cell culture supernatants, serum, plasma, saliva, urine, milk
For the quantitative determination of human Cystatin C concentrations in cell culture supernates, serum, plasma, saliva, urine, and human milk.
Contents of Kit
1. Human Cystatin C Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with a monoclonal antibody specific for human Cystatin C.
2. Human Cystatin C Conjugate: 21 mL of a monoclonal antibody specific for human Cystatin C conjugated to horseradish peroxidase with preservatives.
3. Human Cystatin C Standard: Recombinant human Cystatin C in a buffer with preservatives; lyophilized. Refer to the vial label for reconstitution volume.
4. Assay Diluent CD2-54: 11 mL of a buffered protein base with preservatives. Contains a precipitate. Mix well before and during use.
5. Calibrator Diluent CD6-35 Concentrate: 21 mL of a concentrate buffered protein base with preservatives. Use diluted 1:5 in this assay.
6. Wash Buffer Concentrate: 21 mL of a 25-fold concentrated solution of buffered surfactant with preservative. May turn yellow over time.
7. Color Reagent A: 12 mL of stabilized hydrogen peroxide.
8. Color Reagent B: 12 mL of stabilized chromogen (tetramethylbenzidine).
9. Stop Solution: 6 mL of 2 N sulfuric acid.
10. Plate Sealers: 4 adhesive strips.
Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in forty separate assays to assess inter-assay precision. Assays were performed by at least three technicians using two lots of components.
Fifty assays were evaluated and the minimum detectable dose (MDD) of human Cystatin C ranged from 0.030-0.227 ng/mL. The mean MDD was 0.102 ng/mL.
The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration.
Cystatin C (gene name CST3) is a secreted cysteine protease inhibitor that belongs to the cystatin superfamily (1). It is a protein of 120 amino acids and approximately 13 kDa in its non-glycosylated form; a glycosylated form is reported in rat, but not in mouse (2). Mature human Cystatin C shares 72% amino acid sequence identity with mouse and rat Cystatin C. Cystatin C is susceptible to endoprotease cleavage producing N-terminally truncated forms (1). Cysteine proteases of the papain family, such as Cathepsins B, H, K, L, and S, are the major targets for Cystatin C (3, 4).
Cystatin C is produced in all tissues and is present in all biological fluids, including plasma, urine and cerebrospinal fluid. Cystatin C is freely filtered by the glomeruli. It is then taken up by proximal tubule epithelial cells via megalin-mediated endocytosis, and is metabolized so that it does not return to the bloodstream (1, 5-7). Therefore, Cystatin C serum concentration correlates closely to the glomerular filtration rate (GFR). Its measurement in serum or plasma has been proposed as an indicator of drug nephrotoxicity that is less affected by factors such as gender, age, muscle mass and cirrhosis than creatinine (5, 7, 8). Circulating Cystatin C can, however, be increased during chronic low-level inflammation, in part due to IL-6-mediated increases in Cystatin C production (8). Conversely, the anti-inflammatory cytokines IL-10, IFN-β and IFN-γ can decrease Cystatin C expression and its circulating levels (9-11).
Cystatin C is involved in several disease processes through its regulation of cysteine protease activity (1). In humans, high circulating Cystatin C in the presence of apparently normal kidney function is associated with coronary artery and cardiovascular disease risk (1, 7, 12, 13). In a model of human aortic aneurism, deletion of mouse Cystatin C in ApoE-/- mice promotes inflammation and speeds cathepsin-mediated rupture of the arterial wall tunica elastica (14, 15). Circulating Cystatin C has been reported to influence tumor metastasis. Abnormally low Cystatin C levels allow cathepsin B-mediated degradation of extracellular matrix and promote tumor metastasis, while high Cystatin C levels antagonize TGF-β signaling, slowing cancer invasion and growth (16, 17). Cystatin C is an amyloidogenic protein. In humans, the L68Q variant forms dimers and oligomers more easily than the wild type protein under physiological conditions and is the cause for hereditary Cystatin C amyloid angiopathy (3, 18, 19). Cystatin C also inhibits amyloid-β deposition and protects neuronal cells from toxicity in mouse models of Alzheimer's disease (20-22).
The Quantikine Human Cystatin C Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human Cystatin C in cell culture supernates, serum, plasma, saliva, urine, and human milk. It contains NS0-expressed recombinant human Cystatin C and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Cystatin C showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human Cystatin C.