Product Name:	Human Serpin A8 / Angiotensinogen ELISA Kit
Product Code:	HUFI00397
Size:	96 Assays
Alias:	AGT, Angiotensinogen, ANHU, Serpin A8, SERPINA8, Angiotensin II, Aogen, PAT, alpha-1 antiproteinase, antitrypsin, angiotensin II, pre-angiotensinogen, serine, or cysteine proteinase inhibitor, SERPINA8, angiotensin I
Detection method:	Competitive ELISA, Coated with Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human AGT concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ug/ml
Range:	0.313-20ug/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human AGT and the recovery rates were calculated by comparing the measured value to the expected amount of Human AGT in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	91-104	97
EDTA plasma(n=5)	85-102	92
UFH plasma(n=5)	87-103	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human AGT and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-101%	85-104%	96-105%
EDTA plasma(n=5)	86-95%	85-98%	86-100%
UFH plasma(n=5)	80-100%	82-95%	80-95%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate(Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	60ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipettetips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P01019

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:	AGTÃ‚ Ã‚
NCBI Official Synonym Symbols:	ANHU; SERPINA8Ã‚ Ã‚
NCBI Protein Information:	angiotensinogen
UniProt Protein Name:	Angiotensinogen
UniProt Synonym Protein Names:	Serpin A8
Protein Family:	Angiotensinogen
UniProt Gene Name:	AGTÃ‚ Ã‚
UniProt Entry Name:	ANGT_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!
2.	Add Sample and Biotin-detection antibody: Add 50Ã‚µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 Ã‚µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.	Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.
4.	HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.
5.	Wash: Repeat the aspiration/wash process for five times.
6.	TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.
7.	Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.
8.	OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testing parameters.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.