Mouse TNF-α ELISA Kit,specification,price,image-Bio-Equip in China

Product Detail
Company Profile

Product Specification

style="width: 6.63198%; height: 22px;" ">4

Usage

Need to bring your own test equipment
1. Microplate reader (can measure the absorption value of 450nm detection wavelength and 540nm or 570nm correction wavelength)
2. High precision liquid dispenser and disposable suction head
3. Distilled water or deionized water
4. Washing bottle (spray bottle), multi-channel plate washer or automatic plate washer
5. 500mL cylinder

One, preparation before the experiment
1. Sample collection and storage
① Cell culture supernatant: particles should be removed by centrifugation; Test the samples immediately. If the sample is not tested in time after collection, it is recommended that the sample be divided according to the dosage and stored in the refrigerator at -20 ° C to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
② Serum: Samples were collected using a serum separation tube (SST) and samples were left at room temperature for 30 minutes. The samples were centrifuged at 1000g for 15 min. Serum was immediately removed and tested immediately. If the sample is not tested in time after collection, it is recommended to repack according to a single dosage and freeze in ≤ -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
③ Plasma: Plasma was collected using EDTA, heparin, or citric acid as an anticoagulant, centrifuged at 1000g for 15 min within 30 min of collection, and tested immediately. If the samples are not detected in time after collection, it is recommended to separate the samples according to the single dosage and freeze them in &le. -20℃ refrigerator to avoid repeated freezing and thawing. Samples may need to be diluted (1×) Dilute.
2 Reagent preparation (Place all reagents and samples at room temperature for 15 minutes before use. It is recommended that all experimental samples and standards do double hole detection )
1× Preparation of washing solution: concentrated washing solution in the kit is 20× Mother liquor, diluted to 1× with distilled water before use; Working liquid. Example: Take 10mL concentrated washing solution +190mL distilled water to 200mL, the actual operation can be calculated first, then make up.
②1× Dilution with buffer preparation: concentrate dilution in the kit with buffer 10× Mother liquor, diluted to 1× with distilled water before use; Working solution. example: Take 3mL of concentrated dilution with buffer +27mL of distilled water to a constant volume of 30mL. In practical operation, the required dilution buffer can be calculated according to the dilution multiple of the sample, and then the preparation can be made.
③ Detection of antibody: the dry powder was centrifuged to the bottom of the tube, and 110uL dilution buffer (1×) was used. Dissolve and let stand at room temperature for 5 minutes to obtain 100× Mother liquor; Dilute to 1× before use; Working solution. Calculate the desired volume by using 100uL per well. example: 10 Wells were used, then take 10uL of 100 times the working concentration of the test antibody, using dilution buffer (1×) Constant volume to 1mL, get 1mL of 1× The working concentration of the detected antibody.
④SA-HRP: SA-HRP is 40× Mother liquor, use dilution buffer before use (1×) Dilute to make 1× Working solution, 100uL required per well. example: used 10 holes, then take 25uL of 40× Mother liquor +975uL dilution buffer (1×) Constant volume to 1mL to obtain 1× of 1mL; The working concentration of the detected antibody.
⑤ Chromogenic agent: according to 100uL per well, calculate the amount needed for this test, take out the corresponding volume of chromogenic agent, avoid light; The removed chromogenic agent is only used on the same day.
⑥ Standards: lyophilized standards with dilution buffer (1×) Redissolve, redissolve volume 1000uL, to obtain a concentration of 2000pg/mL standard mother liquor. Gently shake for at least 5 minutes, it is fully dissolved. Add 300uL dilution buffer (1×) to each dilution tube. . The standard mother liquor is diluted according to the picture below, and each tube must be fully mixed before pipetting to the next tube. The standard mother liquor without dilution can be used as the highest point of the standard curve (2000pg/mL), and the dilution buffer (1×) Can be used as the zero point of the standard curve (0pg/mL).

