Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse PCSK9 in samples. No significant cross-reactivity or interference between Mouse PCSK9 and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse PCSK9. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse PCSK9 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse PCSK9, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse PCSK9. The concentration of Mouse PCSK9 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	PCSK9: Crucial player in the regulation of plasma cholesterol homeostasis. Binds to low-density lipid receptor family members: low density lipoprotein receptor (LDLR), very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor (LRP1/APOER) and apolipoprotein receptor 2 (LRP8/APOER2), and promotes their degradation in intracellular acidic compartments. Acts via a non-proteolytic mechanism to enhance the degradation of the hepatic LDLR through a clathrin LDLRAP1/ARH-mediated pathway. May prevent the recycling of LDLR from endosomes to the cell surface or direct it to lysosomes for degradation. Can induce ubiquitination of LDLR leading to its subsequent degradation. Inhibits intracellular degradation of APOB via the autophagosome/lysosome pathway in a LDLR-independent manner. Involved in the disposal of non-acetylated intermediates of BACE1 in the early secretory pathway. Inhibits epithelial Na(+) channel (ENaC)-mediated Na(+) absorption by reducing ENaC surface expression primarily by increasing its proteasomal degradation. Regulates neuronal apoptosis via modulation of LRP8/APOER2 levels and related anti-apoptotic signaling pathways. Defects in PCSK9 are the cause of hypercholesterolemia autosomal dominant type 3 (HCHOLA3). A familial condition characterized by elevated circulating cholesterol contained in either low-density lipoproteins alone or also in very-low-density lipoproteins. Belongs to the peptidase S8 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell development/differentiation; EC 3. 4. 21. -; Protease; Secreted; Secreted, signal peptide Cellular Component: Golgi apparatus; extracellular space; cell surface; rough endoplasmic reticulum; endoplasmic reticulum; lysosome; early endosome; extracellular region; ER to Golgi transport vesicle; perinuclear region of cytoplasm; cytoplasm; late endosome; plasma membrane; endosome Molecular Function:peptidase activity; sodium channel inhibitor activity; very-low-density lipoprotein binding; protein self-association; hydrolase activity; low-density lipoprotein receptor binding; serine-type peptidase activity; serine-type endopeptidase activity; low-density lipoprotein binding; apolipoprotein binding; apolipoprotein receptor binding Biological Process: steroid metabolic process; cholesterol metabolic process; lysosomal transport; apoptosis; positive regulation of receptor internalization; lipoprotein metabolic process; regulation of low-density lipoprotein receptor catabolic process; cellular response to starvation; proteolysis; neuron differentiation; protein autoprocessing; cholesterol homeostasis; triacylglycerol metabolic process; cellular response to insulin stimulus; phospholipid metabolic process; positive regulation of neuron apoptosis; regulation of neuron apoptosis; negative regulation of receptor recycling; low-density lipoprotein receptor catabolic process; protein processing; regulation of receptor activity; lipid metabolic process; low-density lipoprotein receptor metabolic process
UniProt Code:	Q80W65
NCBI GenInfo Identifier:	38257924
NCBI Gene ID:	100102
NCBI Accession:	Q80W65. 2
UniProt Secondary Accession:	Q80W65,Q3UEH7, Q8BXW9, Q8CFT6, B1AZI4,
UniProt Related Accession:	Q80W65
Molecular Weight:	74,823 Da
NCBI Full Name:	Proprotein convertase subtilisin/kexin type 9
NCBI Synonym Full Names:	proprotein convertase subtilisin/kexin type 9
NCBI Official Symbol:	Pcsk9
NCBI Official Synonym Symbols:	FH3; PC9; Narc1; HCHOLA3; AI415265; AI747682
NCBI Protein Information:	proprotein convertase subtilisin/kexin type 9; NARC-1; proprotein convertase 9; proprotein convertase PC9; subtilisin/kexin-like protease PC9; neural apoptosis regulated convertase 1; neural apoptosis-regulated convertase 1
UniProt Protein Name:	Proprotein convertase subtilisin/kexin type 9
UniProt Synonym Protein Names:	Neural apoptosis-regulated convertase 1; NARC-1; Proprotein convertase 9; PC9; Subtilisin/kexin-like protease PC9
Protein Family:	Proprotein convertase subtilisin/kexin
UniProt Gene Name:	Pcsk9
UniProt Entry Name:	PCSK9_MOUSE

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	O.D	Average	Corrected
4000	2.384 2.398	2.391	2.337
2000	1.549 1.577	1.563	1.509
1000	0.878 0.848	0.863	0.809
500	0.426 0.442	0.434	0.38
250	0.215 0.213	0.214	0.16
125	0.161 0.141	0.151	0.097
62.50	0.103 0.105	0.104	0.05
0	0.052 0.056	0.054	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse PCSK9 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse PCSK9 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	196.60	594.41	1776.72	213.36	594.84	1824.11
Standard deviation	12.27	25.62	61.65	13.48	29.44	72.23
C V (%)	6.24	4.31	3.47	6.32	4.95	3.96

Recovery

The recovery of Mouse PCSK9 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	91-105	97
EDTA plasma (n=5)	88-99	93
Cell culture media (n=5)	89-104	96

Linearity

Samples were spiked with high concentrations of Mouse PCSK9 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	93-107	97-113	85-100
1:2	Average (%)	100	103	92
1:4	Range (%)	93-109	84-95	86-102
1:4	Average (%)	99	89	93
1:8	Range (%)	85-101	83-93	82-96
1:8	Average (%)	92	88	88
1:16	Range (%)	88-104	85-97	89-101
1:16	Average (%)	95	91	94

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro ELISA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.