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Human Cystatin C ELISA Kit

SKU:
HUFI00097
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P01034
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich
Synonyms:
Cystatin C, CST3, Cys-C, Cystatin 3, ARMD11, Post-Gamma-Globulin
Reactivity:
Human
$599
Frequently bought together:

Description

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Human Cystatin C ELISA Kit

The Human Cystatin C ELISA Kit is a powerful tool for researchers looking to accurately measure levels of cystatin C in human biological samples. This kit offers high sensitivity and specificity, allowing for precise and reliable results in serum, plasma, and cell culture supernatants.Cystatin C is a vital protein associated with renal function and is commonly used as a biomarker for kidney health. Elevated levels of cystatin C have been linked to various health conditions such as chronic kidney disease, cardiovascular disease, and even mortality risk, making it a valuable marker for monitoring and diagnosing these conditions.

With its easy-to-use format and dependable performance, the Human Cystatin C ELISA Kit is an indispensable asset for researchers studying kidney function, cardiovascular health, and other related fields. Trust in this kit to provide accurate and reproducible results for your research needs.

Product Name:Human Cystatin C ELISA Kit
Product Code:HUFI00097
Size:96 Assays
Alias:Cystatin C, CST3, Cys-C, Cystatin 3, ARMD11, Post-Gamma-Globulin
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human CST3 concentrations in serum plasma and other biological fluids.
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human CST3 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CST3 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)87-10297
EDTA plasma(n=5)87-10196
UFH plasma(n=5)85-10497
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CST3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)86-105%85-102%88-102%
EDTA plasma(n=5)85-101%89-99%83-101%
UFH plasma(n=5)85-100%80-96%90-100%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

Other materials and equipment required:

  • Microplate reader with 450 nm wavelength filter
  • Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
  • Incubator
  • Deionized or distilled water
  • Absorbent paper
  • Buffer resevoir
UniprotP01034
UniProt Protein Function:CST3: As an inhibitor of cysteine proteinases, this protein is thought to serve an important physiological role as a local regulator of this enzyme activity. Defects in CST3 are the cause of amyloidosis type 6 (AMYL6); also known as hereditary cerebral hemorrhage with amyloidosis (HCHWA), cerebral amyloid angiopathy (CAA) or cerebroarterial amyloidosis Icelandic type. AMYL6 is a hereditary generalized amyloidosis due to cystatin C amyloid deposition. Cystatin C amyloid accumulates in the walls of arteries, arterioles, and sometimes capillaries and veins of the brain, and in various organs including lymphoid tissue, spleen, salivary glands, and seminal vesicles. Amyloid deposition in the cerebral vessels results in cerebral amyloid angiopathy, cerebral hemorrhage and premature stroke. Cystatin C levels in the cerebrospinal fluid are abnormally low. Genetic variations in CST3 are associated with age- related macular degeneration type 11 (ARMD11). ARMD is a multifactorial eye disease and the most common cause of irreversible vision loss in the developed world. In most patients, the disease is manifest as ophthalmoscopically visible yellowish accumulations of protein and lipid that lie beneath the retinal pigment epithelium and within an elastin-containing structure known as Bruch membrane. Belongs to the cystatin family.
UniProt Protein Details:Protein type:Secreted, signal peptide; Inhibitor; Secreted

Chromosomal Location of Human Ortholog: 20p11.21

Cellular Component: extracellular space; extracellular region

Molecular Function:protein binding; protease binding; beta-amyloid binding; endopeptidase inhibitor activity; cysteine protease inhibitor activity

Biological Process: negative regulation of proteolysis; fibril organization and biogenesis; negative regulation of peptidase activity; defense response; regulation of tissue remodeling

Disease: Macular Degeneration, Age-related, 11; Cerebral Amyloid Angiopathy, Cst3-related

NCBI Summary:The cystatin superfamily encompasses proteins that contain multiple cystatin-like sequences. Some of the members are active cysteine protease inhibitors, while others have lost or perhaps never acquired this inhibitory activity. There are three inhibitory families in the superfamily, including the type 1 cystatins (stefins), type 2 cystatins and the kininogens. The type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. The cystatin locus on chromosome 20 contains the majority of the type 2 cystatin genes and pseudogenes. This gene is located in the cystatin locus and encodes the most abundant extracellular inhibitor of cysteine proteases, which is found in high concentrations in biological fluids and is expressed in virtually all organs of the body. A mutation in this gene has been associated with amyloid angiopathy. Expression of this protein in vascular wall smooth muscle cells is severely reduced in both atherosclerotic and aneurysmal aortic lesions, establishing its role in vascular disease. In addition, this protein has been shown to have an antimicrobial function, inhibiting the replication of herpes simplex virus. Alternative splicing results in multiple transcript variants encoding a single protein. [provided by RefSeq, Nov 2014]
UniProt Code:P01034
NCBI GenInfo Identifier:118183
NCBI Gene ID:1471
NCBI Accession:P01034.1
UniProt Related Accession:P01034
Molecular Weight:
NCBI Full Name:Cystatin-C
NCBI Synonym Full Names:cystatin C
NCBI Official Symbol:CST3  
NCBI Official Synonym Symbols:ARMD11; HEL-S-2  
NCBI Protein Information:cystatin-C
UniProt Protein Name:Cystatin-C
UniProt Synonym Protein Names:Cystatin-3; Gamma-trace; Neuroendocrine basic polypeptide; Post-gamma-globulin
Protein Family:Cystatin
UniProt Gene Name:CST3  
UniProt Entry Name:CYTC_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample TypeProtocol
SerumIf using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.

PlasmaCollect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal FluidCollect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatantCollect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysatesSolubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenatesThe preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysatesRinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast MilkCollect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

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