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Title: Immunochemical techniques for the detection, isolation and characterization of connective tissue growth factor in diabetic urine and peritoneal dialysis fluid
Author: Wu Shumei, Jocelyn
Awarding Body: King's College London (University of London)
Current Institution: King's College London (University of London)
Date of Award: 2012
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Connective tissue growth factor (CTGF) has been implicated in the pathogenesis of diabetic nephropathy and has been proposed as a possible specific biomarker for the early detection of fibrosis. CTGF is reported to be a 349-amino acid cysteine-rich, heparin binding monomeric polypeptide that is secreted as a 36-38 kDa protein. To date, the natural form of CTGF has not been successfully purified from clinical samples. Commercial test kits for detecting CTGF by ELISA utilize antibodies raised against recombinant human (rh) CTGF. In the present study, assessment of specific analytical methods for the reliable detection of naturally occurring CTGF has been deployed. In order to achieve this, a peptide sequence to CTGF was synthesized and an antibody raised against this peptide. This antibody was characterized using novel chemistry to improve the specificity of the final assay. The antibody raised against a unique peptide sequence in CTGF, and another to rhCTGF, were used, alongside a commercially available anti-rhCTGF antibody to detect this protein in diabetic urine and peritoneal dialysis fluid. The synthetic peptide antibody was also coupled to Sepharose 4B for the purification of CTGF from clinical samples. ELISA performed on the diabetic urine and peritoneal dialysis fluid showed that the response of the antibody against the synthetic peptide was the highest when compared to the rhCTGF and commercial antibodies. Five peritoneal dialysis fluid and five diabetic urine samples were assayed for CTGF. Diabetic urine was found to be a richer source of CTGF. 2-dimemsional electrophoresis and Western blot analyses on the peritoneal dialysis fluid showed various immunoreactive species to the synthetic peptide antibody at isoelectric point 8.0. It can be concluded that these immunoreactive species may be CTGF bound to other proteins or CTGF complexes that have been formed from the oxidation of the cysteine residues.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available