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The interest in diagnostic biochips is growing. However, most biochips are designed for ex vivo applications. Beyond, an elevated serum human cystatin C (hCC) concentration correlates with various diseases. Thus, hCC was suggested as biomarker. To meet the requirements of an implantable biochip system it is essential to develop an hCC-specific bioassay enabling repetitive measurements.Within this PhD thesis an hCC-specific bioassay was developed which is based on the concentration-dependant replacement between hCC and an hCC-fusion protein on an hCCspecific antibody layer thus enabling the repetitive determination of the hCC concentration. Therefore hCC, hCC-fusion proteins and glutathione S-transferase (GST) were successfully cloned, produced and purified. The fusion protein stGST-hCC was further used for the immunization of mice. Thereby an hCCspecific immune response could be induced. With the use of the hybridoma generation technology monoclonal hybridoma cell lines were established, which enabled the selection and production of hCC-specific mAbs. The selection of monoclonal hybridoma cell lines which produced hCC-specific antibodies was successful due to analysis by indirect ELISA, sandwich ELISA and surface plasmon spectroscopy (SPR)-spectroscopy. Selected hybridoma cell lines were used for small scale production of hCC-specific mAbs which without purification were analysed for their hCC association and dissociation characteristics by SPR spectroscopy. From 12 initially potential antibodies, 5 were identified to display specific yet distinct association and dissociation characteristics towards hCC. For the replacement assay the dissociation characteristics were of special interest, thus they were used to rank the antibodies. Whereas mAb CyDI-1 showed no and CyDI-12 showed barely any dissociation, it was clearly seen for mAb CyDI-2, CyDI-4 and to the highest degree for mAb CyDI-3. The five corresponding hybridoma cell lines were used for the large scale production of mAbs, all producing mAbs of the IgG1:κ isotype. All mAbs were purified by Protein G-affinity chromatography, yielding pure protein. Due to their accessibility mAb CyDI-1, CyDI-2 and CyDI-4 were further analysed by SPR spectroscopy. This time hCC-fusion proteins were used beside hCC for the identification of suitable hCC and hCC-fusion protein combinations for the replacement assay. The mAb CyDI-4 was identified as the antibody with the highest potential for enabling replacement. Except for enhanced green fluorescent protein (GFP)-hCC, all fusion proteins and hCC showed exactly identical association and dissociation behaviour towards hCC. The combination of hCC and stGST-hCC was identified to be best by SPR spectroscopy, especially in respect to the end application. However, the evaluation of the hCC-specific bioassay in the predicted chip-based detection system was not possible until the end of this study. In parallel to the chip evolution, a fluorescence-based replacement assay was established for the evaluation of the replacement ability on mAb CyDI-4. Thus, the discrimination between hCC and the hCC-fusion protein was possible without the modification of antigens and the use of secondary antibodies, respectively, which was essential as the former may alter the affinity and the latter is not involved in the final bioassay. Using the mAb CyDI-4 and the antigen combination composed of hCC and GFP-hCC a fluorescence-based replacement assay was developed, which meets the implant requirements regarding the assay duration, the compatibility with human serum and the required sensitivity for physiological and most of the pathological conditions. Additionally, the assay standard deviation decreased upon use of reference material (hCC spiced human serum) while maintaining the sensitivity. However, the repetitive replacement assays revealed the replacement to slightly favour one replacement direction, as was predicted by SPR analysis for hCC and GFP-hCC. Although the combination of GFP-hCC and hCC was not ideal, and was only used to fulfil the detection requirements in plate-based format, the results indicate that mAb CyDI-4 is an excellent antibody for the hCC-specific replacement assay. |
