Description

Nori Canine Endostatin ELISA Kit Summary

Alternative names for endostatin: COL1A2

Alternative names for canine: dog

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Endostatin is a naturally-occurring, 20-kDa C-terminal fragment derived from type XVIII collagen and is a broad-spectrum angiogenesis inhibitor and may interfere with the pro-angiogenic action of growth factors such as  bFGF/FGF-2 and  VEGF.^[1] It was first found secreted in the media of non-metastasizing mouse cells from a hemangioendothelioma cell line in 1997 and was subsequently found in humans.^[2][3] It is produced by proteolytic cleavage of collagen XVIII, a member of the multiplexin family that is characterized by interruptions in the triple helix creating multiple domains, by proteases such as cathepsins.^[4] Endostatin, as a fragment of collagen 18, demonstrates a role of the ECM in suppression of neoangiogenesis.^[2] Overall, endostatin down regulates many signaling cascades like ephrin, TNF-α, and NFκB signaling as well as coagulation and adhesion cascades.^[5]  In-vitro studies have shown endostatin blocks the proliferation and organization of endothelial cells into new blood vessels.^[6] In animal studies endostatin inhibited angiogenesis and growth of both primary tumors and secondary metastasis.^[2] Endostatin is currently being studied as part of cancer research. Prior results indicated that endostatin can be beneficial in combinations with other medicines, but endostatin alone gave no significant improvements in tumor/disease progression. Endostatin may also be useful as a therapeutic for inflammatory diseases like rheumatoid arthritis as well as Crohn’s disease, diabetic retinopathy, psoriasis, and endometriosis by reducing the infiltration of inflammatory cells through invading angiogenesis.^[7] Down’s syndrome patients seem to be protected from diabetic retinopathy due to an additional copy of chromosome 21, and elevated expression of endostatin.^[8]

References

1. Folkman, J. (2006). Exp. Cell. Res. 312 (5, part 2): 594–607.
2. OReilly, M.S.; et al. (1997). Cell 88 (2): 277–85.
3. Standker, L; et al. (1997). FEBS Lett 420 (2–3): 129–33.
4. Felbor, U; et al. (2000). EMBO J 19 (6): 1187–94.
5. Abdollahi, A.; et al. (2004). Mol. Cell 13 (5): 649–63.
6. Folkman, J.; Kalluri, R. (2004). “Cancer without disease”. Nature 427 (6977): 787.
7. Yin, G., et al. (2002). Mol Ther 5 (5 Pt 1): 547–54.
8. Ryeom S, (2009). J Craniofac Surg. 20 Suppl 1: 595–6.

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