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Mouse Endothelin 1 （（ET-1））

（（ ））

ELISA Kit

Catalog No. CSB-E05145m

（96T）

This immunoassay kit allows for the in vitro quantitative determination of mouse

ET-1 concentrations in cell culture supernates, serum, plasma and other

biological fluids.

Expiration date six months from the date of manufacture

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES.

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INTRODUCTION

Endothelin-1（ET-1）, a peptide of 21 amino acid residues, is the most potent

vasoconstrictive substance known. Originally isolated from porcine aortic

endothelial cells, ET-1 is now known to be one of a family of three

mammalian vasoactive peptides that also includes Endothelin-2 （ET-2） and

Endothelin-3 （ET-3）. These related peptides differ from ET-1 at two and six

amino acid residue positions, respectively. A fourth peptide, vasoactive

intestinal contractor （VIC）, is sometimes classified as rat ET-2. All

members of the endothelin family contain two essential disulfide bridges

and six conserved amino acid residues at the C-terminus. Additionally, all of

the endothelin family members are synthesized initially as

prepropolypeptides of approximay 200 amino acid residues encoded by

separate genes. These are proteolytically cleaved to produce

biologically-inactive propolypeptides of approximay 40 amino acid

residues termed big endothelins . Big ET-1 is cleaved by the proteolytic

action of a membrane-bound metalloprotease [endothelin-converting

enzyme （ECE-1）] producing the 21 amino acid residue active peptide. The

biochemistry and biology of the endothelins have been the subject of

several reviews.

PRINCIPLE OF THE ASSAY

The microtiter plate provided in this kit has been pre-coated with an

antibody specific to ET-1. Standards or samples are then added to the

appropriate microtiter plate wells with a biotin-conjugated polyclonal

antibody preparation

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specific for ET-1. and Avidin conjugated to Horseradish Peroxidase （HRP）

is added to each microplate well and incubated. Then a TMB （3,3\'5, 5\'

tetramethyl-benzidine） substrate solution is added to each well. Only those

wells that contain ET-1, biotin-conjugated antibody and enzyme-conjugated

Avidin will exhibit a change in color. The enzyme-substrate reaction is

terminated by the addition of a sulphuric acid solution and the color change

is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The

concentration of ET-1. in the samples is then determined by comparing the

O.D. of the samples to the standard curve.

DETECTION RANGE

1.6 pg/ml-100 pg/ml. The standard curve concentrations used for the

ELISA s were 100 pg/ml, 50 pg/ml, 25 pg/ml, 12.5 pg/ml, 6.2 pg/ml, 3.2

pg/ml,1.6pg/ml.

SPECIFICITY

This assay recognizes recombinant and natural mouse ET-1 No

significant cross-reactivity or interference was observed.

SENSITIVITY

The minimum detectable dose of mouse ET-1 is typically less than 0.8

pg/ml.

The sensitivity of this assay, or Lower Limit of Detection （LLD） was defined

as the lowest protein concentration that could be differentiated from zero.

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MATERIALS PROVIDED

Reagent Quantity

Assay plate 1

Standard 2

Sample Diluent 1 x 20 ml

Biotin-antibody Diluent 1 x 10 ml

HRP-avidin Diluent 1 x 10 ml

Biotin-antibody 1 x 120 l

HRP-avidin 1 x 120 l

1 x 20 ml

Wash Buffer

（25 concentrate）

TMB Substrate 1 x 10 ml

Stop Solution 1 x 10 ml

STORAGE

1. Unopened test kits should be stored at 2-8 C upon receipt and the

microtiter plate should be kept in a sealed bag. The test kit may be used

throughout the expiration date of the kit, provided it is stored as

prescribed above. Refer to the package label for the expiration date.

2. Opened test plate should be stored at 2-8 C in the aluminum foil bag

with desiccants to minimize exposure to damp air. The kits will remain

stable until the expiring date shown, provided it is stored as prescribed

above.

3. A microtiter plate reader with a bandwidth of 10 nm or less and an

optical density range of 0-3 OD or greater at 450nm wavelength is

acceptable for use in absorbance measurement.

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REAGENT PREPARATION

Bring all reagents to room temperature before use.

1. Wash Buffer If crystals have formed in the concentrate, warm up to

room temperature and mix gently until the crystals have compley

dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or

distilled water to prepare 500 ml of Wash Buffer.

2. Standard Centrifuge the standard vial at 6000-10000rpm for 30s.

Reconstitute the Standard with 1.0 ml of Sample Diluent. This

reconstitution produces a stock solution of 100 pg/ml. Allow the

standard to sit for a minimum of 15 minutes with gentle agitation prior to

making serial dilutions. The undiluted standard serves as the high

standard （100 pg/ml）. The Sample Diluent serves as the zero standard

（0 pg/ml）. Prepare fresh for each assay. Use within 4 hours and discard

after use.

3. Biotin-antibody Centrifuge the vial before opening. Dilute to the

working concentration using Biotin-antibody Diluent（1:100）,

respectively.

4. HRP-avidin Centrifuge the vial before opening. Dilute to the working

concentration using HRP-avidin Diluent（1:100）, respectively.

Precaution: The Stop Solution provided with this kit is an acid solution. Wear

eye, hand, face, and clothing protection when using this material.

