Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
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 Rat FLT1/VEGFR1 in samples. No significant cross-reactivity or interference between Rat FLT1/VEGFR1 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 Rat FLT1/VEGFR1. 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 Rat FLT1/VEGFR1 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 Rat FLT1/VEGFR1, 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 Rat FLT1/VEGFR1. The concentration of Rat FLT1/VEGFR1 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	VEGFR1: a receptor tyrosine kinase of the VEGFR family. Receptor for VEGF, VEGFB and PGF. The VEGF-kinase ligand/receptor signaling system plays a key role in vascular development and regulation of vascular permeability. Two splice variant isoforms have been described. Isoform SFlt1 may have an inhibitory role in angiogenesis.
UniProt Protein Details:	Protein type:Protein kinase, tyrosine (receptor); Membrane protein, integral; Protein kinase, TK; EC 2. 7. 10. 1; Kinase, protein; TK group; VEGFR family Cellular Component: focal adhesion; integral to plasma membrane; plasma membrane; receptor complex; endosome Molecular Function:vascular endothelial growth factor receptor activity; identical protein binding; growth factor binding; transmembrane receptor protein tyrosine kinase activity; receptor signaling protein tyrosine kinase activity; ATP binding Biological Process: peptidyl-tyrosine phosphorylation; activation of MAPKK activity; protein amino acid autophosphorylation; positive regulation of smooth muscle cell proliferation; chemotaxis; protein amino acid phosphorylation; positive regulation of vascular endothelial growth factor receptor signaling pathway; positive regulation of MAP kinase activity; monocyte chemotaxis; positive regulation of fibroblast proliferation; positive regulation of cell proliferation; angiogenesis; embryonic morphogenesis; cell differentiation; regulation of smooth muscle contraction; cell migration; intracellular receptor-mediated signaling pathway; positive regulation of phosphoinositide 3-kinase activity; patterning of blood vessels; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of angiogenesis; response to hypoxia; blood vessel morphogenesis; sprouting angiogenesis; vascular endothelial growth factor receptor signaling pathway; transmembrane receptor protein tyrosine kinase signaling pathway; positive regulation of cell migration
NCBI Summary:	tyrosine kinase receptor for vascular endothelial growth factor; may play a role in cell proliferation and cell survival [RGD, Feb 2006]
UniProt Code:	P53767
NCBI GenInfo Identifier:	1718161
NCBI Gene ID:	54251
NCBI Accession:	P53767. 1
UniProt Related Accession:	P53767
Molecular Weight:	150,250 Da
NCBI Full Name:	Vascular endothelial growth factor receptor 1
NCBI Synonym Full Names:	FMS-related tyrosine kinase 1
NCBI Official Symbol:	Flt1
NCBI Official Synonym Symbols:	VEGFR-1
NCBI Protein Information:	vascular endothelial growth factor receptor 1; FLT-1; FMS-like tyrosine kinase 1; tyrosine-protein kinase receptor FLT; vascular endothelial growth factor receptor-1; Fms-related tyrosine kinase 1 (vascular endothelial growth factor/vascular permeability factor receptor)
UniProt Protein Name:	Vascular endothelial growth factor receptor 1
UniProt Synonym Protein Names:	Fms-like tyrosine kinase 1; FLT-1; Tyrosine-protein kinase receptor FLT
Protein Family:	Vascular endothelial growth factor receptor
UniProt Gene Name:	Flt1
UniProt Entry Name:	VGFR1_RAT

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	2.351 2.395	2.373	2.282
5	1.607 1.663	1.635	1.544
2.5	0.994 0.968	0.981	0.89
1.25	0.528 0.568	0.548	0.457
0.63	0.276 0.258	0.267	0.176
0.32	0.209 0.181	0.195	0.104
0.16	0.135 0.153	0.144	0.053
0	0.084 0.098	0.091	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat FLT1/VEGFR1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat FLT1/VEGFR1 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)	0.55	1.28	3.82	0.54	1.24	4.03
Standard deviation	0.04	0.06	0.15	0.04	0.07	0.20
C V (%)	7.27	4.69	3.93	7.41	5.65	4.96

Recovery

The recovery of Rat FLT1/VEGFR1 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)	92-109	99
EDTA plasma (n=5)	85-97	91
Cell culture media (n=5)	89-102	95

Linearity

Samples were spiked with high concentrations of Rat FLT1/VEGFR1 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 (%)	96-109	85-98	90-103
1:2	Average (%)	102	92	95
1:4	Range (%)	88-101	85-98	83-94
1:4	Average (%)	95	92	89
1:8	Range (%)	92-108	84-96	83-94
1:8	Average (%)	99	89	88
1:16	Range (%)	88-102	83-95	86-100
1:16	Average (%)	95	90	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.