Isolation and immunochemical characterization of human cystatin C

Authors

  • Lejla Begić Department of Biochemistry, Faculty of Medicine, University of Tuzla
  • Selma Berbić Department of Biochemistry, Faculty of Medicine, University of Tuzla

DOI:

https://doi.org/10.17305/bjbms.2002.3576

Keywords:

human cystatin C, cystatin C isoforms, ELISA test, chronic renal failure

Abstract

Cystatin C is a natural inhibitor of the cysteine proteinases papain, and mammalian lysosomal cathepsins B, H, L and S. This protein is thought to serve an important physiological role as a local regulator of enzyme activity. The changes of levels of cystatin C in extracellular fluids have shown themselves having potential clinical importance. We have purified cystatin C from urine of patients with chronic renal failure by procedure using affinity chromatography on CM-papain Sepharose, gel filtration on Sephacryl S-200, and ion exchange chromatography on CM-cellulose. After isolation we obtained three inhibitory peaks (pI's from 7.8 to 9.2) which represent isoforms of the same protein. These isoforms are immunologically identical and differ in N-terminal sequence of the molecule. The form with pI 9.2 represents the intact inhibitor form, whereas the form with pI 7.8 is shortened for 8 amino-acid residues at N-terminal end. Purified cystatin C pI 9.2 was used for immunization of rabbits. Polyclonal antibodies, produced in rabbits, were isolated from rabbit sera by affinity chromatography on Protein A Sepharose. Enzyme immunoassay (ELISA) for cystatin C is developed on the basis of purified antibodies. Using ELISA test we determined amount of cystatin C in urine and serum samples of patients with chronic renal failure. The concentration of the inhibitor in the urine of these patients was approximately 100-fold more than in normal urine. In the serum from the same patients we found concentrations of cystatin C to be five times higher in comparison with the serum of healthy individuals.

Isolation and immunochemical characterization of human cystatin C

Published

20-02-2002

How to Cite

1.
Isolation and immunochemical characterization of human cystatin C. Biomol Biomed [Internet]. 2002 Feb. 20 [cited 2024 Feb. 23];2(1-2):12-5. Available from: https://www.bjbms.org/ojs/index.php/bjbms/article/view/3576