Osteopontin (OPN), also known as bone sialoprotein (BSP), is a secreted SIBLING family protein that can be variably modified by O- and N-glycosylation, sulfation, phosphorylation, and transglutamination. OPN is widely expressed and is prominent in mineralized tissues. It inhibits bone mineralization and kidney stone formation and promotes inflammation, cell adÂhesion, and migration. Its expression is upregulatÂed during inflammation, obesity, atherosclerosis, cancer, and tissue damage and contributes to the pathophysiology of these conditions. The central region of OPN contains RGD and non-RGD binding sites for multiple integrins. Adjacent to the RGD motif is the sequence SLAYGLR (SVVYGLR in human) which serves as a cryptic binding site for additional integrins: it is masked in full length OPN but is exposed following OPN cleavage by multiple proteases in tumors and sites of tissue injury.
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Human Osteopontin (OPN) Quantikine ELISA Kit
R&D Systems | Catalog # DOST00
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Key Product Details
Assay Length
4.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (50 µL), EDTA Plasma (10 µL), Heparin Plasma (10 µL), Urine (50 µL), Human Milk (50 µL)
Sensitivity
0.024 ng/mL
Assay Range
0.312-20 ng/mL (Cell Culture Supernates, EDTA Plasma, Heparin Plasma, Urine, Human Milk)
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Product Summary for Human Osteopontin (OPN) Quantikine ELISA Kit
The Quantikine Human Osteopontin Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human Osteopontin in cell culture supernates, plasma, urine, and human milk. It contains NS0-expressed recombinant human Osteopontin and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Osteopontin showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring human Osteopontin.
Product Specifications
Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Measurement
Quantitative ELISA
Detection Method
Colorimetric - 450nm (TMB)
Conjugate
HRP
Species
Human
Specificity
Natural and recombinant human Osteopontin
Cross-reactivity
< 0.5% cross-reactivity observed with available related molecules. < 50% cross-species reactivity observed with species tested.
Interference
No significant interference observed with available related molecules.
Sample Values
Although serum has been validated for use in this assay, it is not a recommended sample
type because of proteolytic cleavage by thrombin during the clotting process. Serum
values are approximately 50% of the plasma values. See references 32-34 for more
details.
Cell Culture Supernates:
HepG2 human hepatocellular carcinoma cells were cultured in MEM supplemented with 5% fetal bovine serum until confluent. Cells were then stimulated with 50 ng/mL PMA for 24 hours. An aliquot of the cell culture supernate was removed, assayed for human OPN and measured 790 ng/mL.
Plasma/Urine - Samples from apparently healthy volunteers were evaluated for the presence
of human OPN in this assay. No medical histories were available for the donors used in this
study.
| Sample Type | Mean (ng/mL) | Range (ng/mL) | Standard Deviation (ng/mL) |
| EDTA plasma (n=40) | 94.8 | 49.2-175 | 24.9 |
| Heparin plasma (n=40) | 96.2 | 53.4-195 | 26.7 |
| Urine (n=21) | 2044 | 122-8796 | 1951 |
Cell Culture Supernates:
Human peripheral blood cells (1 x 106
cells/mL) were cultured in DMEM supplemented with
5% fetal bovine serum, 5 μM beta -mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and
100 μg/mL streptomycin sulfate. Cells were cultured unstimulated or stimulated with
10 μg/mL PHA. Aliquots of the cell culture supernates were removed and assayed for levels of
human OPN.
| Condition | Day 1 (ng/mL) | Day 6 (ng/mL) |
| Unstimulated | 0.675 | 1.85 |
| Stimulated | 1.50 | 2.67 |
HepG2 human hepatocellular carcinoma cells were cultured in MEM supplemented with 5% fetal bovine serum until confluent. Cells were then stimulated with 50 ng/mL PMA for 24 hours. An aliquot of the cell culture supernate was removed, assayed for human OPN and measured 790 ng/mL.
A431 human epithelial carcinoma cells were cultured in DMEM supplemented with
10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin
sulfate. An aliquot of the cell culture supernate was removed, assayed for human OPN and
measured 8.54 ng/mL.
Human Milk - Three human milk samples were assayed for human OPN and ranged from
35,600-248,000 ng/mL.
Precision
Intra-Assay Precision (Precision within an assay) Three samples of known concentration were tested on one plate to assess intra-assay precision.
