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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.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.
Recovery
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
Assay Procedure
Refer to the product 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
Human Osteopontin (OPN) Quantikine ELISA Kit Summary
Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Assay Length
4.5 hours
Sample Type & Volume Required Per Well
Cell Culture Supernates (50 uL), EDTA Plasma (10 uL), Heparin Plasma (10 uL), Urine (50 uL), Human Milk (50 uL)
Sensitivity
0.024 ng/mL
Assay Range
0.3 - 20 ng/mL (Cell Culture Supernates, EDTA Plasma, Heparin Plasma, Urine, Human Milk)
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.
背景
别名
BNSP,Bone sialoprotein 1,Eta-1,MGC110940,Nephropontin,OPN,Osteopontin,secreted phosphoprotein 1bone sialoprotein I, early T-lymphocyteactivation 1),secreted phosphoprotein-1 (osteopontin, bone sialoprotein),Spp1,SPP-1,SPP1/CALPHA1 fusion,Urinary stone protein,uropontin,Secreted Phosphoprotein 1 [BNSP]
背景
Background: Osteopontin/OPN
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 ad¬hesion, and migration. Its expression is upregulat¬ed 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.
Long Name:
Secreted Phosphoprotein 1 [BNSP]
Entrez Gene IDs:
6696 (Human); 20750 (Mouse); 25353 (Rat); 281499 (Bovine)
Alternate Names:
BNSP; Bone sialoprotein 1; Eta-1; MGC110940; Nephropontin; OPN; Osteopontin; secreted phosphoprotein 1bone sialoprotein I, early T-lymphocyteactivation 1); secreted phosphoprotein-1 (osteopontin, bone sialoprotein); Spp1; SPP-1; SPP1/CALPHA1 fusion; Urinary stone protein; uropontin
研究领域
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