Development of a Competitive Cystatin C-Specific Bioassay Suitable for Repetitive Measurements

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（cystatin C，hCC）作为一种半胱氨酸蛋白酶抑制剂，已被提议作为诊断标志物，因其血清水平与部分心血管及肾脏疾病存在关联。目前所有的hCC检测方法均基于离体检测（ex vivo）。本文描述了可实现hCC及hCC融合蛋白重复结合与解离的抗体的制备与评价——这是实现长期检测的前提条件，而长期检测是可植入生物芯片设备兼容所需，也是开发适用于持续体内（in vivo）及体外（in vitro）监测的新型抗体类检测方法的必要条件。研究人员在大肠杆菌（Escherichia coli）及HEK293T细胞中重组表达了hCC与hCC融合蛋白，并通过杂交瘤技术（hybridoma technology）制备抗体。通过间接ELISA及夹心ELISA筛选后，共获得12株可分泌hCC特异性单克隆抗体的杂交瘤细胞系。为探究这些抗体的hCC结合与解离特性，研究人员采用表面等离子体共振光谱法（surface plasmon resonance spectroscopy）对其进行分析，最终筛选出3株具备理想的快速结合及中速至快速解离特性的单克隆抗体。通过基于荧光的置换试验（fluorescence-based replacement assays）对hCC及hCC融合蛋白存在条件下的结合与解离过程进行分析后发现，单克隆抗体（monoclonal antibody，mAb）CyDI-4最适合用于后续研究。实验结果表明，可在单克隆抗体CyDI-4上实现重复置换，且绝大多数信号强度变化发生在20至30分钟内。此外，研究人员采用参考物质与测量研究所（Institute for Reference Materials and Measurements，ERM-DA471/IFCC）的系列稀释参考血清开展基于荧光的置换试验，证实了单克隆抗体CyDI-4用于血清hCC检测的适用性。本研究结果表明，该检测方法可覆盖hCC的生理及病理浓度范围。