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Human Interleukin 12 (IL-12 / P70)
ELISA Kit
(96 tests)
This immunoassay kit allows for the in vitro quantitative determination of human
IL-12 / P70 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
Interleukin 12 (IL-12) is an interleukin that is naturally produced by dendritic
cells, macrophages and human B-lymphoblastoid cells (NC-37) in response
to antigenic stimulation.
IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric
cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40).
The active heterodimer, and a homodimer of p40 are formed following
protein synthesis. IL-12 is involved in the differentiation of naive T cells into
Th1 cells, which is important in resistance against pathogens. It is known as
a T cell stimulating factor, which can stimulate the growth and function of T
cells. It stimulates the production of interferon-gamma (IFN-γ) and tumor
necrosis factor-alpha (TNF-α) from T and natural killer (NK) cells, and
reduces IL-4 mediated suppression of IFN-γ. T cells which produce IL-12
have a coreceptor, CD30, which is associated with IL-12 activity.
IL-12 plays an important role in the activities of natural killer cells and T
lymphocytes. IL-12 mediates enhancement of the cytotoxic activity of NK
cells and CD8 + cytotoxic T lymphocytes. There also seems to be a link
between IL-2 and the signal transduction of IL-12 in NK cells. IL-2
stimulates the expression of two IL-12 receptors, IL-12R-β1 and IL-12R-β2,
maintaining the expression of a critical protein involved in IL-12 signaling in
NK cells. Enhanced functional response is demonstrated by IFN-γ
production and killing of target cells.
IL-12 also has anti-angiogenic activity, which means it can block the
formation of new blood vessels. It does this by increasing production of
interferon gamma, which in turn increases the production of a chemokine
called inducible protein-10 (IP-10 or CXCL10). IP-10 then mediates this
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anti-angiogenic effect. Because of its ability to induce immune responses
and its anti-angiogenic activity, there has been an interest in testing IL-12
as a possible anti-cancer drug. However, it has not been shown to have
substantial activity in the tumors tested to this date. There is a link that may
be useful in treatment between IL-12 and the diseases psoriasis and
inflammatory bowel disease.
PRINCIPLE OF THE ASSAY
The microtiter plate provided in this kit has been pre-coated with an
antibody specific to IL-12 / P70. Standards or samples are then added to the
appropriate microtiter plate wells with a biotin-conjugated polyclonal
antibody preparation specific for IL-12 / P70 and Avidin conjugated to
Horseradish Peroxidase (HRP) is added to each microplate well and
initiate. Then a TMB (3,35, 5 tetramethyl-benzidine) substrate solution
is added to each well. Only those wells that contain IL-12 / P70,
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 IL-12 / P70 in the samples is then determined by comparing
the OD of the samples to the standard curve.
DETECTION RANGE
4.7 pg / ml-300 pg / ml. The standard curve concentrations used for the
ELISA's were 300 pg / ml, 150 pg / ml, 75 pg / ml, 37.5 pg / ml, 18.8 pg / ml, 9.4
pg / ml, 4.7 pg / ml.
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SPECIFICITY
This assay recognizes recombinant and natural human IL-12 / P70. No
significant cross-reactivity or interference was observed.
SENSITIVITY
The minimum detectable dose of human IL-12 / P70 is typically less than 1.2
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.
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 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.
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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.
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 300 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 (300 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.
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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.
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 100μl 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 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.
4. 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.
5. 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.
6. Repeat the aspiration and wash three times as step 4.
7. 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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8. 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.
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) 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
IL-12 / P70 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.
LIMITATIONS OF THE PROCEDURE
The kit should not be used beyond the expiration date on the kit label.
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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.
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.
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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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Enzyme-linked immunoassay of human interleukin 12 (IL-12 / P70)
Kit instruction manual
This kit is for research use only
Product Numbers: CSB-E04599h
Detection range: 4.7 pg / ml-300 pg / ml
Minimum detection limit: 1.2 pg / ml
Specificity: This kit can detect natural or recombinant human IL-12 / P70 at the same time, and is related to other
There is no cross-reactivity of the protein.
Validity: 6 months
Intended application: ELISA method for quantitative determination of human serum, plasma, cell culture supernatant or other related
The content of IL-12 / P70 in the liquid.
Explanation
1 Storage of the kit: unopened kits should be stored at 2-8 ° C; the microplate after opening should be dried
Store the agent together in an aluminum foil bag and store at 2-8 ° C. Only under this storage condition, the product has
It 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 There may be a little water-like substance in the well of the enzyme-linked plate just opened. This is a normal phenomenon and will not be wrong.
The test results have no effect.
