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.

A monoclonal antibody for the detection of SNAP/CLIP-tagged proteins.

SNAP/CLIP-tag technology is a novel approach that allows tagged proteins to be covalently coupled to diverse labels, such as fluorochromes and particles, using a convenient and specific enzymatic reaction. A monoclonal antibody (mAb) that binds to the SNAP/CLIP-tag would be useful to determine labeling efficiency, and to achieve reproducible detection in a variety of experimental formats. We therefore generated the murine mAb M2D11 by standard immunization and hybridoma technology. M2D11 binds to both the SNAP- and the CLIP-tag in either the coupled or uncoupled configurations and can be detected in the context of ELISA, flow cytometry, immunohistochemistry and western blot. The new antibody increases the versatility of the SNAP-tag technology by enabling the detection of tagged proteins using conventional immunological methods and widely available secondary antibodies.