Inter-Assay Precision (Precision between assays) Three samples of known concentration were tested in separate assays to assess inter-assay precision.
Cell Culture Supernates, EDTA Plasma, Heparin Plasma, Human Milk, Urine
| Intra-Assay Precision | Inter-Assay Precision | |||||
|---|---|---|---|---|---|---|
| Sample | 1 | 2 | 3 | 1 | 2 | 3 |
| n | 20 | 20 | 20 | 40 | 40 | 40 |
| Mean (ng/mL) | 2.30 | 4.90 | 9.30 | 2.36 | 4.81 | 9.17 |
| Standard Deviation | 0.092 | 0.126 | 0.267 | 0.155 | 0.274 | 0.499 |
| CV% | 4.0 | 2.6 | 2.9 | 6.6 | 5.7 | 5.4 |
Recovery for Human Osteopontin (OPN) Quantikine ELISA Kit
The recovery of Osteopontin spiked to three different levels throughout the range of the assay in various matrices was evaluated.
| Sample Type | Average % Recovery | Range % |
|---|---|---|
| Cell Culture Media (n=4) | 104 | 86-113 |
| EDTA Plasma (n=4) | 102 | 92-114 |
| Heparin Plasma (n=4) | 101 | 95-112 |
Linearity
To assess the linearity of the assay, samples containing and/or spiked with high concentrations of Osteopontin were serially diluted with the Calibrator Diluent to produce samples with values within the dynamic range of the assay.
Scientific Data Images for Human Osteopontin (OPN) Quantikine ELISA Kit
Human Osteopontin/OPN ELISA Standard Curve
Preparation and Storage
Shipping
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.
Background: Osteopontin/OPN
Long Name
Secreted Phosphoprotein 1 [BNSP]
Alternate Names
Eta-1, OPN, Spp1
Gene Symbol
SPP1
Additional Osteopontin/OPN Products
Product Documents for Human Osteopontin (OPN) Quantikine ELISA Kit
Certificate of Analysis
To download a Certificate of Analysis, please enter a lot or batch number in the search box below.
Note: Certificate of Analysis not available for kit components.
Product Specific Notices for Human Osteopontin (OPN) Quantikine ELISA Kit
For research use only
⚠ WARNING: This product can expose you to chemicals including N,N-Dimethylforamide, which is known to the State of California to cause cancer. For more information, go to www.P65Warnings.ca.gov.Citations for Human Osteopontin (OPN) Quantikine ELISA Kit
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Sample Tested: EDTA PlasmaVerified Customer | Posted 05/15/2024
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Sample Tested: Plasma and UrineVerified Customer | Posted 11/03/2023Measured plasma at 25-fold dilution and urine at 200-fold dilution. We were able to obtain values in range for both biofluids.
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Sample Tested: SerumVerified Customer | Posted 02/18/2021
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Sample Tested: Human cell conditioned mediumVerified Customer | Posted 12/01/2017
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Protocols
View specific protocols for Human Osteopontin (OPN) Quantikine ELISA Kit (DOST00):
Refer to the product for complete assay procedure.
Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.
- Prepare all reagents, standard dilutions, and samples as directed in the product insert.
- Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.
- Add 100 µL of Assay Diluent to each well.
- Add 50 µL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.
- Aspirate each well and wash, repeating the process 3 times for a total of 4 washes.
- Add 200 µL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.
- Aspirate and wash 4 times.
- Add 200 µL Substrate Solution to each well. Incubate at room temperature for 30 minutes. PROTECT FROM LIGHT.
- Add 50 µL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.
100 µL Assay Diluent
50 µL Standard, Control, or Sample
200 µL Conjugate
200 µL Substrate Solution
50 µL Stop Solution
Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.
- ELISA Sample Preparation & Collection Guide
- ELISA Troubleshooting Guide
- How to Run an R&D Systems DuoSet ELISA
- How to Run an R&D Systems Quantikine ELISA
- How to Run an R&D Systems Quantikine™ QuicKit™ ELISA
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- Quantikine HS ELISA Kit Principle, Streptavidin-HRP Polymer
- Sandwich ELISA (Colorimetric) – Biotin/Streptavidin Detection Protocol
- Sandwich ELISA (Colorimetric) – Direct Detection Protocol
- Troubleshooting Guide: ELISA
- View all Protocols, Troubleshooting, Illustrated assays and Webinars
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