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Human Osteopontin (OPN) DuoSet ELISA Summary

Assay Type
Solid Phase Sandwich ELISA
Format
96-well strip plate
Sample Volume Required
100 µL
Assay Range
62.5 - 4,000 pg/mL
Sufficient Materials
For fifteen 96-well plates*
Specificity
Please see the product datasheet

* Provided that the recommended microplates, buffers, diluents, substrates and solutions are used, and the assay is run as summarized in the Assay Procedure provided.

This DuoSet ELISA Development kit contains the basic components required for the development of sandwich ELISAs to measure natural and recombinant human Osteopontin (OPN). The suggested diluent is suitable for the analysis of most cell culture supernate samples. Diluents for complex matrices, such as serum and plasma, should be evaluated prior to use in this DuoSet.

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)

Data Example

Human Osteopontin / OPN ELISA Standard Curve

Product Datasheets

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

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

Assay Procedure

GENERAL ELISA PROTOCOL

Plate Preparation

  1. 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.
  2. 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.
  3. Block plates by adding 300 μL Reagent Diluent to each well. Incubate at room temperature for a minimum of 1 hour.
  4. Repeat the aspiration/wash as in step 2. The plates are now ready for sample addition.

Assay Procedure

  1. 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.
  2. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  3. 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.
  4. Repeat the aspiration/wash as in step 2 of Plate Preparation.
  5. 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.
  6. Repeat the aspiration/wash as in step 2.
  7. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light.
  8. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  9. 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 Osteopontin (OPN) 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.

