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.

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.

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