Human Pro-Collagen II 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
DuoSet Ancillary Reagent Kit 2 (5 plates): (Catalog # DY008) containing 96 well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and Reagent Diluent Concentrate 2.
The components listed above may be purchased separately:
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 0.05% Tween® 20 in PBS, pH 7.2-7.4
Reagent Diluent: (Catalog # DY995), or 1% BSA in PBS, pH 7.2-7.4, 0.2 µm filtered
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)
Plate Sealers: ELISA Plate Sealers (Catalog # DY992)
Scientific Data
Product Datasheets
Preparation and Storage
Background: Collagen II
Collagens comprise a large family of insoluble extracellular glycoproteins that are essential components of connective tissues such as tendons, ligaments, cartilage, bone and skin. The mature polypeptides are secreted as coiled, left-handed helices that subsequently assemble into rope-like collagen fibers. Collagen I is a fibril-forming collagen that requires N- and C- terminal processing. Collagen IV is a network forming collagen whose C-terminus forms dimers and N-terminus forms tetramers.
Assay Procedure
GENERAL ELISA PROTOCOL
Plate Preparation
- Dilute the Capture Antibody to the working concentration 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 plates by adding 300 μL of Block Buffer to each well. Incubate at room temperature for a minimum of 1 hour.
- Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.
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, 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 Pro-Collagen II 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.
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Citations: Showing 1 - 10
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Differential Effects of Hypoxia versus Hyperoxia or Physoxia on Phenotype and Energy Metabolism in Human Chondrocytes from Osteoarthritic Compared to Macroscopically Normal Cartilage
Authors: L Jain, SM Bolam, AP Monk, JT Munro, E Chen, J Tamatea, N Dalbeth, RC Poulsen
International Journal of Molecular Sciences, 2023-04-19;24(8):.
Species: Human
Sample Types: Cell Culture Supernates
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Syringaresinol attenuates osteoarthritis via regulating the NF-kappaB pathway
Authors: X Wang, D Wang, B Deng, L Yan
International immunopharmacology, 2023-03-28;118(0):109982.
Species: Mouse
Sample Types: Cell Culture Supernates
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Formononetin inhibits IL-1beta-induced inflammation in human chondrocytes and slows the progression of osteoarthritis in rat model via the regulation of PTEN/AKT/NF-kappaB pathway
Authors: C Jia, F Hu, D Lu, H Jin, H Lu, E Xue, D Wu
International immunopharmacology, 2022-10-25;113(0):109309.
Species: Human, Rat
Sample Types: Cell Culture Supernates
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Physiologic Mechanical Stress Directly Induces Bone Formation by Activating Glucose Transporter 1 (Glut 1) in Osteoblasts, Inducing Signaling via NAD+-Dependent Deacetylase (Sirtuin 1) and Runt-Related Transcription Factor 2 (Runx2)
Authors: S Somemura, T Kumai, K Yatabe, C Sasaki, H Fujiya, H Niki, K Yudoh
International Journal of Molecular Sciences, 2021-08-23;22(16):.
Species: Human
Sample Types: Cell Culture Supernates
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Concerted actions by MMPs, ADAMTS and serine proteases during remodeling of the cartilage callus into bone during osseointegration of hip implants
Authors: J Cassuto, A Folestad, J Göthlin, H Malchau, J Kärrholm
Bone Rep, 2020-09-11;13(0):100715.
Species: Human
Sample Types: Plasma
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Nomilin targets the Keap1-Nrf2 signalling and ameliorates the development of osteoarthritis
Authors: XH Xue, JX Xue, W Hu, FL Shi, Y Yang
J. Cell. Mol. Med., 2020-06-21;0(0):.
Species: Mouse
Sample Types: Cell Culture Supernates
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Regulation of differentiation of annulus fibrosus-derived stem cells using heterogeneous electrospun fibrous scaffolds
Authors: P Zhou, G Chu, Z Yuan, H Wang, W Zhang, Y Mao, X Zhu, W Chen, H Yang, B Li
Journal of Orthopaedic Translation, 2020-03-03;26(0):171-180.
Species: Rabbit
Sample Types: Cell Culture Supernates
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Pharmacological blockade of PCAF ameliorates osteoarthritis development via dual inhibition of TNF-alpha-driven inflammation and ER stress
Authors: D Chen, D Lu, H Liu, E Xue, Y Zhang, P Shang, X Pan
EBioMedicine, 2019-11-14;0(0):.
Species: Human
Sample Types: Cell Culture Supernates
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Knockout of Apolipoprotein E in rabbit promotes premature intervertebral disc degeneration: A new in vivo model for therapeutic approaches of spinal disc disorders
Authors: A Beierfu beta, H Dietrich, C Kremser, M Hunjadi, A Ritsch, T Rülicke, C Thomé, DS Mern
PLoS ONE, 2017-11-03;12(11):e0187564.
Species: Rabbit
Sample Types: Tissue Homogenates
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Self-complementary adeno-associated virus serotype 6 mediated knockdown of ADAMTS4 induces long-term and effective enhancement of aggrecan in degenerative human nucleus pulposus cells: A new therapeutic approach for intervertebral disc disorders
Authors: DS Mern, A Tschugg, S Hartmann, C Thomé
PLoS ONE, 2017-02-16;12(2):e0172181.
Species: Human
Sample Types: Cell Extracts
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