Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse GRO alpha/CXCL1 in samples. No significant cross-reactivity or interference between Mouse GRO alpha/CXCL1 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 GRO alpha/CXCL1. 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 GRO alpha/CXCL1 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 GRO alpha/CXCL1, 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 GRO alpha/CXCL1. The concentration of Mouse GRO alpha/CXCL1 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	CXCL2: Produced by activated monocytes and neutrophils and expressed at sites of inflammation. Hematoregulatory chemokine, which, in vitro, suppresses hematopoietic progenitor cell proliferation. GRO-beta(5-73) shows a highly enhanced hematopoietic activity. Belongs to the intercrine alpha (chemokine CxC) family.
UniProt Protein Details:	Protein type:Secreted; Motility/polarity/chemotaxis; Secreted, signal peptide Cellular Component: extracellular space; cell; extracellular region; cytosol Molecular Function:growth factor activity; chemokine activity; CXCR chemokine receptor binding; cytokine activity Biological Process: neutrophil chemotaxis; G-protein coupled receptor protein signaling pathway; elevation of cytosolic calcium ion concentration; response to molecule of bacterial origin; positive regulation of superoxide release; positive regulation of potassium ion transport; response to lipopolysaccharide; immune response; positive regulation of leukocyte chemotaxis; inflammatory response; acute inflammatory response; regulation of cell proliferation
NCBI Summary:	This gene encodes a protein that is a member of the CXC subfamily of chemokines. Chemokines, which recruit and activate leukocytes, are classified by function (inflammatory or homeostatic) or by structure. This secretory protein is proposed to bind the G-protein coupled receptor chemokine (C-X-C motif) receptor 2 to recruit neutrophils. In mouse, deficiency of this gene is associated with colitis and with defects in immune cell recruitment to the lung. [provided by RefSeq, Apr 2013]
UniProt Code:	P12850
NCBI GenInfo Identifier:	121623
NCBI Gene ID:	14825
NCBI Accession:	P12850. 1
UniProt Related Accession:	P12850
Molecular Weight:	10,254 Da
NCBI Full Name:	Growth-regulated alpha protein
NCBI Synonym Full Names:	chemokine (C-X-C motif) ligand 1
NCBI Official Symbol:	Cxcl1
NCBI Official Synonym Symbols:	KC; Fsp; N51; gro; Gro1; Mgsa; Scyb1
NCBI Protein Information:	growth-regulated alpha protein; KC/GR)-alpha; KC/GRO-alpha; GRO1 oncogene; alpha-chemokine; secretory protein N51; C-X-C motif chemokine 1; platelet-derived growth factor-inducible protein KC
UniProt Protein Name:	Growth-regulated alpha protein
UniProt Synonym Protein Names:	C-X-C motif chemokine 1; Platelet-derived growth factor-inducible protein KC; Secretory protein N51Cleaved into the following chain:KC(5-72)Alternative name(s):Hematopoietic synergistic factor; HSF; KC-T
Protein Family:	KCNQ1 downstream neighbor protein
UniProt Gene Name:	Cxcl1
UniProt Entry Name:	GROA_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
1000	2.323 2.379	2.351	2.276
500	1.635 1.643	1.639	1.564
250	0.997 0.961	0.979	0.904
125	0.441 0.477	0.459	0.384
62.5	0.245 0.231	0.238	0.163
31.25	0.175 0.175	0.175	0.1
15.63	0.126 0.128	0.127	0.052
0	0.067 0.083	0.075	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse GRO alpha/CXCL1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse GRO alpha/CXCL1 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)	52.10	153.40	344.90	55.00	140.70	312.60
Standard deviation	2.80	8.30	16.20	3.50	5.90	10.00
C V (%)	5.37	5.41	4.70	6.36	4.19	3.20

Recovery

The recovery of Mouse GRO alpha/CXCL1 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)	87-101	93
EDTA plasma (n=5)	89-102	95
Cell culture media (n=5)	94-106	100

Linearity

Samples were spiked with high concentrations of Mouse GRO alpha/CXCL1 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 (%)	91-106	95-108	93-108
1:2	Average (%)	98	101	98
1:4	Range (%)	100-112	80-92	94-108
1:4	Average (%)	106	87	101
1:8	Range (%)	100-115	86-100	92-109
1:8	Average (%)	107	92	99
1:16	Range (%)	99-115	86-98	93-106
1:16	Average (%)	107	91	100

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.