25 Citations: Showing 1 - 10
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  1. Chemerin Overexpression in the Liver Protects against Inflammation in Experimental Non-Alcoholic Steatohepatitis
    Authors: R Pohl, S Feder, EM Haberl, L Rein-Fisch, TS Weiss, M Spirk, A Bruckmann, N McMullen, CJ Sinal, C Buechler
    Biomedicines, 2022;10(1):.
    Species: Human
    Sample Types: Cell Culture Supernates
  2. Zinc Ameliorates the Osteogenic Effects of High Glucose in Vascular Smooth Muscle Cells
    Authors: LA Henze, M Estepa, B Pieske, F Lang, KU Eckardt, I Alesutan, J Voelkl
    Cells, 2021;10(11):.
    Species: Human
    Sample Types: Cell Culture Supernates
  3. Osteopontin levels are associated with late-time lower regional brain volumes in multiple sclerosis
    Authors: G Orsi, Z Hayden, T Cseh, T Berki, Z Illes
    Scientific Reports, 2021;11(1):23604.
    Species: Human
    Sample Types: Serum
  4. Strong Associations between Plasma Osteopontin and Several Inflammatory Chemokines, Cytokines, and Growth Factors
    Authors: A Larsson, J Helmersson, L Lind, J Ärnlöv, TR Feldreich
    Biomedicines, 2021;9(8):.
    Species: Human
    Sample Types: Urine
  5. High Levels of the Cleaved Form of Galectin-9 and Osteopontin in the Plasma Are Associated with Inflammatory Markers That Reflect the Severity of COVID-19 Pneumonia
    Authors: G Bai, D Furushima, T Niki, T Matsuba, Y Maeda, A Takahashi, T Hattori, Y Ashino
    International Journal of Molecular Sciences, 2021;22(9):.
    Species: Human
    Sample Types: Plasma
  6. Serum protein triplet TGF-&beta1, TIMP-1, and YKL-40 serve as diagnostic and prognostic profile for astrocytoma
    Authors: R Urbanavi?i, R Zabitait?, A Kriš?iukai, VP Deltuva, D Skiriut?
    Scientific Reports, 2021;11(1):13100.
    Species: Human
    Sample Types: Serum
  7. MiR-181 Family Modulates Osteopontin in Glioblastoma Multiforme
    Authors: A Marisetty, J Wei, LY Kong, M Ott, D Fang, A Sabbagh, AB Heimberger
    Cancers, 2020;12(12):.
    Species: Human
    Sample Types: Cell Culture Supernates
  8. Circulating IL-13 Is Associated with De Novo Development of HCC in HCV-Infected Patients Responding to Direct-Acting Antivirals
    Authors: Z Macek Jílk, A Seigneurin, C Coppard, L Ouaguia, C Aspord, PN Marche, V Leroy, T Decaens
    Cancers, 2020;12(12):.
    Species: Human
    Sample Types: Serum
  9. Stimulation of THP-1 Macrophages with LPS Increased the Production of Osteopontin-Encapsulating Exosome
    Authors: G Bai, T Matsuba, T Niki, T Hattori
    Int J Mol Sci, 2020;21(22):.
    Species: Human
    Sample Types: Cell Culture Supernates
  10. Plasma Levels of a Cleaved Form of Galectin-9 Are the Most Sensitive Biomarkers of Acquired Immune Deficiency Syndrome and Tuberculosis Coinfection
    Authors: ST Padilla, T Niki, D Furushima, G Bai, H Chagan-Yas, EF Telan, RJ Tactacan-A, Y Maeda, R Solante, T Hattori
    Biomolecules, 2020;10(11):.
    Species: Human
    Sample Types: Plasma
  11. Co-culture of human fibroblasts, smooth muscle and endothelial cells promotes osteopontin induction in hypoxia
    Authors: N Sadaghianl, J Contenti, M Dufies, J Parola, M Rouleau, S Lee, JF Peyron, L Fabbri, R Hassen-Kho, J Pouysségur, F Bost, E Jean-Bapti, A Dardik, NM Mazure
    J. Cell. Mol. Med., 2020;0(0):.
    Species: Human
    Sample Types: Cell Culture Supernates
  12. Osteopontin Expression in Small Airway Epithelium in Copd is Dependent on Differentiation and Confined to Subsets of Cells
    Authors: MN Ali, M Mori, TCJ Mertens, P Siddhuraj, JS Erjefält, P Önnerfjord, PS Hiemstra, A Egesten
    Sci Rep, 2019;9(1):15566.
    Species: Human
    Sample Types: Cell Culture Supernates
  13. Osteopontin in the Cerebrospinal Fluid of Patients with Severe Aneurysmal Subarachnoid Hemorrhage
    Authors: MG Abate, L Moretto, I Licari, T Esposito, L Capuano, C Olivieri, A Benech, M Brucoli, GC Avanzi, G Cammarota, U Dianzani, N Clemente, G Panzarasa, G rio, F Carfagna, G Cappellano, F Della Cort, R Vaschetto
    Cells, 2019;8(7):.
    Species: Human
    Sample Types: Plasma
  14. Synthesis of a Cleaved Form of Osteopontin by THP-1 Cells and Its Alteration by Phorbol 12-Myristate 13-Acetate and BCG Infection
    Authors: G Bai, H Motoda, R Ozuru, H Chagan-Yas, T Hattori, T Matsuba
    Int J Mol Sci, 2018;19(2):.
