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

PloS One
Tatjana DammJoerg Naehring

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 repl...Continue Reading

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Datasets Mentioned

BETA
CAA36497.1

Methods Mentioned

BETA
enzyme-linked immunosorbent assay
ELISA
surface plasmon resonance
electrophoresis
chip
sELISA
ELISAs
biosensors
transfection
FCS

Software Mentioned

ExPASy
AIDA
BiaEval

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