Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human AGT in samples. No significant cross-reactivity or interference between Human AGT 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 AGT. 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 AGT 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 AGT, 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 AGT. The concentration of Human AGT in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	angiotensin: Essential component of the renin-angiotensin system (RAS), a potent regulator of blood pressure, body fluid and electrolyte homeostasis. In response to lowered blood pressure, the enzyme renin cleaves angiotensinogen to produce angiotensin-1 (angiotensin 1-10). Angiotensin-1 is a substrate of ACE (angiotensin converting enzyme) that removes a dipeptide to yield the physiologically active peptide angiotensin-2 (angiotensin 1- 8). Angiotensin-1 and angiotensin-2 can be further processed to generate angiotensin-3 (angiotensin 2-8), angiotensin-4 (angiotensin 3-8). Angiotensin 1-7 is cleaved from angiotensin-2 by ACE2 or from angiotensin-1 by MME (neprilysin). Angiotensin 1-9 is cleaved from angiotensin-1 by ACE2. Genetic variations in AGT are a cause of susceptibility to essential hypertension (EHT). Essential hypertension is a condition in which blood pressure is consistently higher than normal with no identifiable cause. Defects in AGT are a cause of renal tubular dysgenesis (RTD). RTD is an autosomal recessive severe disorder of renal tubular development characterized by persistent fetal anuria and perinatal death, probably due to pulmonary hypoplasia from early-onset oligohydramnios (the Potter phenotype). Belongs to the serpin family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 1q42. 2 Cellular Component: extracellular region; extracellular space Molecular Function:growth factor activity; hormone activity; protein binding; serine-type endopeptidase inhibitor activity; type 1 angiotensin receptor binding; type 2 angiotensin receptor binding Biological Process: activation of NF-kappaB transcription factor; angiotensin maturation; blood vessel remodeling; cell-cell signaling; G-protein coupled receptor protein signaling pathway; G-protein signaling, coupled to cGMP nucleotide second messenger; kidney development; negative regulation of nerve growth factor receptor signaling pathway; nitric oxide mediated signal transduction; positive regulation of cellular protein metabolic process; positive regulation of cytokine production; positive regulation of epidermal growth factor receptor signaling pathway; positive regulation of fibroblast proliferation; positive regulation of inflammatory response; positive regulation of NAD(P)H oxidase activity; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of transcription, DNA-dependent; regulation of blood pressure; renal system process; renin-angiotensin regulation of blood vessel size; response to muscle activity involved in regulation of muscle adaptation Disease: Hypertension, Essential; Renal Tubular Dysgenesis
NCBI Summary:	The protein encoded by this gene, pre-angiotensinogen or angiotensinogen precursor, is expressed in the liver and is cleaved by the enzyme renin in response to lowered blood pressure. The resulting product, angiotensin I, is then cleaved by angiotensin converting enzyme (ACE) to generate the physiologically active enzyme angiotensin II. The protein is involved in maintaining blood pressure and in the pathogenesis of essential hypertension and preeclampsia. Mutations in this gene are associated with susceptibility to essential hypertension, and can cause renal tubular dysgenesis, a severe disorder of renal tubular development. Defects in this gene have also been associated with non-familial structural atrial fibrillation, and inflammatory bowel disease. [provided by RefSeq, Jul 2008]
UniProt Code:	P01019
NCBI GenInfo Identifier:	113880
NCBI Gene ID:	183
NCBI Accession:	P01019. 1
UniProt Secondary Accession:	P01019,Q16358, Q16359, Q96F91,
UniProt Related Accession:	P01019
Molecular Weight:	53,154 Da
NCBI Full Name:	Angiotensinogen
NCBI Synonym Full Names:	angiotensinogen
NCBI Official Symbol:	AGT
NCBI Official Synonym Symbols:	ANHU; SERPINA8
NCBI Protein Information:	angiotensinogen
UniProt Protein Name:	Angiotensinogen
UniProt Synonym Protein Names:	Serpin A8
Protein Family:	Angiotensinogen
UniProt Gene Name:	AGT
UniProt Entry Name:	ANGT_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 (ng/mL)	RLU	Average	Corrected
20	32040 35924	33982	33950
10	12302 12638	12470	12438
5	5356 5144	5250	5218
2.5	2416 2632	2524	2492
1.25	1398 1368	1383	1351
0.63	891 843	867	835
0.31	590 656	623	591
0	32 32	32	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human AGT 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)	0.96	2.83	7.58	0.92	2.68	6.97
Standard deviation	0.10	0.28	0.86	0.08	0.23	0.70
C V (%)	10.42	9.89	11.35	8.70	8.58	10.04

Recovery

The recovery of Human AGT 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	94
EDTA plasma (n=5)	100-115	108
Cell culture media (n=5)	100-113	107

Linearity

Samples were spiked with high concentrations of Human AGT 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 (%)	98-116	90-103	87-101
1:2	Average (%)	106	97	94
1:4	Range (%)	89-103	89-101	102-114
1:4	Average (%)	95	94	108
1:8	Range (%)	84-95	90-105	89-103
1:8	Average (%)	90	97	95
1:16	Range (%)	86-102	87-102	102-118
1:16	Average (%)	93	93	108

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.