Abstract A microplate-based sandwich assay for the determination of α-human thrombin (HTb) was developed. Fluorescein-modified 29-mer thrombin binding aptamer (FAM-TBA29) and biotinylated 15-mer thrombin binding aptamer (Bio-TBA15) reacting with different exosites of HTb were used as the biorecognition components in the assay. FAM-TBA29 (capture aptamer) was immobilized using its interaction with anti-fluorescein antibody adsorbed on the microplate surface. As a sensitive signaling system, a combination of Bio-TBA15 and streptavidin-polyHRP conjugate was used. Under the optimized conditions, the detection limit for HTb was 1.4 nM; this value was the same in both the colorimetric and the chemiluminescent assays. The replacement of colorimetry for HRP measurement with chemiluminescence increased the assay sensitivity from 0.06 to 1.7 × 106 nM−1 that clearly demonstrated advantage of the latter approach.

中文翻译：

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基于化学发光和比色适配体的人 α-凝血酶检测

摘要 开发了一种用于测定 α-人凝血酶 (HTb) 的基于微孔板的夹心法。荧光素修饰的 29 聚体凝血酶结合适体 (FAM-TBA29) 和生物素化的 15 聚体凝血酶结合适体 (Bio-TBA15) 与不同的 HTb 外位点反应用作该测定中的生物识别组分。FAM-TBA29（捕获适体）使用其与吸附在微孔板表面上的抗荧光素抗体的相互作用进行固定。作为一种灵敏的信号系统，使用了 Bio-TBA15 和链霉亲和素-polyHRP 偶联物的组合。在优化条件下，HTb的检测限为1.4 nM；该值在比色和化学发光分析中是相同的。用化学发光代替 HRP 测量的比色法将检测灵敏度从 0.06 提高到 1。