（VCAM-1）人血管内皮细胞粘附分子1(VCAM-1)Elisa试剂盒说明书

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Human Vascuolar cell adhesion

molecule 1,VCAM-1

ELISA kit

Catalog No. CSB-E04753h

(96 T)

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

VCAM-1 concentrations in 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

VCAM-1 (vascular cell adhesion molecule-1), also known as

CD106, is the name of both a human gene and its protein

product which have a considerable role in the human immune

system.

The VCAM-1 protein mediates the adhesion of lymphocytes,

monocytes, eosinophils, and basophils to vascular endothelium.

It also functions in leukocyte-endothelial cell signal transduction,

and it may play a role in the development of atherosclerosis and

rheumatoid arthritis.

Upregulation of VCAM-1 in endothelial cells by cytokines occurs

as a result of increased gene transcription (e.g., in response to

Tumor necrosis factor-alpha (TNF-α) and Interleukin-1 (IL-1))

and through stabilization of Messenger RNA (mRNA) (e.g.,

Interleukin-4 (IL-4)). The promoter region of the VCAM-1 gene

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contains functional tandem NF-κB (nuclear factor-kappa B) sites.

The sustained expression of VCAM-1 lasts over 24 hours.

Primarily, the VCAM-1 protein is an endothelial ligand for VLA-4

(Very Late Antigen-4 or α4β1) of the β1 subfamily of integrins,

and for integrin α4β7. VCAM-1 expression has also been

observed in other cell types (e.g., smooth muscle cells). It has

also been shown to interact with EZR and Moesin.

PRINCIPLE OF THE ASSAY

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

an antibody specific to VCAM-1. Standards or samples are then

added to the appropriate microtiter plate wells with a

biotin-conjugated antibody preparation specific for VCAM-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.

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Only those wells that contain VCAM-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 VCAM-1 in the samples is then

determined by comparing the O.D. of the samples to the

standard curve.

DETECTION RANGE

15.6 ng/ml-1000 ng/ml. The standard curve concentrations used

for the ELISA’s were 1000 ng/ml, 500 ng/ml, 250 ng/ml, 125

ng/ml, 62.5 ng/ml, 31.2 ng/ml, 15.6 ng/ml.

SPECIFICITY

This assay recognizes recombinant and natural human VCAM-1.

No significant cross-reactivity or interference was observed.

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SENSITIVITY

The minimum detectable dose of human VCAM-1 is typically less

than 3.9 ng/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.

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 120l

HRP-avidin 1 x 120l

1 x 20 ml

Wash Buffer

(25×concentrate)

TMB Substrate 1 x 10 ml

Stop Solution 1 x 10 ml

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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.

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 completely dissolved. Dilute 20 ml of Wash

Buffer Concentrate into deionized or distilled water to prepare

500 ml of Wash Buffer.

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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 1000

ng/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 (1000 ng/ml).

The Sample Diluent serves as the zero standard (0 ng/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 immediately 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 immediately 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 assay.

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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.

1. Add 100l 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 100l 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 (200l) 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.

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5. Add 100l 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 five times as step 4.

7. Add 90l 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 50l 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.

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)

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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 VCAM-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.

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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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Notes

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