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Human P-selectin ELISA Kit
(96 T)
? This immunoassay kit allows for the in vitro quantitative determination of human
P-selectin 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
P-selectin is a cell adhesion molecule (CAM) on the surfaces of activated
endothelial cells, which line the inner surface of blood vessels, and
activated platelets. In unactivated endothelial cells, it is stored in granules
Weibel-Palade bodies, and α-granules in unactivated platelets. P-selectin
plays an essential role in the initial recruitment of leukocytes (white blood
cells) to the site of injury during inflammation.
Thrombin is one trigger which can stimulate endothelial-cell release of
P-selectin and recent studies suggest an additional Ca2 + -independent
pathway involved in release of P-selectin. Ligands for P-selectin on
eosinophils and neutrophils are similar sialylated, protease-sensitive,
endo-beta-galactosidase-resistant structures, clearly different than those
reported for E-selectin, and suggest disparate roles for P-selectin and
E-selectin during recruitment during inflammatory responses. P-selectin
attaches to the actin cytoskeleton through anchor proteins that are still
poorly characterized.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to P-selectin. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for P-selectin and Avidin conjugated to
Horseradish Peroxidase (HRP) is added to each microplate well and
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initiate. Then a TMB (3,3,5,5 tetramethyl-benzidine) substrate solution
is added to each well. Only those wells that contain P-selectin,
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 P-selectin in the samples is then determined by comparing
the OD of the samples to the standard curve.
DETECTION RANGE
0.94 ng / ml-60 ng / ml. The standard curve concentrations used for the
ELISA's were 60 ng / ml, 30 ng / ml, 15 ng / ml, 7.5 ng / ml, 3.75 ng / ml, 1.88
ng / ml, 0.94 ng / ml.
SPECIFICITY
This assay recognizes recombinant and natural human P-selectin. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human P-selectin is typically less than
0.24 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.
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MATERIALS PROVIDED
Reagent Quantity
Assay plate 1
Standard 2
Sample Diluent 2 x 20 ml
Biotin-antibody Diluent 1 x 10 ml
HRP-avidin Diluent 1 x 10 ml
Biotin-antibody 1 x 120μl
HRP-avidin 1 x 120μl
Wash Buffer
1 x 20 ml
(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 completely
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 60 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 (60
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.
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.
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. Serum and plasma samples require a 20-fold dilution into Sample
Diluent. The suggested 20-fold dilution can be achieved by adding 15μl
sample to 285μl of Sample Diluent. The recommended dilution factor is
for reference only. The optimal dilution factor should be determined by
users according to their particular experiments.
2. Add 100μl of Standard, Blank, or Sample per well. Cover with the
adhesive strip. Incubate for 2 hours at 37 ° C.
3. Remove the liquid of each well, don't wash.
4. Add 100μl 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.
5. Aspirate each well and wash, repeating the process three times for a
total of three washes. Wash: Fill each well with Wash Buffer (200μl) 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.
6. Add 100μl of HRP-avidin working solution to each well. Cover the
microtiter plate with a new adhesive strip. Incubate for 1 hour at 37 ° C.
7. Repeat the aspiration and wash three times as step 4.
8. Add 90μl 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.
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9. Add 50μl 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.
10. 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) curve-fit. As an alternative, construct a standard
curve by plotting the mean absorbance for each standard on the x-axis
against the concentration on the y-axis and draw a best fit curve through the
points on the graph. The data may be linearized by plotting the log of the
P-selectin concentrations versus the log of the OD 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.
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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.
? 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 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.
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? 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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Human P-Selectin (P-Selectin) ELISA kit instruction manual This kit is for research use only Detection range: 0.94 ng / ml – 60 ng / ml
Minimum detection limit: 0.24 ng / ml
Specificity: This kit can detect natural or recombinant human P-selectin at the same time, and does not cross-react with other related proteins.
Validity: 6 months Expected application: ELISA method for quantitative determination of human serum, plasma or other related biological fluids
P-selectin content.