2, operation steps
1. Prepare all required reagents and standards;
2 Remove the microplate from the sealed bag that has been balanced to room temperature, and put the unused slat back into the aluminum foil bag and re-seal it;
3. Add 300uL washing solution to the microplate, let it soak for 30 seconds, discard the washing solution and pat the microplate dry on absorbent paper, please use immediately do not let the microplate dry;
4. Add different concentrations of standard, experimental samples or quality control into the corresponding Wells, 100uL for each well. The Wells were sealed with plate adhesive and incubated at room temperature for 2 hours.
5 Suck the liquid out of the plate and wash the plate using a bottle washer, a multi-channel plate washer, or an automatic plate washer. Add 300uL of washing liquid to each well, and then suck the washing liquid out of the plate. Repeat 3 times. Every time you wash the plate, try to absorb the residual liquid to help you get a good test result. At the end of the last plate wash, please blot all the liquid in the plate or invert the plate and pat all the residual liquid in the absorbent paper;
6. Add 100uL detection antibody to each microwell. Seal the reaction Wells with sealer tape and incubate for 2 hours at room temperature;
7. Repeat the plate washing operation of step 5;
8. Add 100 ULSA-HRP to each microwell and incubate for 20 minutes at room temperature. Be careful to avoid light;
9. Repeat step 5 to wash the plate;
10. Add 100uL of color development solution to each microwell, incubate at room temperature for 5-30 minutes, pay attention to avoid light;
11. Add 50uL of termination solution to each microwell, and the color of the solution in the well will change from blue to yellow. If the color of the solution turns green or the color change is inconsistent, tap the microplate to mix the solution evenly;
12. Within 30 minutes after the termination solution is added, the absorbance value at 450nm is measured using a microplate reader and 540nm or 570nm is set as the correction wavelength. If the dual wavelength correction is not used, the accuracy of the results may be affected;
13 Calculation results: The corrected absorbance values (OD450-OD540/OD570), the compound reading were averaged for each standard and sample, and then the average zero standard OD value was subtracted. Standard curves were created by 4-parameter logic (4-PL) curve fitting using computer software. Alternatively, a curve can be generated by plotting the logarithm of the concentration of the standard against the logarithm of the corresponding OD value, and the best fit line can be determined by regression analysis. This process produces an adequate but less accurate fit to the data. If the sample is diluted, the concentration should be multiplied by the dilution.
Note: The standard curve data provided in are for reference only, and the sample content should be calculated according to the standard curve drawn in the same test.

three, kit parameter
1. Recovery: Different levels of mouse TNF-&alpha were incorporated into the cell culture medium samples; And the recovery rate was determined. The recovery ranges from 88 to 107%, with an average recovery of 94%.
2. Sensitivity: mouse TNF-α The minimum detectable dose (MDD) of murine TNF - & alpha was generally less than 5.5pg/mL. The lowest detectable value is the corresponding concentration calculated from the mean of the zero absorbance values of 20 standard curves plus two standard deviations.
3. Correction: the ELISA kit by e. coli expression of TNF - high purity recombinant mice & alpha; Protein corrected.
4. Linearity: four different samples were mixed with high concentrations of mouse TNF-α And then use thinner (1 & times) Measure linearity by dilating the sample to within the detection range.
range (%)
1:2 94-103
1:4
99-111
1:8 97-114
1:16 104-111

5. Specificity: The ELISA can detect native and recombinant mouse TNF-&alpha. Protein. Combine the following factors with diluent (1×) Formulated to a concentration of 50ng/mL to detect the difference with mouse TNF-α The cross-reaction with the mouse TNF - & alpha. 50ng/mL of interfering factor was incorporated into the intermediate range of recombinant mouse TNF-α Reference substance, to detect TNF - in mice & alpha; Interference of TNF - & alpha. No significant cross-reaction or interference was observed.
human recombinant protein style="width: 48.1332%; height: 22px;" >TNF-α style="width: 48.1332%; height: 22px;" ">if the temperature is too low, extend the incubation time and drink color development time
Lymphotoxin α 1/β 2 TNF RII
Lymphotoxin α 2/β 1

TNF RI
4, common problem resolution
1. border-collapse: collapse; border-collapse: collapse; border-collapse: collapse; border-collapse: collapse; border-collapse: collapse; width: 98.562%; height: 164px; margin: 0 auto; page-break-inside: avoid;" border="1">
1 Improperly stored kit; Buy a new kit and pay attention to storage conditions;
2
3
4 The container used to configure the solution is not clean, or there is a problem with the water use clean containers and qualified distilled water
5
6 All reagents should be equilibrated at room temperature for 30 minutes
7 detect antibody and/or HRP concentration too low Refer to the instructions, do not arbitrarily dilute

2.
style="border-collapse: collapse; width: 97.9085%; height: 164px; margin: 0 auto; page-break-inside: avoid;" border="1">

The substrate 3,3',5,5' -tetramethylbenzidine (TMB) was contaminated or exposed to metal ions or oxidants.

Use clean containers and qualified distilled water for preparation;

High incubation temperature and/or too long

Control the temperature and time of incubation and final enzymatic reaction

sample hemolysis, storage for a long time, incomplete agglutination, bacterial contamination, effect of adding in the collection vein

avoid hemolysis, pollution, the phenomenon such as too long storage
five, the flow chart of experimental

Theory

Double antibody sandwich enzyme-linked immunosorbent assay was used in this kit. Specific anti mouse antibody of TNF alpha pre package is on the high affinity enzyme label plate. The standard substance, test sample and biotinylated detection antibody were added into the microplate Wells. After incubation, the TNF-α present in the sample combined with the solid-phase antibody and detection antibody to form immune complexes. After washing to remove the unbound material, the horseradish peroxidase-labeled Streptavidin-HRP was added. After washing, the chromogenic substrate was added and the color was developed in the dark. Join terminated liquid termination reaction, in the 450 - nm wavelength (reference calibration wavelength of 540 nm or 570 nm) determination of absorbance values.