OTHER SUPPLIES REQUIRED

Microplate reader capable of measuring absorbance at 450 nm, with

the correction wavelength set at 540 nm or 570 nm.

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Pipettes and pipette tips.

Deionized or distilled water.

Squirt bottle, manifold dispenser, or automated microplate washer.

An incubator which can provide stable incubation conditions up to

37 C±0.5 C.

SAMPLE COLLECTION AND STORAGE

Serum Use a serum separator tube （SST） and allow samples to clot

for 30 minutes before centrifugation for 15 minutes at 1000 g. Remove

serum and assay immediay or aliquot and store samples at -20 C.

Centrifuge the sample again after thawing before the assay. Avoid

repeated freeze-thaw cycles.

Plasma Collect plasma using citrate, EDTA, or heparin as an

anticoagulant. Centrifuge for 15 minutes at 1000 g within 30 minutes of

collection. Assay immediay or aliquot and store samples at -20 C.

Centrifuge the sample again after thawing before the assay. Avoid

repeated freeze-thaw cycles.

Note: Grossly hemolyzed samples are not suitable for use in this as

ASSAY PROCEDURE

Bring all reagents and samples to room temperature before use. It is

recommended that all samples, standards, and controls be assayed in duplicate.

All the reagents should be added directly to the liquid level in the well. The

pipette should avoid contacting the inner wall of the well.

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1. Add 100 l of Standard, Blank, or Sample per well. Cover with the

adhesive strip. Incubate for 2 hours at 37 C.

2. Remove the liquid of each well, don t wash.

3. Add 100 l of Biotin-antibody working solution to each well. Incubate

for 1 hour at 37 C. Biotin-antibody working solution may appear

cloudy. Warm up to room temperature and mix gently until solution

appears uniform.

4. Aspirate each well and wash, repeating the process three times for a

total of three washes. Wash: Fill each well with Wash Buffer （200 l） and

let it stand for 2 minutes, then remove the liquid by flicking the plate

over a sink. The remaining drops are removed by patting the plate on a

paper towel. Complete removal of liquid at each step is essential to

good performance.

5. Add 100 l of HRP-avidin working solution to each well. Cover the

microtiter plate with a new adhesive strip. Incubate for 1 hour at 37 C.

6. Repeat the aspiration and wash three times as step 4.

7. Add 90 l of TMB Substrate to each well. Incubate for 10-30 minutes at

37 C. Keeping the plate away from drafts and other temperature

fluctuations in the dark.

8. Add 50 l of Stop Solution to each well when the first four wells

containing the highest concentration of standards develop obvious blue

color. If color change does not appear uniform, gently tap the plate to

ensure thorough mixing.

9. Determine the optical density of each well within 30 minutes, using a

microplate reader set to 450 nm.

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CALCULATION OF RESULTS

Using the professional soft \"Curve Exert 1.3\" to make a standard curve is

recommended, which can be downloaded from our web.

Average the duplicate readings for each standard, control, and sample and

subtract the average zero standard optical density. Create a standard curve

by reducing the data using computer software capable of generating a four

parameter logistic （4-PL）curve-fit. As an alternative, construct a standard

curve by plotting the mean absorbance for each standard on the y-axis

against the concentration on the x-axis and draw a best fit curve through the

points on the graph. The data may be linearized by plotting the log of the

ET-1. concentrations versus the log of the O.D. and the best fit line can be

determined by regression analysis. This procedure will produce an

adequate but less precise fit of the data. If samples have been diluted, the

concentration read from the standard curve must be multiplied by the

dilution factor.

LIMITATIONS OF THE PROCEDURE

The kit should not be used beyond the expiration date on the kit label.

Do not mix or substitute reagents with those from other lots or sources.

It is important that the Standard Diluent selected for the standard curve

be consistent with the samples being assayed.

If samples generate values higher than the highest standard, dilute the

samples with the appropriate Standard Diluent and repeat the assay.

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Any variation in Standard Diluent, operator, pipetting technique,

washing technique, incubation time or temperature, and kit age can

cause variation in binding.

This assay is designed to eliminate interference by soluble receptors,

binding proteins, and other factors present in biological samples. Until

all factors have been tested in the Quantikine Immunoassay, the

possibility of interference cannot be excluded.

TECHNICAL HINTS

Centrifuge vials before opening to collect contents.

When mixing or reconstituting protein solutions, always avoid foaming.

To avoid cross-contamination, change pipette tips between additions of

each standard level, between sample additions, and between reagent

additions. Also, use separate reservoirs for each reagent.

When using an automated plate washer, adding a 30 second soak

period following the addition of wash buffer, and/or rotating the plate

180 degrees between wash steps may improve assay precision.

To ensure accurate results, proper adhesion of plate sealers during

incubation steps is necessary.

Substrate Solution should remain colorless or light blue until added to

the plate. Keep Substrate Solution protected from light. Substrate

Solution should change from colorless or light blue to gradations of

blue.

Stop Solution should be added to the plate in the same order as the

Substrate Solution. The color developed in the wells will turn from blue

to yellow upon addition of the Stop Solution. Wells that are green in

color indicate that the Stop Solution has not mixed thoroughly with the

Substrate Solution.

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