Explanation
1 Storage of the kit: unopened kits should be stored at 2-8 ° C; the opened microplate should be stored in an aluminum foil bag together with desiccant at 2-8 ° C. Only under this storage condition, the product can be used normally within the validity period.
2 When the concentrated washing liquid is stored at low temperature, salt will precipitate out. When diluted, it can be heated and dissolved in a water bath.
3 There may be inconsistencies between the Chinese and English manuals, please refer to the English manual.
4 The well of the ELISA plate just opened may contain a little water-like substance. This is normal and will not have any impact on the experimental results.
Experimental principle The microtiter plate is coated with purified antibody to make a solid phase carrier, and the specimen or standard, biotinylated anti-P-selectin antibody, HRP-labeled antibody are added to the microwell coated with anti-P-selectin antibody Avidin, after thorough washing, is developed with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The shade of the color and the
P-selectin was positively correlated. Measure the absorbance (OD value) at 450nm with a microplate reader to calculate the sample concentration.
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Kit composition and reagent preparation
1. Assay plate: one piece (96 wells).
2. Standard product (Standard): 2 bottles (lyophilized product).
3. Sample Diluent: 2 × 20ml / bottle.
4. Biotin-antibody Diluent: 1 × 10ml / bottle.
5. Horseradish peroxidase labeled avidin diluent (HRP-avidin Diluent) 1 × 10ml / bottle.
6. Biotin-antibody: 1 × 120μl / bottle (1: 100).
7. Horseradish peroxidase labeled avidin (HRP-avidin): 1 × 120μl / vial (1: 100).
8. Substrate solution (TMB Substrate): 1 × 10ml / bottle.
9. Wash Buffer 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water.
10. Stop Solution: 1 × 10ml / bottle.
Reagents and equipment needed but not provided
1. Standard Specification Microplate Reader
2. High-speed centrifuge
3. Electric heating thermostat incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips. When testing more samples at one time, it is best to use multi-channel pipettes
6. Collection and preservation of specimens such as distilled water and volumetric flasks
1. Serum: Whole blood samples should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000g for 20 minutes.
Take the supernatant for immediate detection; or aliquot and store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing. The thawed samples should be centrifuged again and then tested.
2. Plasma: EDTA or heparin can be used as anticoagulant, within 2-8 ° C within 30 minutes after specimen collection
Centrifuge at 1000 g for 15 minutes, take the supernatant for immediate detection; or aliquot and place the specimen in
Store at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing. The thawed samples should be centrifuged again and then tested.
Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested.
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Standard dilution principle: 2 bottles, centrifuge at 6000-10000rpm for 30 seconds before use. Before use, dilute each bottle with sample diluent to 1ml, cover and let stand for more than 10 minutes, then repeatedly invert
/ Twist to help dissolve, its concentration is 60 ng / ml, after serial dilution, diluted 60 ng / ml,
30 ng / ml, 15 ng / ml, 7.5 ng / ml, 3.75 ng / ml, 1.88 ng / ml, 0.94 ng / ml, the sample dilution is directly used as the standard concentration 0 ng / ml, prepared within 15 minutes before use Discard after use, and use freshly configured standards for the next test.
For example, to prepare a 30 ng / ml standard: take 0.5 ml (not less than 0.5 ml) of the above standard at 60 ng / ml and add it to an Eppendorf tube containing 0.5 ml of sample dilution.
Dilution principle of biotinylated antibody:
Before opening the cap, centrifuge to collect the solution on the bottle wall. Before use, dilute with biotin-labeled antibody diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). In actual preparation, 0.1-0.2ml should be prepared. For example, prepare 10μl biotin-labeled antibody plus 990μl biotin-labeled antibody dilution, mix gently, and prepare within one hour before use.
