Vashist S K, Schneider E M, Luong J H T
HSG-IMIT - Institut für Mikro - und Informationstechnik, Georges-Koehler Allee 103, 79110 Freiburg, Germany.
Analyst. 2014 May 7;139(9):2237-42. doi: 10.1039/c4an00149d.
This article describes a highly-sensitive surface plasmon resonance (SPR)-based immunoassay (IA) for human fetuin A (HFA), a specific biomarker for atherosclerosis and hepatocellular carcinoma. The assay is based on a novel immobilization procedure that simply involves the dilution of an anti-HFA capture antibody (Ab) in 1% (v/v) 3-aminopropyltriethoxysilane (APTES), followed by its dispensing on a KOH-treated gold (Au)-coated SPR chip and incubation for 30 min. The developed SPR IA detected 0.3-20 ng mL(-1) of HFA with a limit of detection and sensitivity of 0.7 ng mL(-1) and 1 ng mL(-1), respectively. The highly-simplified Ab immobilization procedure is also 5-fold more rapid than conventional procedures. It leads to the leach-proof binding of the capture Ab, which means that the developed SPR IA is highly cost-effective, as the Ab-bound SPR chip could be reused for many repeated HFA IAs after regeneration with 10 mM glycine-HCl, pH 2.0. The Ab-bound SPR chip, stored at 4 °C, lost only 18% of its original activity after 4 months. For the detection of HFA spiked in diluted human whole blood and plasma, the results obtained by the developed SPR IA agreed well with the commercial HFA sandwich ELISA.
本文描述了一种基于高灵敏度表面等离子体共振（SPR）的免疫分析方法（IA），用于检测人胎球蛋白A（HFA），它是动脉粥样硬化和肝细胞癌的一种特异性生物标志物。该分析方法基于一种新颖的固定化程序，该程序只需将抗HFA捕获抗体（Ab）稀释于1%（v/v）的3-氨丙基三乙氧基硅烷（APTES）中，然后将其滴加在经氢氧化钾处理的金（Au）涂层SPR芯片上并孵育30分钟。所开发的SPR免疫分析方法检测HFA的浓度范围为0.3 - 20 ng mL⁻¹，检测限和灵敏度分别为0.7 ng mL⁻¹和1 ng mL⁻¹。这种高度简化的抗体固定化程序也比传统程序快5倍。它能使捕获抗体实现防渗漏结合，这意味着所开发的SPR免疫分析方法具有很高的成本效益，因为用10 mM甘氨酸 - 盐酸（pH 2.0）再生后，结合了抗体的SPR芯片可重复用于多次重复的HFA免疫分析。结合了抗体的SPR芯片在4℃储存4个月后，其原始活性仅损失了18%。对于检测加样于稀释人全血和血浆中的HFA，所开发的SPR免疫分析方法得到的结果与市售的HFA夹心ELISA法结果吻合良好。