Experimental principle
The microtiter plate is coated with purified antibody to make a solid phase carrier, and the anti-IL-12 / P70 antibody is coated
Add samples or standards, biotinylated anti-IL-12 / P70 antibody, HRP-labeled
Avidin, after thorough washing, is developed with the substrate TMB. TMB is catalyzed by peroxidase
It turns into blue and turns into the final yellow under the action of acid. The shade of the color and the
IL-12 / P70 was positively correlated. Measure the absorbance (OD value) at 450nm with a microplate reader and calculate
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: 1 × 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. Distilled water, volumetric flask, etc.
Collection and preservation of specimens
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 sample should be centrifuged again, then
Detection.
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. each
Dilute the sample bottle to 1ml with the sample diluent before use, cover and let stand for more than 10 minutes, then invert repeatedly
/ Twist to help dissolve, the concentration is 300 pg / ml, after serial dilution, dilute 300 pg / ml,
150 pg / ml, 75 pg / ml, 37.5 pg / ml, 18.8 pg / ml, 9.4 pg / ml, 4.7 pg / ml, sample dilution
The solution is directly used as the standard concentration of 0 pg / ml, prepared within 15 minutes before use, discarded after use, the next test
Use freshly configured standards.
If preparing 150 pg / ml standard: take 0.5ml (not less than 0.5ml) 300 pg / ml of the above standard
The quasi product is added to the Eppendorf tube containing 0.5ml of sample diluent and mixed well.
analogy.
Dilution principle of biotinylated antibody:
Before opening the cap, centrifuge to collect the solution on the bottle wall. Diluted with biotin-labeled antibody dilution before use
Before the dilution, according to the pre-calculated total amount required for each experiment (100μl per well), the actual
When preparing, 0.1-0.2ml should be prepared. For example, 10μl biotin-labeled antibody plus 990μl biotin-labeled antibody
Prepare the ratio of diluent, 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. Labeled affinity with horseradish peroxidase before use
Diluent dilution, 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. Such as 10μl horseradish peroxidase labeled avidin
Add 990μl of horseradish peroxidase-labeled avidin dilution, mix gently, and use
Prepared within the first hour.
Steps
Before starting the experiment, please configure all reagents in advance. When the reagents or samples are diluted, they must be mixed well.
Try to avoid blistering. A standard curve should be made for each test. If the sample concentration is too high, use the sample
The dilution solution is diluted so that the sample conforms to the detection range of the kit. When loading samples, the gun head should be directly
On the quasi-liquid surface, do not apply sample along the hole wall.
1 Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100μl of sample diluent to blank wells,
Add 100μl of the standard product or the sample to be tested to the remaining hole, be careful not to have bubbles, add the sample to the sample
At the bottom of the well of the microtiter plate, try not to touch the wall of the well, gently shake and mix well.
Incubate at 37 ° C for 120 minutes.
To ensure the validity of the experimental results, please use a new standard solution for each experiment.
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2 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.
3 After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 1-2 minutes each time,
200μl / well, spin dry.
4 Add horseradish peroxidase-labeled avidin working solution to each well (same as biotin-labeled antibody working solution)
100 μl, 37 ° C, 60 minutes.
5 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.
6 Add 90μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, the standard is visible to the naked eye
The first 3-4 holes of the quasi product have a clear gradient blue, and the latter 3-4 holes are not obvious, and can be terminated).
7 Add 50μl of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). Stop solution
The order of addition should be the same as the order of addition of substrate solution. In order to ensure the accuracy of the experimental results, the bottom
The stop solution should be added as soon as possible after the reaction time.
8 Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Stop solution
Test within 15 minutes.
Experimental remarks
1. When using the kit for the first time, the user should centrifuge various reagent tubes for a few minutes
Tube bottom.
2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate is added at the end to dissolve
Solution and stop solution. 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.
Cover or cover.
4. Store unused microplates or reagents at 2-8 ° C. Standards, biotinylated antibodies
Working solution, horseradish peroxidase-labeled avidin working solution, please use according to the required amount. please
Do not reuse diluted standards, biotinylated antibody working solution, or horseradish peroxide
Enzyme-labeled avidin working solution.
5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results.
Washing method
Manual plate washing method: aspirate (do not touch the wall) or shake off the liquid in the enzyme plate; on the experimental table
Paved with several layers of absorbent paper, and slamming the microtiter plate down several times; put the recommended wash buffer 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.
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Calculation
Please download the professional software "Curve Exert 1.3" from our website and follow the prompts to make a standard curve.
Taking the concentration of the standard as the abscissa (logarithmic coordinate), the OD value is the ordinate (common coordinate),
Draw a standard curve on the graph paper, and find out 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 of the standard curve
Formula, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply it by the dilution factor, that is
The actual concentration of the sample.
Precautions
1. These operating instructions also apply to 48T kits, enzyme-linked plates, standards, and biologicals in 48T kits
Halogen-labeled antibodies and horseradish peroxidase-labeled avidin are 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 before measuring, please multiply it by the dilution when calculating
multiple.
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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