Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.16-10 ng/mL
Sensitivity:	0.10 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human SORT1 in samples. No significant cross-reactivity or interference between Human SORT1 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 Human SORT1. 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 Human SORT1 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 Human SORT1, 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 Human SORT1. The concentration of Human SORT1 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	SORT1: Functions as a sorting receptor in the Golgi compartment and as a clearance receptor on the cell surface. Required for protein transport from the Golgi apparatus to the lysosomes by a pathway that is independent of the mannose-6-phosphate receptor (M6PR). Also required for protein transport from the Golgi apparatus to the endosomes. Promotes neuronal apoptosis by mediating endocytosis of the proapoptotic precursor forms of BDNF (proBDNF) and NGFB (proNGFB). Also acts as a receptor for neurotensin. May promote mineralization of the extracellular matrix during osteogenic differentiation by scavenging extracellular LPL. Probably required in adipocytes for the formation of specialized storage vesicles containing the glucose transporter SLC2A4/GLUT4 (GLUT4 storage vesicles, or GSVs). These vesicles provide a stable pool of SLC2A4 and confer increased responsiveness to insulin. May also mediate transport from the endoplasmic reticulum to the Golgi. A common polymorphism located in a non-coding region between CELSR2 and PSRC1 alters a CEBP transcription factor binding site and is responsible for changes in hepatic expression of SORT1. Altered SORT1 expression in liver affects low density lipoprotein cholesterol levels in plasma and is associated with susceptibility to myocardial infarction. Belongs to the VPS10-related sortilin family. SORT1 subfamily.
UniProt Protein Details:	Protein type:Membrane protein, integral; Receptor, misc. Chromosomal Location of Human Ortholog: 1p13. 3\|1p21. 3-p13. 1 Cellular Component: Golgi apparatus; endoplasmic reticulum membrane; nuclear membrane; cell surface; clathrin-coated vesicle; lysosomal membrane; early endosome; cytoplasmic membrane-bound vesicle; integral to membrane; coated pit; trans-Golgi network transport vesicle; perinuclear region of cytoplasm; plasma membrane; endosome membrane Molecular Function:protein binding; nerve growth factor receptor activity; enzyme binding; nerve growth factor binding; neurotensin receptor activity, non-G-protein coupled Biological Process: ossification; nerve growth factor receptor signaling pathway; vesicle organization and biogenesis; multicellular organismal development; endosome transport via multivesicular body sorting pathway; myotube differentiation; Golgi to endosome transport; endocytosis; response to insulin stimulus; G-protein coupled receptor protein signaling pathway; induction of apoptosis via death domain receptors; neuropeptide signaling pathway; regulation of gene expression; glucose import; endosome to lysosome transport; plasma membrane to endosome transport; negative regulation of lipoprotein lipase activity Disease: Low Density Lipoprotein Cholesterol Level Quantitative Trait Locus 6
NCBI Summary:	This gene encodes a member of the VPS10-related sortilin family of proteins. The encoded preproprotein is proteolytically processed by furin to generate the mature receptor. This receptor plays a role in the trafficking of different proteins to either the cell surface, or subcellular compartments such as lysosomes and endosomes. Expression levels of this gene may influence the risk of myocardial infarction in human patients. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Oct 2015]
UniProt Code:	Q99523
NCBI GenInfo Identifier:	84028263
NCBI Gene ID:	6272
NCBI Accession:	Q99523. 3
UniProt Related Accession:	Q99523
Molecular Weight:	92kDa
NCBI Full Name:	Sortilin
NCBI Synonym Full Names:	sortilin 1
NCBI Official Symbol:	SORT1
NCBI Official Synonym Symbols:	NT3; Gp95; NTR3; LDLCQ6
NCBI Protein Information:	sortilin
UniProt Protein Name:	Sortilin
UniProt Synonym Protein Names:	100 kDa NT receptor; Glycoprotein 95; Gp95; Neurotensin receptor 3; NT3; NTR3
Protein Family:	Sortilin
UniProt Gene Name:	SORT1
UniProt Entry Name:	SORT_HUMAN

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 (ng/mL)	O.D	Average	Corrected
10	2.368 2.372	2.37	2.304
5	1.614 1.628	1.621	1.555
2.5	0.916 0.902	0.909	0.843
1.25	0.426 0.462	0.444	0.378
0.63	0.273 0.253	0.263	0.197
0.32	0.166 0.162	0.164	0.098
0.16	0.112 0.12	0.116	0.05
0	0.066 0.066	0.066	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human SORT1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human SORT1 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 (ng/mL)	0.54	1.20	3.82	0.50	1.24	3.84
Standard deviation	0.04	0.06	0.15	0.03	0.05	0.21
C V (%)	7.41	5.00	3.93	6.00	4.03	5.47

Recovery

The recovery of Human SORT1 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)	93-107	99
EDTA plasma (n=5)	94-106	101
Cell culture media (n=5)	88-104	96

Linearity

Samples were spiked with high concentrations of Human SORT1 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 (%)	89-105	91-108	95-109
1:2	Average (%)	96	98	100
1:4	Range (%)	90-102	81-92	85-97
1:4	Average (%)	95	87	91
1:8	Range (%)	92-106	83-93	87-100
1:8	Average (%)	99	88	93
1:16	Range (%)	92-104	85-97	86-100
1:16	Average (%)	99	91	92

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.