Cat No.: 31241 ★Download Datasheet★
Other Names: Cystatin 3, post-gamma-globulin, neuroendocrine basic polypeptide, CST3
Introduction
Human Cystatin C (or cystatin 3), which is composed of 120 amino acid residues, belongs to the cystatins superfamilly that
inactivates lysosomal cysteine proteinases. As a strongly cationic and low-molecular weight (13.4 kDa) protein, it is almost freely
filtered across the glomerular membrane, and is mainly used as a biomarker of kidney function. A growing body of evidence
suggests that cystatin C is a more reliable biomarker of glomerular filtration rate than creatinine [1-3]. In addition to kidney disease,
altered serum levels of cystatin C are associated with several types of cardiovascular disease, including myocardial infarction,
stroke, heart failure, peripheral arterial disease and metabolic syndrome [4-7]. It also seems to play a role in brain disorders
involving amyloid, such as Alzheimer's disease [8, 9]. Furthermore, Cystatin C has also been investigated as a prognostic marker in several forms of cancer.
Principle of the Assay
This assay is a sandwich ELISA designed for the quantitative detection of human cystatin c in samples in 1 hour. The immunoplate is pre-coated with antibody specific to human cystatin c. Standards and samples are pipetted into the wells and any human cystatin c
present is sandwiched by the immobilised antibody and a second horseradish peroxidase (HRP)-linked antibody specific to human
cystatin c that is co-incubated with the samples. After wash step to remove any unbound substances, the HRP substrate solution is
added and colour develops in proportion to the amount of human cystatin c bound initially. The assay is stopped and the optical
density of the wells determined using a micro-plate reader. Since the increases in absorbance are directly proportional to the amount
of captured human cystatin c, the unknown sample concentration can be interpolated from a reference curve included in each assay.
Assay Performance
A. Typical representation of standard curve
The following standard curve is provided for demonstration only. A standard curve should be generated for each set of sample
assay.
Cystatin C (ng/mL) | Absorbance (450 nm) | Blanked Absorbance |
0 | 0.069 | 0 |
0.23 | 0.127 | 0.058 |
0.46 | 0.187 | 0.118 |
0.93 | 0.309 | 0.24 |
1.87 | 0.539 | 0.47 |
3.75 | 0.946 | 0.877 |
7.5 | 1.685 | 1.616 |
15 | 2.551 | 2.482 |
B. Sensitivity
The lowest level of human cystatin C that can be detected by this assay is 0.23 ng/mL.
C. Specificity
The antibodies used in this assay are specific to human cystatin c and do not cross-react with mouse and rat cystatin C, and other
cytokine or hormone molecules.
D. Precision
Intra-assay Precision (Precision within an assay)
Three samples of known concentration were tested 12 times on one plate.
Sample | Mean (ng/mL) | SD (ng/mL) | CV (%) |
1 | 507.3 | 30.1 | 5.9 |
2 | 377.2 | 23.1 | 6.1 |
3 | 137.4 | 10.8 | 7.9 |
Inter-assay Precision (Precision between assays)
Three samples of known concentration were tested in 10 separate assays.
Sample | Mean (ng/mL) | SD (ng/mL) | CV (%) |
1 | 499.3 | 39.0 | 7.8 |
2 | 377.2 | 23.1 | 6.1 |
3 | 137.4 | 10.8 | 7.9 |
E. Recovery
Serum samples were spiked with different amounts of human cystatin C and assayed.
Sample | Average % Recovery | Range % |
Serum | 99 | 94-107 |