Human Cystatin C DuoSet ELISA Summary
* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.
Product Features
- Optimized capture and detection antibody pairings with recommended concentrations save lengthy development time
- Development protocols are provided to guide further assay optimization
- Assay can be customized to your specific needs
- Economical alternative to complete kits
Kit Content
- Capture Antibody
- Detection Antibody
- Recombinant Standard
- Streptavidin conjugated to horseradish-peroxidase (Streptavidin-HRP)
Other Reagents Required
PBS: (Catalog # DY006), or 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.2 - 7.4, 0.2 µm filtered
Wash Buffer: (Catalog # WA126), or equivalent
Reagent Diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).
Blocking Buffer: 1% BSA in PBS, pH 7.2-7.4, 0.2 m filtered (R&D Systems Catalog # DY995). Quality of BSA is critical (see Technical Hints).
Substrate Solution: 1:1 mixture of Color Reagent A (H2O2) and Color Reagent B (Tetramethylbenzidine) (Catalog # DY999)
Stop Solution: 2 N H2SO4 (Catalog # DY994)
Microplates: R&D Systems (Catalog # DY990), or equivalent
Plate Sealers: ELISA Plate Sealers (Catalog # DY992), or equivalent
Scientific Data
Product Datasheets
Preparation and Storage
Background: Cystatin C
Cystatin C is a secreted cysteine protease inhibitor that inhibits Cathepsins B, H, K, L, and S. Cystatin C serum concentration correlates closely to the glomerular filtration rate (GFR); elevated levels are associated with coronary artery and cardiovascular disease risk. Dysregulation of Cystatin C can modulate tumor growth and metastasis. In humans, the L68Q variant forms dimers and oligomers more easily than wild type protein and is the cause for hereditary Cystatin C amyloid angiopathy.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody (to the working concentration stated in the product datasheet ) in PBS without carrier protein. Immediately coat a 96-well microplate with 100 µL per well of the diluted Capture Antibody. Seal the plate and incubate overnight at room temperature.
- Aspirate each well and wash with Wash Buffer, repeating the process two times for a total of three washes. Wash by filling each well with Wash Buffer (400 µL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Buffer by aspirating or by inverting the plate and blotting it against clean paper towels.
- Block each well of the microplate as recommended in the product datasheet. Incubate at room temperature for a minimum of 1 hour.
Note: The recommended Reagent Diluent typically contains 1% BSA. Some DuoSet Development Kits require alternative blocking agents, or for plates to be blocked overnight with a higher percentage of BSA, please see the product datasheet for details.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
PRECAUTION
The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.
Assay Procedure
- Add 100 µL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 µL of the Detection Antibody, diluted in Reagent Diluent (as recommended in the product datasheet), to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature.
- Repeat the aspiration/wash as in step 2 of Plate Preparation.
- Add 100 µL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Repeat the aspiration/wash as in step 2.
- Add 100 µL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
- Add 50 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
- Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Citations for Human Cystatin C DuoSet ELISA
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
8
Citations: Showing 1 - 8
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Longitudinal Follow-Up of Serum and Urine Biomarkers Indicative of COVID-19-Associated Acute Kidney Injury: Diagnostic and Prognostic Impacts
Authors: Lablad, Y;Vanhomwegen, C;De Prez, E;Antoine, MH;Hasan, S;Baudoux, T;Nortier, J;
International journal of molecular sciences
Species: Human
Sample Types: Plasma
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Flash Characterization of Smartphones Used in Point-of-Care Diagnostics
Authors: BV Vu, R Lei, C Mohan, K Kourentzi, RC Willson
Biosensors, 2022-11-22;12(12):.
Species: Human
Sample Types: Complex Sample Type
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Binding of heparan sulfate to human cystatin C modulates inhibition of cathepsin L: Putative consequences in mucopolysaccharidosis
Authors: S Denamur, T Chazeirat, M Maszota-Zi, RR Vivès, A Saidi, F Zhang, RJ Linhardt, F Labarthe, SA Samsonov, G Lalmanach, F Lecaille
Carbohydrate polymers, 2022-06-18;293(0):119734.
Species: Human
Sample Types: Cell Culture Supernates
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Chitinase-3-like 1 is a biomarker of acute kidney injury and mortality in paediatric severe malaria
Authors: AL Conroy, MT Hawkes, R Elphinston, RO Opoka, S Namasopo, C Miller, CC John, KC Kain
Malar. J., 2018-02-15;17(1):82.
Species: Human
Sample Types: Plasma
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Creatinine, arsenic metabolism, and renal function in an arsenic-exposed population in Bangladesh.
Authors: Peters B, Hall M, Liu X, Neugut Y, Pilsner J, Levy D, Ilievski V, Slavkovich V, Islam T, Factor-Litvak P, Graziano J, Gamble M
PLoS ONE, 2014-12-01;9(12):U1429.
Species: Human
Sample Types: Plasma
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Regulation of TGF-beta1-driven differentiation of human lung fibroblasts: emerging roles of cathepsin B and cystatin C.
Authors: Kasabova M, Joulin-Giet A, Lecaille F, Gilmore B, Marchand-Adam S, Saidi A, Lalmanach G
J Biol Chem, 2014-04-30;289(23):16239-51.
Species: Human
Sample Types: Cell Culture Supernates
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Plasma cathepsin S and cystatin C levels and risk of abdominal aortic aneurysm: a randomized population-based study.
Authors: Lv B, Lindholt J, Cheng X, Wang J, Shi G
PLoS ONE, 2012-07-23;7(7):e41813.
Species: Human
Sample Types: Plasma
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Characterization of the seminal plasma proteome in men with prostatitis by mass spectrometry.
Authors: Kagedan D, Lecker I, Batruch I
Clin Proteomics, 2012-02-06;9(1):2.
Species: Human
Sample Types: Seminal Plasma
FAQs
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Serum ran at 1:500 dilution and urine ran at 1:300 dilution. Great kit.
