Mouse Proprotein Convertase 9/PCSK9 Quantikine ELISA Kit Summary

Product Summary

The Quantikine Mouse PCSK9 Immunoassay is a 4.5 hour solid-phase ELISA designed to measure mouse PCSK9 in cell culture supernates, cell lysates, serum, and plasma. It contains NS0-expressed recombinant mouse PCSK9 and antibodies raised against the recombinant factor. This immunoassay has been shown to accurately quantitate the recombinant factor. Results obtained using natural mouse PCSK9 showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring mouse PCSK9.

Preparation and Storage

Background: Proprotein Convertase 9/PCSK9

PCSK9 (proprotein convertase subtilisin kexin 9), also called proprotein convertase 9 or NARC-1 (neural apoptosis-regulated convertase 1), is a member of the proteinase K subfamily of subtilisinrelated serine endoproteases. Mouse PCSK9 cDNA encodes 694 amino acids, including a signal peptide, a prodomain, and a catalytic domain. PCSK9 is highly expressed in the liver, intestine, and kidney. It is initially synthesized as a soluble 74 kDa precursor protein. In the endoplasmic reticulum, it undergoes autocatalytic intramolecular cleavage to generate a 14 kDa prodomain and a 60 kDa catalytic domain. While within the secretion pathway, the prodomain remains associated and functions as a chaperone for the catalytic domain (1-4). During secretion, a portion of active PCSK9 may undergo additional N-terminal proteolysis by furin or proprotein convertase 5/6A, creating an inactive 53 kDa form (5). This cleavage site is conserved between mouse and human or rat PCSK9, which share 78% or 93% amino acid sequence identity, respectively. While the 60 kDa protein is the major form, its ratio with the 53 kDa forms is variable in humans (5, 6). 

The primary physiologic function of PCSK9 is to mediate the degradation of low density lipoprotein receptor (LDLR). Early observations indicated that gain-of-function missense mutations in the human PCSK9 gene can cause an autosomal dominant form of hypercholesterolemia (7, 8). The expression of PCSK9 is also upregulated by the sterol regulatory element binding proteins (SREBPs), a family of transcription factors that are responsible for the upregulation of genes involved in cholesterol and fatty acid metabolism, such as the LDLR gene (9, 10). Further experimental evidence revealed that when the mouse PCSK9 gene is deleted, LDLR expression in hepatocytes is increased. Conversely, PCSK9 over-expression decreases liver LDLR protein expression (11, 12). In humans, genetic analyses have shown that individuals who have nonsense or loss-of-function mutations in the PCSK9 gene have significantly lower plasma LDL cholesterol levels, while in mouse, administration of a PCSK9 neutralizing antibody or antisense oligonucleotides lowers serum cholesterol (1, 13-15). These investigations clearly indicate that PCSK9 plays a key role in reducing the hepatic LDLR levels. Paradoxically, administration of cholesterol-lowering drugs such as statins appear to enhance production of PCSK9 (6). 

The underlying mechanism of cholesterol regulation by PCSK9 is as follows: under normal physiologic conditions, the LDLR is internalized at the cell surface and directed to the endosomes in order to be recycled back to the cell surface. PCSK9 binds to the EGF domain of the LDLR and prevents LDLR from being sorted to the endosomes. Instead, the PCSK9/LDLR complex is redistributed to the lysosomes for degradation (16-18). As such, PCSK9 regulates the amount of LDLR in the circulation and hence, modulates cholesterol levels. Serum PCSK9 concentrations have been found to be directly associated with cholesterol levels (19, 20). Since PCSK9 loss-of-function mutations strikingly reduce risk of coronary heart diseases, PCSK9 has become an attractive drug target (1, 21, 22). One approach is to generate small molecules that are able to interfere with PCSK9 autoactivation and its interaction with LDLR. Other approaches aiming to reduce the amount of PCSK9 in the circulation, such as small interfering RNAs (siRNAs), have also shown promise (23, 24).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1、Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2、Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3、Add 50 μL of Assay Diluent to each well.

4、Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5、Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6、Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7、Aspirate and wash 5 times.

8、Add 100 μL Substrate Solution to each well.

9、Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.