Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	15.63-1000 pg/mL
Sensitivity:	9.38 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human NOS3/eNOS in samples. No significant cross-reactivity or interference between Human NOS3/eNOS and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human NOS3/eNOS. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human NOS3/eNOS 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 NOS3/eNOS, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human NOS3/eNOS. The concentration of Human NOS3/eNOS in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	eNOS: endothelial constitutive nitric oxide synthase. Synthesizes nitric oxide (NO) from arginine and oxygen, which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets. Induced in humans by caloric restriction. Stimulated by calcium/calmodulin. Inhibited by NOSIP and NOSTRIN.
UniProt Protein Details:	Protein type:Amino Acid Metabolism - arginine and proline; Oxidoreductase; EC 1. 14. 13. 39 Chromosomal Location of Human Ortholog: 7q36 Cellular Component: caveola; cytoplasm; cytoskeleton; cytosol; Golgi membrane; nucleus; plasma membrane Molecular Function:actin monomer binding; cadmium ion binding; calmodulin binding; FAD binding; FMN binding; heme binding; iron ion binding; NADP binding; nitric-oxide synthase activity; protein binding Biological Process: angiogenesis; arginine catabolic process; blood coagulation; blood vessel remodeling; cell redox homeostasis; endothelial cell migration; in utero embryonic development; lipopolysaccharide-mediated signaling pathway; lung development; mitochondrion organization and biogenesis; negative regulation of blood pressure; negative regulation of calcium ion transport; negative regulation of cell proliferation; negative regulation of hydrolase activity; negative regulation of muscle hyperplasia; negative regulation of potassium ion transport; nitric oxide biosynthetic process; nitric oxide mediated signal transduction; nitric oxide metabolic process; ovulation from ovarian follicle; positive regulation of angiogenesis; positive regulation of guanylate cyclase activity; positive regulation of vasodilation; regulation of blood pressure; regulation of blood vessel size; regulation of nitric-oxide synthase activity; regulation of sodium ion transport; regulation of systemic arterial blood pressure by endothelin; regulation of the force of heart contraction by chemical signal; removal of superoxide radicals; response to heat; smooth muscle hyperplasia; vascular endothelial growth factor receptor signaling pathway Disease: Alzheimer Disease; Hypertension, Essential; Preeclampsia/eclampsia 1; Stroke, Ischemic
NCBI Summary:	Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2009]
UniProt Code:	P29474
NCBI GenInfo Identifier:	266648
NCBI Gene ID:	4846
NCBI Accession:	P29474. 3
UniProt Secondary Accession:	P29474,Q13662, Q14251, Q14434, Q548C1, Q6GSL5, Q9UDC6 A0S0A7, A0S0A8, A8KA63, B2RCQ1, E9PFR2,
UniProt Related Accession:	P29474
Molecular Weight:
NCBI Full Name:	Nitric oxide synthase, endothelial
NCBI Synonym Full Names:	nitric oxide synthase 3
NCBI Official Symbol:	NOS3
NCBI Official Synonym Symbols:	eNOS; ECNOS
NCBI Protein Information:	nitric oxide synthase, endothelial
UniProt Protein Name:	Nitric oxide synthase, endothelial
UniProt Synonym Protein Names:	Constitutive NOS; cNOS; EC-NOS; Endothelial NOS; eNOS; NOS type III; NOSIII
Protein Family:	Nitric oxide synthase
UniProt Gene Name:	NOS3
UniProt Entry Name:	NOS3_HUMAN

As the RLU 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)	RLU	Average	Corrected
1000	51725 55625	53675	53650
500	21321 23657	22489	22464
250	10842 9580	10211	10186
125	4568 5234	4901	4876
62.5	2503 2403	2453	2428
31.25	1329 1233	1281	1256
15.63	663 753	708	683
0	25 25	25	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human NOS3/eNOS were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human NOS3/eNOS 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)	49.99	142.23	366.47	45.51	148.76	366.50
Standard deviation	6.46	14.22	22.32	5.18	13.92	29.72
C V (%)	12.92	10.00	6.09	11.38	9.36	8.11

Recovery

The recovery of Human NOS3/eNOS 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)	92-102	97
EDTA plasma (n=5)	98-116	106
Cell culture media (n=5)	90-105	96

Linearity

Samples were spiked with high concentrations of Human NOS3/eNOS 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 (%)	89-102	101-113	91-102
1:2	Average (%)	96	107	96
1:4	Range (%)	89-103	102-115	87-99
1:4	Average (%)	97	108	94
1:8	Range (%)	99-116	100-116	85-95
1:8	Average (%)	106	107	90
1:16	Range (%)	96-112	91-103	97-109
1:16	Average (%)	104	98	103

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 CLIA 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 A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
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 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.