Damm Tatjana, Spiegel Holger, Barth Stefan, Fischer Rainer, Naehring Joerg
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
Institute for Molecular Biology, RWTH Aachen University, Aachen, Germany.
PLoS One. 2016 Jan 22;11(1):e0147177. doi: 10.1371/journal.pone.0147177. eCollection 2016.
Human cystatin C (hCC), a cysteine protease inhibitor, has been proposed as a diagnostic marker because its serum levels correlate with certain cardiovascular and kidney diseases. All current hCC assays are based on ex vivo detection. Here we describe the generation and evaluation of antibodies that allow the repetitive binding and release of hCC and hCC-fusion proteins, a prerequisite for long-term measurement, which is required for compatibility with implantable biochip devices and for the development of innovative antibody-based assays suitable for continuous in vivo and in vitro monitoring. Recombinant hCC and hCC-fusion proteins were produced in Escherichia coli and HEK293T cells and were used to generate antibodies by hybridoma technology. After screening by indirect and sandwich ELISAs, 12 monoclonal hybridoma cell lines producing hCC-specific monoclonal antibodies were identified. To determine their hCC association and dissociation properties, the antibodies were analysed by surface plasmon resonance spectroscopy, revealing three with the desired fast binding and moderate-to-fast release characteristics. The analysis of binding and dissociation in the presence of hCC and hCC-fusion proteins using fluorescence-based replacement assays showed that mAb CyDI-4 was the most suitable for further analysis. The results showed that repetitive replacement on mAb CyDI-4 was possible and that most of the change in signal intensity occurred after 20-30 min. Furthermore, the suitability of mAb CyDI-4 for serum hCC measurement was confirmed by a fluorescence-based replacement assay using serially-diluted reference serum from the Institute for Reference Materials and Measurements (ERM-DA471/IFCC). Our results suggest that the assay covers the physiological and pathological ranges of hCC.
人胱抑素C（hCC），一种半胱氨酸蛋白酶抑制剂，已被提议作为一种诊断标志物，因为其血清水平与某些心血管和肾脏疾病相关。目前所有的hCC检测方法都是基于体外检测。在此，我们描述了能够实现hCC和hCC融合蛋白的重复结合与释放的抗体的产生及评估，这是长期测量的前提条件，对于与可植入生物芯片设备的兼容性以及开发适用于体内和体外连续监测的基于抗体的创新检测方法而言是必需的。重组hCC和hCC融合蛋白在大肠杆菌和HEK293T细胞中产生，并通过杂交瘤技术用于产生抗体。通过间接和夹心ELISA筛选后，鉴定出12个产生hCC特异性单克隆抗体的单克隆杂交瘤细胞系。为了确定它们与hCC的结合和解离特性，通过表面等离子体共振光谱对抗体进行分析，发现其中三个具有所需的快速结合和中等到快速释放的特性。使用基于荧光的置换分析在hCC和hCC融合蛋白存在的情况下对结合和解离进行分析，结果表明单克隆抗体CyDI-4最适合进一步分析。结果表明，单克隆抗体CyDI-4上的重复置换是可能的，并且信号强度的大部分变化发生在20 - 30分钟后。此外，使用来自参考物质和测量研究所（ERM-DA471/IFCC）的系列稀释参考血清进行的基于荧光的置换分析证实了单克隆抗体CyDI-4用于血清hCC测量的适用性。我们的结果表明该检测方法涵盖了hCC的生理和病理范围。
