Product Name:	Human ACE / Angiotensin Converting Enzyme 1 ELISA Kit
Product Code:	HUFI00025
Size:	96 Assays
Alias:	ACE, CD143, DCP1, Kininase II, ACE1angiotensin converting enzyme, somatic isoform, angiotensin I converting enzyme, peptidyl-dipeptidase A 1, carboxycathepsin, CD143, CD143 antigen, DCP, DCP1angiotensin-converting enzyme, dipeptidyl carboxypeptidase 1, Dipeptidyl carboxypeptidase I, Kininase II, MVCD3, peptidase P, testicular ECA
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human ACE concentrations in serum plasma and other biological fluids.
Sensitivity:	0.469ng/ml
Range:	0.781-50ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

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

Matrix	Recovery range(%)	Average(%)
serum(n=5)	96-103	99
EDTA plasma(n=5)	85-103	91
UFH plasma(n=5)	96-104	100

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ACE 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)	85-102%	85-101%	88-101%
EDTA plasma(n=5)	85-99%	86-100%	82-98%
UFH plasma(n=5)	90-96%	80-100%	84-95%

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)	120ul	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 pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P12821

UniProt Protein Function:	ACE: Converts angiotensin I to angiotensin II by release of the terminal His-Leu, this results in an increase of the vasoconstrictor activity of angiotensin. Also able to inactivate bradykinin, a potent vasodilator. Has also a glycosidase activity which releases GPI-anchored proteins from the membrane by cleaving the mannose linkage in the GPI moiety. Genetic variations in ACE may be a cause of susceptibility to ischemic stroke (ISCHSTR); also known as cerebrovascular accident or cerebral infarction. A stroke is an acute neurologic event leading to death of neural tissue of the brain and resulting in loss of motor, sensory and/or cognitive function. Ischemic strokes, resulting from vascular occlusion, is considered to be a highly complex disease consisting of a group of heterogeneous disorders with multiple genetic and environmental risk factors. Defects in ACE 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). Genetic variations in ACE are associated with susceptibility to microvascular complications of diabetes type 3 (MVCD3). These are pathological conditions that develop in numerous tissues and organs as a consequence of diabetes mellitus. They include diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease, and diabetic neuropathy. Diabetic retinopathy remains the major cause of new- onset blindness among diabetic adults. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis. Defects in ACE are a cause of susceptibility to intracerebral hemorrhage (ICH). A pathological condition characterized by bleeding into one or both cerebral hemispheres including the basal ganglia and the cerebral cortex. It is often associated with hypertension and craniocerebral trauma. Intracerebral bleeding is a common cause of stroke. Belongs to the peptidase M2 family. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 3.4.15.1; Protease; Membrane protein, integral Chromosomal Location of Human Ortholog: 17q23.3 Cellular Component: extracellular space; lysosome; extracellular region; integral to membrane; plasma membrane; endosome; external side of plasma membrane Molecular Function:tripeptidyl-peptidase activity; peptidyl-dipeptidase activity; protein binding; metallopeptidase activity; carboxypeptidase activity; zinc ion binding; mitogen-activated protein kinase kinase binding; endopeptidase activity; bradykinin receptor binding; exopeptidase activity; mitogen-activated protein kinase binding; drug binding; actin binding; chloride ion binding Biological Process: mononuclear cell proliferation; regulation of vasodilation; angiotensin mediated regulation of renal output; neutrophil mediated immunity; regulation of angiotensin metabolic process; angiotensin maturation; arachidonic acid secretion; proteolysis; regulation of systemic arterial blood pressure by renin-angiotensin; antigen processing and presentation of peptide antigen via MHC class I; regulation of smooth muscle cell migration; cellular protein metabolic process; heart contraction; regulation of blood pressure; peptide catabolic process; beta-amyloid metabolic process; regulation of vasoconstriction; angiotensin catabolic process in blood; spermatogenesis; blood vessel remodeling; hormone catabolic process; kidney development Disease: Microvascular Complications Of Diabetes, Susceptibility To, 3; Renal Tubular Dysgenesis; Alzheimer Disease; Hemorrhage, Intracerebral, Susceptibility To
NCBI Summary:	This gene encodes an enzyme involved in catalyzing the conversion of angiotensin I into a physiologically active peptide angiotensin II. Angiotensin II is a potent vasopressor and aldosterone-stimulating peptide that controls blood pressure and fluid-electrolyte balance. This enzyme plays a key role in the renin-angiotensin system. Many studies have associated the presence or absence of a 287 bp Alu repeat element in this gene with the levels of circulating enzyme or cardiovascular pathophysiologies. Multiple alternatively spliced transcript variants encoding different isoforms have been identified, and two most abundant spliced variants encode the somatic form and the testicular form, respectively, that are equally active. [provided by RefSeq, May 2010]
UniProt Code:	P12821
NCBI GenInfo Identifier:	113045
NCBI Gene ID:	1636
NCBI Accession:	P12821.1
UniProt Secondary Accession:	P12821,P22966, Q53YX9, Q59GY8, Q7M4L4, B0LPF0, B4DXI3 E7EU16,
UniProt Related Accession:	P12821
Molecular Weight:	1306
NCBI Full Name:	Angiotensin-converting enzyme
NCBI Synonym Full Names:	angiotensin I converting enzyme
NCBI Official Symbol:	ACE
NCBI Official Synonym Symbols:	DCP; ICH; ACE1; DCP1; CD143; MVCD3
NCBI Protein Information:	angiotensin-converting enzyme; kininase II; peptidase P; CD143 antigen; testicular ECA; carboxycathepsin; dipeptidyl carboxypeptidase 1; dipeptidyl carboxypeptidase I; angiotensin converting enzyme, somatic isoform; angiotensin I converting enzyme (peptidyl-dipeptidase A) 1; angiotensin I converting enzyme peptidyl-dipeptidase A 1 transcript
UniProt Protein Name:	Angiotensin-converting enzyme
UniProt Synonym Protein Names:	Dipeptidyl carboxypeptidase I; Kininase II; CD_antigen: CD143Angiotensin-converting enzyme, soluble form
Protein Family:	Acetylcholinesterase
UniProt Gene Name:	ACE
UniProt Entry Name:	ACE_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. 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-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37 °C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37 °C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 µL of TMB substrate into each well, cover the plate and incubate at 37 °C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 µL of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

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.