Driskell Jeremy D, Uhlenkamp Jill M, Lipert Robert J, Porter Marc D
Institute for Combinatorial Discovery, Department of Chemistry, Iowa State University, and Ames Laboratory-U.S. DOE, Ames, Iowa 50011-3020, USA.
Anal Chem. 2007 Jun 1;79(11):4141-8. doi: 10.1021/ac0701031. Epub 2007 May 8.
A rapid, sensitive format for immunosorbent assays has been developed to meet the increasing levels of performance (i.e., reduction of incubation times and detection limits) demanded in the medical, veterinary, and bioterrorism prevention arenas. This paper introduces the concept of a rotating capture substrate as a facile means to increase the flux of antigen and label to the solid-phase surface and thereby reduce assay time. To this end, a sandwich-type assay is carried out that couples the specificity of antibody-antigen interactions with the high sensitivity of surface-enhanced Raman scattering detection. To investigate this strategy, polyclonal anti-rabbit IgG was immobilized on a gold capture substrate via a thiolate coupling agent. The capture substrate, capable of controlled rotation, was then immersed in a sample solution containing rabbit IgG, which served as a model analyte. After binding the target IgG, the substrates were immersed and rotated in an extrinsic Raman label (ERL) labeling solution, which is composed of gold nanoparticles (60 nm) coated with an aromatic moiety as the Raman scatterer and an antibody as the biospecific recognition element. The effect of substrate rotation on both the antigen binding and ERL labeling steps was investigated. Implementation of optimized rotation conditions resulted in the reduction of assay times from 24 h to 25 min and a 10-fold improvement in the limit of detection. Finally, the developed protocol was applied to the detection of rabbit IgG suspended in goat serum, which served to assess performance in a biological matrix.
为满足医学、兽医和生物恐怖主义预防领域对检测性能不断提高的要求（即缩短孵育时间和降低检测限），已开发出一种快速、灵敏的免疫吸附测定方法。本文介绍了旋转捕获基质的概念，这是一种增加抗原和标记物向固相表面通量从而缩短测定时间的简便方法。为此，进行了一种夹心型测定，将抗体-抗原相互作用的特异性与表面增强拉曼散射检测的高灵敏度相结合。为研究该策略，通过硫醇盐偶联剂将多克隆抗兔IgG固定在金捕获基质上。然后将能够控制旋转的捕获基质浸入含有兔IgG的样品溶液中，兔IgG用作模型分析物。结合目标IgG后，将基质浸入由涂有芳香部分作为拉曼散射体的金纳米颗粒（60 nm）和作为生物特异性识别元件的抗体组成的外在拉曼标记（ERL）标记溶液中并旋转。研究了基质旋转对抗原结合和ERL标记步骤的影响。实施优化的旋转条件可将测定时间从24小时缩短至25分钟，并将检测限提高10倍。最后，将所开发的方案应用于检测悬浮在山羊血清中的兔IgG，以评估在生物基质中的性能。
