Description

Nori Porcine PCSK9 ELISA Kit Summary

Alternative names for PCSK9: Proprotein convertase subtilisin/kexin type 9 (PCSK9)

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: 

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme encoded by the PCSK9 gene.^[1] It is the 9th member of the proprotein convertase family that activate other proteins.^[2] PCSK9 becomes activated when a section of peptide chains is removed.^[3] The PCSK9 gene also contains one of 27 loci associated with increased risk of coronary artery disease.^[4] PCSK9 is ubiquitously expressed in many tissues and cell types but mainly in the liver, the intestine, the kidney, and the central nervous system.^[5] PCSK9 is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. After being processed in the ER, PCSK9 co-localizes with the protein sortilin on its way through the Golgi and trans-Golgi complex. A PCSK9-sortilin interaction is proposed to be required for cellular secretion of PCSK9.^[6] The protein may function as a proprotein convertase^[3] and plays a major regulatory role in cholesterol homeostasis, mainly by reducing LDLR levels on the plasma membrane.  Reduced LDLR levels result in decreased metabolism of LDL-particles, which could lead to hypercholesterolemia.  PCSK9 also plays an important role in intestinal triglyceride-rich apoB lipoprotein production in small intestine and postprandial lipemia.^[7] PCSK9 binds to the receptor for low-density lipoprotein particles (LDL). The LDL receptor (LDLR), on liver and other cell membranes, binds and initiates ingestion of LDL-particles from extracellular fluid into cells, thus reducing LDL particle concentrations. If PCSK9 is blocked, more LDLRs are recycled and are present on the surface of cells to remove LDL-particles from the extracellular fluid.^[8] Therefore, blocking PCSK9 can lower blood LDL-particle concentrations.^[9] In healthy humans, plasma PCSK9 levels directly correlate with plasma sortilin levels, following a diurnal rhythm similar to cholesterol synthesis.^[10] The plasma PCSK9 concentration is higher in women compared to men, and the PCSK9 concentrations decrease with age in men but increase in women, suggesting that estrogen level most likely plays a role.^[11] PCSK9 gene expression can be regulated by sterol-response element binding proteins (SREBP-1/2), which also controls LDLR expression.^[10]

References

1. Seidah NG, et al. (2003). Proc. Natl. Acad. Sci. U.S.A. 100 (3): 928–33.
2. Zhang L, et al. (2016) International Journal of Neuroscience. 126 (6): 675–680.
3. Lagace TA (2014). Current Opinion in Lipidology. 25 (5): 387–93.
4. Mega JL, et al. (2015). Lancet. 385 (9984): 2264–71.
5. Norata GD, et al. (2014). Annual Review of Pharmacology and Toxicology. 54: 273–93.
6. Gustafsen C, et al. (2014). Cell Metabolism. 19 (2): 310–8.
7. Rashid S, et al. (2014). Circulation. 130 (5): 431–41.
8. Weinreich M, Frishman WH (2014) Cardiology in Review. 22 (3): 140–6.
9. Joseph L, Robinson JG (2015). Progress in Cardiovascular Diseases. 58 (1): 19–31.
10. Schulz R, et al. (2015). Basic Research in Cardiology. 110 (2): 4.
11. Baass A, et al. (2009). Clinical Chemistry. 55 (9): 1637–45.

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