Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
Detection method:	Chemiluminescence
Detection range:	7.81-500 ng/mL
Sensitivity:	4.69 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Rat AGT in samples. No significant cross-reactivity or interference between Rat 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 Rat 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 Rat 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 Rat 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 Rat AGT. The concentration of Rat 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 space; cytoplasm; extracellular region Molecular Function:serine-type endopeptidase inhibitor activity; protein binding; growth factor activity; sodium channel regulator activity; hormone activity; type 2 angiotensin receptor binding; superoxide-generating NADPH oxidase activator activity; type 1 angiotensin receptor binding Biological Process: renal system process; extracellular matrix organization and biogenesis; positive regulation of nitric oxide biosynthetic process; establishment of blood-nerve barrier; negative regulation of nerve growth factor receptor signaling pathway; positive regulation of transcription, DNA-dependent; stress-activated MAPK cascade; female pregnancy; positive regulation of multicellular organism growth; positive regulation of vasodilation; activation of NF-kappaB transcription factor; ovarian follicle rupture; positive regulation of fibroblast proliferation; cell-cell signaling; positive regulation of superoxide release; negative regulation of neuron apoptosis; kidney development; positive regulation of NAD(P)H oxidase activity; positive regulation of cytokine production; angiotensin mediated regulation of renal output; response to muscle activity involved in regulation of muscle adaptation; regulation of calcium ion transport; regulation of norepinephrine secretion; negative regulation of tissue remodeling; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of peptidyl-tyrosine phosphorylation; phospholipase C activation; angiotensin mediated vasoconstriction involved in regulation of systemic arterial blood pressure; regulation of vasoconstriction; regulation of transmission of nerve impulse; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); smooth muscle cell differentiation; cytokine secretion; nitric oxide mediated signal transduction; regulation of long-term neuronal synaptic plasticity; peristalsis; cell-matrix adhesion; renin-angiotensin regulation of aldosterone production; positive regulation of cellular protein metabolic process; smooth muscle cell proliferation; cellular lipid metabolic process; excretion; angiotensin maturation; vasodilation; response to salt stress; negative regulation of cell proliferation; positive regulation of MAPKKK cascade; fibroblast proliferation; renin-angiotensin regulation of blood vessel size; regulation of blood pressure; positive regulation of epidermal growth factor receptor signaling pathway; renin-angiotensin regulation of blood volume; regulation of cell growth; angiotensin mediated drinking behavior; artery smooth muscle contraction; aging; positive regulation of fatty acid biosynthetic process; blood vessel development; cellular sodium ion homeostasis; renal response to blood flow during renin-angiotensin regulation of systemic arterial blood pressure; activation of NF-kappaB-inducing kinase; positive regulation of peptidyl-serine phosphorylation; positive regulation of organ growth; regulation of cell proliferation; G-protein coupled receptor protein signaling pathway; negative regulation of angiogenesis; cellular protein metabolic process; ureteric bud branching; G-protein signaling, coupled to cGMP nucleotide second messenger; blood vessel remodeling; negative regulation of cell growth; response to cold; astrocyte activation; positive regulation of inflammatory response Disease: Renal Tubular Dysgenesis; Hypertension, Essential
NCBI Summary:	plays a role in regulation of blood pressure [RGD, Feb 2006]
UniProt Code:	P01015
NCBI GenInfo Identifier:	19705570
NCBI Gene ID:	24179
NCBI Accession:	NP_602308. 1
UniProt Secondary Accession:	P01015,Q16358, Q16359, Q96F91,
UniProt Related Accession:	P01015
Molecular Weight:	~61-65,000
NCBI Full Name:	angiotensinogen
NCBI Synonym Full Names:	angiotensinogen (serpin peptidase inhibitor, clade A, member 8)
NCBI Official Symbol:	Agt
NCBI Official Synonym Symbols:	Ang; PAT; ANRT; AngII
NCBI Protein Information:	angiotensinogen; serpin A8; angiotensin II; angiotensinogen (PAT)
UniProt Protein Name:	Angiotensinogen
UniProt Synonym Protein Names:	Serpin A8Cleaved into the following 8 chains:Angiotensin-1Alternative name(s):Angiotensin 1-10; Angiotensin I; Ang I
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
500	51442 51632	51537	51508
250	19281 22715	20998	20969
125	10130 8662	9396	9367
62.5	4194 4830	4512	4483
31.25	2351 2247	2299	2270
15.63	1343 1157	1250	1221
7.81	731 749	740	711
0	29 29	29	--

Precision

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

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat 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)	26.93	78.00	189.83	25.89	75.88	189.94
Standard deviation	3.39	7.53	21.49	2.75	8.97	16.22
C V (%)	12.59	9.65	11.32	10.62	11.82	8.54

Recovery

The recovery of Rat 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)	98-111	105
EDTA plasma (n=5)	89-104	96
Cell culture media (n=5)	97-111	103

Linearity

Samples were spiked with high concentrations of Rat 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 (%)	95-110	102-120	99-117
1:2	Average (%)	103	109	107
1:4	Range (%)	91-109	94-107	97-109
1:4	Average (%)	99	100	103
1:8	Range (%)	94-105	93-107	101-119
1:8	Average (%)	99	100	109
1:16	Range (%)	100-114	101-118	85-98
1:16	Average (%)	108	108	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 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.