFA in less than 10 min.
研究了抗体固定策略（随机、共价、定向以及每种策略的组合），以确定它们在以人胎球蛋白A（HFA）作为模型抗原系统的基于表面等离子体共振的免疫分析中的性能。所选择的随机抗体固定策略是基于抗HFA抗体被动吸附在3-氨丙基三乙氧基硅烷（APTES）功能化的金（Au）芯片上。共价策略采用1-乙基-3-[3-二甲氨基丙基]碳二亚胺（EDC）和磺基-N-羟基琥珀酰亚胺（SNHS）将抗HFA抗体共价交联在APTES功能化的芯片上。定向策略是将蛋白A（PrA）被动吸附在Au芯片上，随后抗HFA抗体通过其可结晶片段（Fc）区域以定向方式结合。在共价-定向策略中，PrA首先共价结合到表面，进而以定向方式结合抗HFA抗体。最后，在最常用的策略中，采用抗HFA抗体与羧甲基葡聚糖（CM5-葡聚糖）的共价结合。这种固定策略产生了最高的抗HFA抗体固定密度，而共价-定向固定策略获得了最高的HFA响应。因此，共价-定向策略对于基于表面等离子体共振的HFA免疫分析是最佳的，并且能够在不到10分钟的时间内检测出0.6 - 20.0 ng/mL的HFA。
