Mouse NOS2/iNOS (Nitric Oxide Synthase 2, Inducible) CLIA Kit (MOES00381)

Name: Mouse NOS2/iNOS (Nitric Oxide Synthase 2, Inducible) CLIA Kit (MOES00381)
Brand: Mouse Cell Signalling ELISA Kits 2
SKU: MOES00381
Availability: OutOfStock

Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection method:	Chemiluminescence
Detection range:	62.50-4000 pg/mL
Sensitivity:	37.50 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Mouse NOS2/iNOS in samples. No significant cross-reactivity or interference between Mouse NOS2/iNOS 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 Mouse NOS2/iNOS. 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 Mouse NOS2/iNOS 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 NOS2/iNOS, 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 Mouse NOS2/iNOS. The concentration of Mouse NOS2/iNOS in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	Produces nitric oxide (NO) which is a messenger molecule with diverse functions throughout the body (PubMed:7503239). In macrophages, NO mediates tumoricidal and bactericidal actions. Also has nitrosylase activity and mediates cysteine S-nitrosylation of cytoplasmic target proteins such PTGS2/COX2 (PubMed:16373578). As component of the iNOS-S100A8/9 transnitrosylase complex involved in the selective inflammatory stimulus-dependent S-nitrosylation of GAPDH implicated in regulation of the GAIT complex activity and probably multiple targets including ANXA5, EZR, MSN and VIM. Involved in inflammation, enhances the synthesis of proinflammatory mediators such as IL6 and IL8.
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. This gene encodes a nitric oxide synthase that is inducible by a combination of lipopolysaccharide and certain cytokines. Three transcript variants encoding two different isoforms have been found for this gene. [provided by RefSeq, Sep 2015]
UniProt Code:	P29477
NCBI GenInfo Identifier:	266649
NCBI Gene ID:	18126
NCBI Accession:	P29477. 1
UniProt Secondary Accession:	P29477,O70515, O70516, Q5SXT3, Q6P6A0, Q8R410,
UniProt Related Accession:	P29477
Molecular Weight:	130,575 Da
NCBI Full Name:	Nitric oxide synthase, inducible
NCBI Synonym Full Names:	nitric oxide synthase 2, inducible
NCBI Official Symbol:	Nos2
NCBI Official Synonym Symbols:	iNOS; Nos-2; Nos2a; i-NOS; NOS-II; MAC-NOS
NCBI Protein Information:	nitric oxide synthase, inducible
UniProt Protein Name:	Nitric oxide synthase, inducible
UniProt Synonym Protein Names:	Inducible NO synthase; Inducible NOS; iNOS; Macrophage NOS; MAC-NOS; NOS type II; Peptidyl-cysteine S-nitrosylase NOS2
Protein Family:	Inositol 3-kinase
UniProt Gene Name:	Nos2

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
4000	55607 62555	59081	59046
2000	26106 29612	27859	27824
1000	14583 12385	13484	13449
500	6573 6637	6605	6570
250	3299 3187	3243	3208
125	1636 1526	1581	1546
62.50	727 783	755	720
0	35 35	35	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse NOS2/iNOS were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse NOS2/iNOS 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)	213.23	560.81	1523.99	192.81	556.72	1413.08
Standard deviation	19.30	40.88	134.11	21.40	48.82	153.46
C V (%)	9.05	7.29	8.80	11.10	8.77	10.86

Recovery

The recovery of Mouse NOS2/iNOS 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)	88-102	95
EDTA plasma (n=5)	89-102	96
Cell culture media (n=5)	90-104	96

Linearity

Samples were spiked with high concentrations of Mouse NOS2/iNOS 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 (%)	90-103	103-118	88-101
1:2	Average (%)	97	110	94
1:4	Range (%)	99-116	93-104	101-117
1:4	Average (%)	107	99	109
1:8	Range (%)	97-110	89-102	98-115
1:8	Average (%)	104	96	105
1:16	Range (%)	94-111	92-105	101-116
1:16	Average (%)	101	99	109

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.