Dreymann Nico, Sabrowski Wiebke, Danso Jennifer, Menger Marcus M
Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalytics and Bioprocesses (IZI-BB), D-14476 Potsdam, Germany.
Institute for Biochemistry and Biology, University of Potsdam, D-14476 Potsdam, Germany.
Cancers (Basel). 2022 Oct 25;14(21):5222. doi: 10.3390/cancers14215222.
Urokinase-type plasminogen activator (urokinase, uPA) is a frequently discussed biomarker for prognosis, diagnosis, and recurrence of cancer. In a previous study, we developed ssDNA aptamers that bind to different forms of human urokinase, which are therefore assumed to have different binding regions. In this study, we demonstrate the development of aptamer-based sandwich assays that use different combinations of these aptamers to detect high molecular weight- (HMW-) uPA in a micro titer plate format. By combining aptamers and antibodies, it was possible to distinguish between HMW-uPA and low molecular weight- (LMW-) uPA. For the best performing aptamer combination, we calculated the limit of detection (LOD) and limit of quantification (LOQ) in spiked buffer and urine samples with an LOD up to 50 ng/mL and 138 ng/mL, respectively. To show the specificity and sequence dependence of the reporter aptamer uPAapt-02-FR, we have identified key nucleotides within the sequence that are important for specific folding and binding to uPA using a fluorescent dye-linked aptamer assay (FLAA). Since uPA is a much-discussed marker for prognosis and diagnosis in various types of cancers, these aptamers and their use in a micro titer plate assay format represent a novel, promising tool for the detection of uPA and for possible diagnostic applications.
尿激酶型纤溶酶原激活剂（尿激酶，uPA）是一种常用于癌症预后、诊断和复发的生物标志物。在之前的一项研究中，我们开发了与不同形式的人尿激酶结合的单链DNA适配体，因此推测它们具有不同的结合区域。在本研究中，我们展示了基于适配体的夹心检测方法的开发，该方法使用这些适配体的不同组合以微量滴定板形式检测高分子量（HMW）-uPA。通过结合适配体和抗体，可以区分HMW-uPA和低分子量（LMW）-uPA。对于性能最佳的适配体组合，我们计算了加标缓冲液和尿液样本中的检测限（LOD）和定量限（LOQ），LOD分别高达50 ng/mL和138 ng/mL。为了展示报告适配体uPAapt-02-FR的特异性和序列依赖性，我们使用荧光染料连接适配体检测法（FLAA）确定了序列中对uPA特异性折叠和结合重要的关键核苷酸。由于uPA是各类癌症中常用于预后和诊断的标志物，这些适配体及其在微量滴定板检测形式中的应用代表了一种用于检测uPA及可能的诊断应用的新型、有前景的工具。
