Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
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 Mouse SELP in samples. No significant cross-reactivity or interference between Mouse SELP 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 SELP. 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 SELP 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 SELP, 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 SELP. The concentration of Mouse SELP in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	SELP: Ca(2+)-dependent receptor for myeloid cells that binds to carbohydrates on neutrophils and monocytes. Mediates the interaction of activated endothelial cells or platelets with leukocytes. The ligand recognized is sialyl-Lewis X. Mediates rapid rolling of leukocyte rolling over vascular surfaces during the initial steps in inflammation through interaction with PSGL1. Defects in SELP may be a cause of susceptibility to ischemic stroke (ISCHSTR); also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors. Belongs to the selectin/LECAM family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cell adhesion Cellular Component: nucleoplasm; extracellular space; platelet alpha granule membrane; membrane; cytoplasm; integral to membrane; external side of plasma membrane Molecular Function:heparin binding; protein binding; lipopolysaccharide binding; carbohydrate binding; glycoprotein binding; calcium-dependent protein binding; sialic acid binding; fucose binding Biological Process: heterophilic cell adhesion; positive regulation of phosphoinositide 3-kinase cascade; cell-cell adhesion; leukocyte adhesion; positive regulation of cell adhesion; regulation of integrin activation; positive regulation of leukocyte migration; regulation of cellular extravasation; blood vessel morphogenesis; cell adhesion; leukocyte tethering or rolling; inflammatory response; leukocyte migration
UniProt Code:	Q01102
NCBI GenInfo Identifier:	266459
NCBI Gene ID:	20344
NCBI Accession:	Q01102. 1
UniProt Secondary Accession:	Q01102,Q32MF1,
UniProt Related Accession:	Q01102
Molecular Weight:	83,099 Da
NCBI Full Name:	P-selectin
NCBI Synonym Full Names:	selectin, platelet
NCBI Official Symbol:	Selp
NCBI Official Synonym Symbols:	Grmp; CD62P; LECAM3; PADGEM; GMP-140
NCBI Protein Information:	P-selectin; granule membrane protein 140; CD62 antigen-like family member P; leukocyte-endothelial cell adhesion molecule 3; platelet activation dependent granule-external membrane protein
UniProt Protein Name:	P-selectin
UniProt Synonym Protein Names:	CD62 antigen-like family member P; Granule membrane protein 140; GMP-140; Leukocyte-endothelial cell adhesion molecule 3; LECAM3; Platelet activation dependent granule-external membrane protein; PADGEM; CD_antigen: CD62P
Protein Family:	P-selectin
UniProt Gene Name:	Selp
UniProt Entry Name:	LYAM3_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 (ng/mL)	O.D	Average	Corrected
10	2.393 2.441	2.417	2.354
5	1.613 1.653	1.633	1.57
2.5	0.958 0.94	0.949	0.886
1.25	0.502 0.532	0.517	0.454
0.63	0.281 0.255	0.268	0.205
0.31	0.17 0.168	0.169	0.106
0.16	0.118 0.12	0.119	0.056
0	0.056 0.07	0.063	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse SELP 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)	0.50	1.30	4.30	0.50	1.20	4.20
Standard deviation	0.03	0.07	0.18	0.03	0.06	0.16
C V (%)	6.00	5.38	4.19	6.00	5.00	3.81

Recovery

The recovery of Mouse SELP 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)	91-109	99
EDTA plasma (n=5)	90-104	95
Cell culture media (n=5)	92-103	98

Linearity

Samples were spiked with high concentrations of Mouse SELP 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 (%)	89-103	98-110	85-100
1:2	Average (%)	95	104	92
1:4	Range (%)	93-109	84-97	98-109
1:4	Average (%)	100	91	104
1:8	Range (%)	97-112	86-98	96-111
1:8	Average (%)	105	91	103
1:16	Range (%)	96-111	87-99	97-111
1:16	Average (%)	104	92	105

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.