Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
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 Human DUOX2 in samples. No significant cross-reactivity or interference between Human DUOX2 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 Human DUOX2. 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 Human DUOX2 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 DUOX2, 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 Human DUOX2. The concentration of Human DUOX2 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	Function: Generates hydrogen peroxide which is required for the activity of thyroid peroxidase/TPO and lactoperoxidase/LPO. Plays a role in thyroid hormones synthesis and lactoperoxidase-mediated antimicrobial defense at the surface of mucosa. May have its own peroxidase activity through its N-terminal peroxidase-like domain. Ref. 7
UniProt Protein Details:	Catalytic activity: NAD(P)H + O2 = NAD(P)+ + H2O2. Ref. 11 Enzyme regulation: Peroxidase activity is inhibited by aminobenzohydrazide By similarity. The NADPH oxidase activity is calcium-dependent. Ref. 11 Pathway: Hormone biosynthesis; thyroid hormone biosynthesis. Subunit structure: Interacts with TXNDC11, TPO and CYBA. Ref. 10 Subcellular location: Apical cell membrane; Multi-pass membrane protein. Note: Localizes to the apical membrane of epithelial cells. Ref. 9 Tissue specificity: Expressed in colon, small intestine, duodenum and tracheal surface epithelial cells (at protein level). Expressed in thyrocytes. Also detected in kidney, liver, lung, pancreas, prostate, salivary glands, rectum and testis. Ref. 1 Ref. 2 Ref. 7 Ref. 8 Ref. 9 Developmental stage: Widely expressed in fetal tissues. Ref. 2 Induction: By forskolin, thyrotropin and the Th1-specific cytokine IFNG/IFN-gamma. Ref. 1 Ref. 11 Post-translational modification: N-glycosylated. Ref. 5 Involvement in Disease: Thyroid dyshormonogenesis 6 (TDH6) [MIM:607200]: A disorder due to a defective conversion of accumulated iodide to organically bound iodine. The iodide organification defect can be partial or complete. Note: The disease is caused by mutations affecting the gene represented in this entry. Ref. 6 Ref. 12 Ref. 13 Ref. 14 Sequence similarities: In the N-terminal section; belongs to the peroxidase family. Contains 3 EF-hand domains. Contains 1 FAD-binding FR-type domain. Contains 1 ferric oxidoreductase domain.
NCBI Summary:	The protein encoded by this gene is a glycoprotein and a member of the NADPH oxidase family. The synthesis of thyroid hormone is catalyzed by a protein complex located at the apical membrane of thyroid follicular cells. This complex contains an iodide transporter, thyroperoxidase, and a peroxide generating system that includes this encoded protein and DUOX1. This protein is known as dual oxidase because it has both a peroxidase homology domain and a gp91phox domain. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9NRD8
NCBI GenInfo Identifier:	132566532
NCBI Gene ID:	50506
NCBI Accession:	NP_054799. 4
UniProt Secondary Accession:	Q9NRD8,Q9NR02, Q9UHF9, A8MQ13,
UniProt Related Accession:	Q9NRD8
Molecular Weight:	175,364 Da
NCBI Full Name:	dual oxidase 2
NCBI Synonym Full Names:	dual oxidase 2
NCBI Official Symbol:	DUOX2
NCBI Official Synonym Symbols:	TDH6; LNOX2; THOX2; NOXEF2; P138-TOX
NCBI Protein Information:	dual oxidase 2; long NOX 2; large NOX 2; thyroid oxidase 2; p138 thyroid oxidase; NADPH thyroid oxidase 2; flavoprotein NADPH oxidase; dual oxidase-like domains 2; NADPH oxidase/peroxidase DUOX2; NADH/NADPH thyroid oxidase p138-tox; nicotinamide adenine dinucleotide phosphate oxidase
UniProt Protein Name:	Dual oxidase 2
UniProt Synonym Protein Names:	Large NOX 2; Long NOX 2; NADH/NADPH thyroid oxidase p138-tox; NADPH oxidase/peroxidase DUOX2; NADPH thyroid oxidase 2; Thyroid oxidase 2; p138 thyroid oxidase
Protein Family:	Dual oxidase
UniProt Gene Name:	DUOX2
UniProt Entry Name:	DUOX2_HUMAN

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	2.32 2.37	2.345	2.255
5	1.482 1.518	1.5	1.41
2.5	0.845 0.821	0.833	0.743
1.25	0.401 0.427	0.414	0.324
0.63	0.29 0.264	0.277	0.187
0.32	0.189 0.173	0.181	0.091
0.16	0.129 0.147	0.138	0.048
0	0.08 0.1	0.09	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human DUOX2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human DUOX2 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)	0.52	1.46	3.90	0.57	1.53	3.61
Standard deviation	0.04	0.09	0.16	0.03	0.08	0.16
C V (%)	7.69	6.16	4.10	5.26	5.23	4.43

Recovery

The recovery of Human DUOX2 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)	87-98	93
EDTA plasma (n=5)	92-107	98
Cell culture media (n=5)	94-108	101

Linearity

Samples were spiked with high concentrations of Human DUOX2 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 (%)	97-111	93-107	91-105
1:2	Average (%)	104	99	96
1:4	Range (%)	89-104	78-92	82-97
1:4	Average (%)	95	85	89
1:8	Range (%)	90-105	85-100	84-98
1:8	Average (%)	98	92	90
1:16	Range (%)	90-104	86-97	82-96
1:16	Average (%)	97	91	88

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.