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Development of a Competitive Cystatin C-Specific Bioassay Suitable for Repetitive Measurements
Tatjana Damm1, Holger Spiegel1, Stefan Barth2,3
1Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Aachen, Germany.
Insights
Researchers developed novel antibodies for continuous monitoring of human cystatin C (hCC), a marker for kidney and cardiovascular diseases. These antibodies enable repetitive binding and release, paving the way for implantable biochip devices and advanced in vivo/in vitro assays.
Area of Science:
- Biotechnology
- Immunology
- Medical Diagnostics
Background:
- Human cystatin C (hCC) is a cysteine protease inhibitor linked to cardiovascular and kidney diseases.
- Current hCC detection methods are limited to ex vivo analysis.
- Long-term and continuous monitoring requires antibodies with specific binding and release kinetics.
Purpose of the Study:
- To generate and evaluate antibodies capable of repetitive binding and release of hCC.
- To develop antibodies suitable for implantable biochip devices and continuous in vivo/in vitro monitoring.
- To assess antibody performance for serum hCC measurement across physiological and pathological ranges.
Main Methods:
- Production of recombinant hCC and hCC-fusion proteins in E. coli and HEK293T cells.
- Generation of hCC-specific monoclonal antibodies using hybridoma technology.
- Screening via ELISAs, characterization by surface plasmon resonance (SPR) and fluorescence-based replacement assays.
Main Results:
- Identified 12 hCC-specific monoclonal antibodies; three exhibited desired fast binding and moderate-to-fast release kinetics.
- mAb CyDI-4 demonstrated suitability for repetitive binding/release with signal changes within 20-30 minutes.
- mAb CyDI-4 successfully measured serum hCC in reference samples, covering physiological and pathological ranges.
Conclusions:
- Developed novel monoclonal antibodies, particularly mAb CyDI-4, with properties suitable for continuous hCC monitoring.
- These antibodies are promising for next-generation diagnostic assays, including implantable devices.
- The findings support the potential for improved in vivo and in vitro diagnostics of hCC-related diseases.
Abstract:
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.