Dilution principle of horseradish peroxidase-labeled avidin:
Before opening the cap, centrifuge to collect the solution on the bottle wall. Dilute with horseradish peroxidase-labeled avidin diluent before use, and prepare according to the pre-calculated total amount required for each experiment before dilution (per well)
100μl), more 0.1-0.2ml should be prepared in actual preparation. For example, 10 μl horseradish peroxidase-labeled avidin plus 990 μl horseradish peroxidase-labeled avidin dilution is prepared, mix gently, and prepare within one hour before use.
Before starting the experiment, please configure all reagents in advance. When diluting the reagents or samples, they should be mixed evenly. Try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit. When adding a sample, the pipette tip should be directed at the liquid surface, and do not apply sample along the hole wall.
1. Serum and plasma samples are diluted 1:20 times with the sample diluent for detection. The specific operation is as follows: take 15μl of the sample and add it to 285μl of the sample diluent (1:20 dilution) and mix it. The result is 1: 20-fold diluted sample. This recommended dilution factor is for reference only, and users should determine their optimal dilution factor based on experiments.
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2. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100μl of sample diluent to blank wells,
Add 100 μl of the standard or the sample to be tested to the remaining wells. Be careful not to have air bubbles. Add the sample to the bottom of the well of the microtiter plate. Try not to touch the wall of the well. Shake gently to mix. ,
Incubate at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
3. Discard the liquid and spin dry without washing. Add 100μl of biotinylated antibody working solution to each well (take 1μl
Prepare a ratio of biotin-labeled antibody plus 99μl of biotin-labeled antibody dilution, mix gently,
Prepared within one hour before use), 37 ℃, 60 minutes.
4. After incubating for 60 minutes, discard the liquid in the hole, spin dry, wash the plate 3 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
5. Add horseradish peroxidase-labeled avidin working solution to each well (same as biotin-labeled antibody working solution)
100 μl, 37 ° C, 60 minutes.
6. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
7. Add 90μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (20-30 minutes, at this time, the first 3-4 wells of the standard product have a clear blue gradient, and the last 3-4 wells show If the color is not obvious, you can terminate).
8. Add 50μl of stop solution to each well in sequence to stop the reaction (the blue color turns to yellow at this time). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires.
9. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution.
Experimental remarks
1. When using the reagent kit for the first time, the user should centrifuge various reagent tubes for several minutes so that the reagents are concentrated to the bottom of the tube.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and the stop solution are added last. Use this hole to adjust the OD value to zero first during measurement.
3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate.
4. Store unused microplates or reagents at 2-8 ° C. Standards, biotin-labeled antibody working solution, horseradish peroxidase-labeled avidin working solution, please use according to the required amount. Do not reuse diluted standard, biotin-labeled antibody working solution, or horseradish peroxidase-labeled avidin working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
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Plate washing method Manual plate washing method: suck (do not touch the wall) or shake off the liquid in the microplate; place a few layers of absorbent paper on the experimental table, and force the microplate down several times; pat the recommended wash buffer Liquid at least 0.3ml
Fill the hole and soak for 1-2 minutes. Repeat this process several times as needed.
Automatic plate washing: If there is an automatic plate washing machine, it should be used in the formal experiment process after being used skillfully.
For calculation, please download the professional software "Curve Exert 1.3" from our website and make a standard curve according to the prompts.
Taking the concentration of the standard as the ordinate (logarithmic coordinate) and the OD value as the abscissa (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding concentration from the standard curve according to the OD value of the sample;
Multiply by the dilution factor; or use the standard concentration and OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor to obtain the actual concentration of the sample.
Precautions
1. These operating instructions also apply to the 48T kit. The 48T kit contains enzyme-linked plates, standards, biotin-labeled antibodies, and horseradish peroxidase-labeled avidin halved.
2. When mixing the protein solution, it should be as gentle as possible to avoid foaming.
3. The washing process is very important. Insufficient washing can easily cause false positives.
4. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples.
5. Please make a standard curve at the same time every measurement.
6. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor.
7. When preparing standard products and testing solution working fluids, please prepare with corresponding diluent, not to be confused.
8. Please keep the substrate away from light.
9. Do not replace the reagents in the kit with reagents from other manufacturers.
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