Nori Human Hemopexin ELISA Kit Summary

Alternative names for hemopexin: HPX, HX, haemopexin, beta-1B-glycoprotein

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: 

Hemopexin (haemopexin; Hpx; Hx), also known as beta-1B-glycoprotein, is a glycoprotein that is encoded by the HPX gene^[1] and belongs to the hemopexin family of proteins. Hemopexin is a plasma protein with the highest binding affinity for heme and is the major vehicle for the transportation of heme in the plasma.^[2] Human plasma Hx consists of a single polypeptide chain of 439 amino acids residues with six intrachain disulfide bridges and has a molecular mass of approximately 63 kD. Its main function is scavenging the heme released or lost by the turnover of heme proteins such as hemoglobin and thus protects the body from the oxidative damage that free heme can cause. In addition, Hx releases its bound ligand for internalisation upon interacting with CD91.^[3] Hx preserves the body’s iron.^[2] Hx-dependent uptake of extracellular heme can lead to the deactivation of Bach1 repression which leads to the transcriptional activation of antioxidant heme oxygenase-1 gene. Hemoglobin, haptoglobin (Hp) and Hx associate with HDL and influence the inflammatory properties of HDL.^[4] Hx can downregulate the angiotensin II Type 1 receptor (AT1-R) in vitro.^[5] The predominant source of circulating Hx is the liver with a plasma concentration of 1–2 mg/ml.^[6] Serum Hx level reflects how much heme is present in the blood. Therefore, a low Hx level indicates that there has been significant degradation of heme containing compounds. A low Hx level is one of the diagnostic features of an intravascular hemolytic anemia. Hx has been implicated in cardiovascular disease, septic shock, cerebral ischemic injury, and experimental autoimmune encephalomyelitis.^[7] The circulating level of Hx is associated with prognosis in patients with septic shock.^[7] HPX is produced in the brain.^[8] Deletion of the HPX gene can aggravate brain injury followed by stroma-free hemoglobin-induced intracerebral haemorrhage.^[9] High Hx level in the cerebrospinal fluid is associated with poor outcome after subarachnoid hemorrhage.^[8] *Both Hp and Hx are acute-phase proteins, the synthesis of which are induced during infection and after inflammatory states to minimize tissue injury and facilitate tissue repair.^[2] Hp and Hx prevent heme toxicity by binding themselves to heme prior to monocyte or macrophage‘s arrivals and ensuing clearances.^[2]

References

1. Altruda F, et al. (1985). Nucleic Acids Research. 13 (11): 3841–59.
2. Tolosano E, Altruda F (April 2002). “Hemopexin: structure, function, and regulation”. DNA and Cell Biology. 21 (4): 297–306.
3. Hvidberg V, et al. (2005). Blood. 106 (7): 2572–9.
4. Watanabe J, et al. (2009). The Journal of Biological Chemistry. 284 (27): 18292–301.
5. Krikken JA, et al. (2013). Journal of Hypertension. 31 (3): 537–41.
6. Muller-Eberhard U, et al. (1968). Blood. 32 (5): 811–5.
7. Mehta NU, Reddy ST (2015). Current Opinion in Lipidology. 26 (5): 384–7.
8. Garland P, et al. (2016). Stroke. 47 (3): 872–6.
9. ^Ma B, et al. (2016). Journal of Neuroinflammation. 13: 26.