    Species: Human
    Sample Types: Plasma
  15. Stem cell functionality is microenvironmentally defined during tumour expansion and therapy response in colon cancer
    Authors: KJ Lenos, DM Miedema, SC Lodestijn, LE Nijman, T van den Bo, X Romero Ros, FC Lourenço, MC Lecca, M van der He, SM van Neerve, A van Oort, N Leveille, RS Adam, F de Sousa E, J Otten, P Veerman, G Hypolite, L Koens, SK Lyons, G Stassi, DJ Winton, JP Medema, E Morrissey, MF Bijlsma, L Vermeulen
    Nat. Cell Biol., 2018;0(0):.
    Species: Xenograft
    Sample Types: Cell Culture Supernates
  16. Periodontal disease influences osteoclastogenic bone markers in subjects with and without rheumatoid arthritis
    Authors: J Panezai, A Ghaffar, M Altamash, PE Engström, A Larsson
    PLoS ONE, 2018;13(6):e0197235.
    Species: Human
    Sample Types: Serum
  17. Osteopontin regulates macrophage activation and osteoclast formation in hypertensive patients with vascular calcification
    Authors: Q Ge, CC Ruan, Y Ma, XF Tang, QH Wu, JG Wang, DL Zhu, PJ Gao
    Sci Rep, 2017;7(0):40253.
    Species: Human
    Sample Types: Serum
  18. Longitudinal Assessment of Transorbital Sonography, Visual Acuity, and Biomarkers for Inflammation and Axonal Injury in Optic Neuritis
    Authors: P Lochner, R Cantello, K Fassbender, M Lesmeister, R Nardone, A Siniscalch, N Clemente, A Naldi, L Coppo, F Brigo, C Comi
    Dis. Markers, 2017;2017(0):5434310.
    Species: Human
    Sample Types: Serum
  19. The association between osteopontin gene polymorphisms, osteopontin expression and sarcoidosis
    Authors: H Lavi, M Assayag, A Schwartz, N Arish, ZG Fridlender, N Berkman
    PLoS ONE, 2017;12(3):e0171945.
    Species: Human
    Sample Types: Plasma
  20. Secretion of IFN-? Associated with Galectin-9 Production by Pleural Fluid Cells from a Patient with Extrapulmonary Tuberculosis
    Authors: J Zhao, B Shiratori, H Chagan-Yas, M Matsumoto, T Niki, M Tanaka, Y Takahashi, O Usami, Y Ashino, T Hattori
    Int J Mol Sci, 2017;18(7):.
    Species: Human
    Sample Types: Plasma
  21. Osteopontin is a strong predictor of incipient diabetic nephropathy, cardiovascular disease, and all-cause mortality in patients with type 1 diabetes.
    Authors: Gordin D, Forsblom C, Panduru N, Thomas M, Bjerre M, Soro-Paavonen A, Tolonen N, Sandholm N, Flyvbjerg A, Harjutsalo V, Groop P
    Diabetes Care, 2014;37(9):2593-600.
    Species: Human
    Sample Types: Serum
  22. Localization of microfibrillar-associated protein 4 (MFAP4) in human tissues: clinical evaluation of serum MFAP4 and its association with various cardiovascular conditions.
    Authors: Wulf-Johansson H, Lock Johansson S, Schlosser A, Trommelholt Holm A, Rasmussen L, Mickley H, Diederichsen A, Munkholm H, Poulsen T, Tornoe I, Nielsen V, Marcussen N, Vestbo J, Saekmose S, Holmskov U, Sorensen G
    PLoS ONE, 2013;8(12):e82243.
    Species: Human
    Sample Types: Serum
  23. Elevated serum osteopontin levels and genetic polymorphisms of osteopontin are associated with Vogt-Koyanagi-Harada disease.
    Authors: Chu M, Yang P, Hu R, Hou S, Li F, Chen Y, Kijlstra A
    Invest. Ophthalmol. Vis. Sci., 2011;52(10):7084-9.
    Species: Human
    Sample Types: Serum
  24. Osteopontin expression and relation to disease severity in human asthma.
    Authors: Samitas K, Zervas E, Vittorakis S, Semitekolou M, Alissafi T, Bossios A, Gogos H, Economidou E, Lotvall J, Xanthou G, Panoutsakopoulou V, Gaga M
    Eur. Respir. J., 2011;37(2):331-41.
    Species: Human
    Sample Types: Serum
  25. Plasma decorin predicts the presence of esophageal squamous cell carcinoma.
    Authors: Wu IC, Wu DC, Huang CC
    Int. J. Cancer, 2010;127(9):2138-46.
    Species: Human
    Sample Types: Plasma

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Reviews for Human Osteopontin (OPN) DuoSet ELISA

Average Rating: 4.7 (Based on 3 Reviews)

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Human Osteopontin (OPN) DuoSet ELISA
By Anonymous on 05/16/2022
Sample Tested: Serum and Plasma

We used this kit to quantify Osteopontin in human serum and plasma, works very well at higher dilution.


Human Osteopontin (OPN) DuoSet ELISA
By Kebin Liu on 01/08/2021
Sample Tested: Humna serum

Human Osteopontin (OPN) DuoSet ELISA
By Abby Sukarto on 05/03/2017
Sample Tested: Human tissue