Description

detection principle:

this kit uses double antibody sandwich enzyme-linked immunosorbent assay technology. Specific mouse tnf-& Alphathe antibody was pre coated on a high affinity enzyme plate. Add the standard, sample to be tested and biotinylated detection antibody into the wells of the enzyme plate, and after incubation, the tnf-& existing in the sample; Alphacombine with solid-phase antibody and detection antibody to form immune complexes. After washing to remove unbound material, horseradish peroxidase labeled streptavidin (streptavidin HRP) was added. After washing, the chromogenic substrate was added to avoid light for color development. Stop the reaction by adding stop solution, and determine the absorbance value at 450 nmwavelength (reference correction wavelength 540nmor 570nm)

detection type: double antibody sandwich method

form: pre coated 96orifice plate

detection sample type: cell supernatant, serum, Plasma

loading amount: 100ul

kit components:

pre coated 96well plates, standards, anti mouse tnf-& Alphaa copy of antibody detection, dilution buffer, chromogenic solution (a, b), washing solution, stop solution, sa-hrp, plate sealing membrane and instructions

sensitivity: 5.5 pg/ml

detection range: 31.2 - 2000 pg/ml

recovery range: 88-107%

storage method: 2-8℃

standard songLine diagram

background:

tumor necrosis factor alpha(tnf-& alpha; ), also known as cachectinand tnfsf1a, are prototypical ligands of the tumor necrosis factor superfamily. It is a pleiotropic factor that plays a central role in inflammatory response, immune system development, apoptosis and lipid metabolismtnf-& Alphait is also involved in many pathological processes, including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, iitype diabetes mellitus, septic shock, autoimmunity and cancer. Human tnf-& Alphais a 26 kdatype iitransmembrane protein, consisting of an 35amino acid (aa) intracellular domain, 21 aatransmembrane segment, and an 177 aaextracellular domain (ecd). In the ecdzone, human tnf-& Alphaand macaque have 97% amino acid sequence homology, 71-92% amino acid sequence homology with cattle, dogs, cotton rats, horses, cats, mice, pigs, rats. It can be expressed by a variety of different cells, such as immune cells, epithelial cells, endothelial cells, tumor cellstnf-& Alphacan induce the lysis of tumor cells and virus-infected cells, and combine with soluble tnf rito produce transmembrane turn signals of downstream cellstace/adam17can cause TNF - & amp; Alphacytokine, which is composed of TNF - & amp; Alphatrimer composed of extracellular soluble structure, with a molecular weight of 55 kda

tnf-& Alphathere are two receptors: tnf riand tnf riiTNF rihas a molecular weight of 55-60 kdaand is widely expressedthe molecular weight of TNF riiis 78-80 kda, which is only expressed by hematopoietic cells. Both are expressed as homotrimerstnf-& Alphaand tnf riand tnf riibind with similar affinity, which can promote nf& Kappa; Activation of b. However, only tnf rihas a cell death domain that can trigger apoptosis. These two soluble receptors are released into human serum and urine, and can neutralize tnf-& Alphaactivity

Composition

compose After dissolving, calculate the amount to repack, which can be at -20° Mouse TNF-α Concentrated volume after dissolving, can be in 2-8° 40× 40× Concentrations can be in 2-8° C store; 1× 1 bottle 1 bottle


Mouse TNF-α		The unused lath can be stored at 2-8 ° C for 30 days after placing it back in an aluminum foil bag with desiccant and sealing it.
Mouse TNF-α Standard

10×	1	After opening, can be in 2-8°


20×	1 bottle
	Storage at room temperature, to avoid contamination, cannot be reused

Background

Tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF1A, is the prototypical ligand of the tumor necrosis factor superfamily. It is a pleiotropic factor that plays a central role in inflammatory responses, immune system development, apoptosis, and lipid metabolism. TNF-α is also involved in many pathological processes, including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, type II diabetes, septic shock, autoimmunity, and cancer. Human TNF-α is a 26-kda type II transmembrane protein consisting of a 35-amino acid (aa) intracellular domain, a 21-aa transmembrane segment, and a 177aa extracellular domain (ECD). "In the ECD region, human TNF-α shares 97% amino acid sequence identity with macaque and 71-92% amino acid sequence identity with bovine, dog, cotton rat, horse, cat, mouse, pig, and rat." It can be expressed and produced by a variety of different cells such as immune cells, epithelial cells, endothelial cells, and tumor cells. TNF-α induces lysis of tumor cells and virus-infected cells and binds to soluble TNFRI to generate transmembrane trafficking signals from downstream cells. TACE/ADAM17 can cause the membrane shedding of TNF-α containing cells and release of active TNF-α cytokine, which is a trimer composed of extracellular soluble structure of TNF-α with a molecular weight of 55kDa.
TNF-α has two receptors: TNFRI and TNFRII. TNFRI has a molecular weight of 55-60 kda and is widely expressed. TNFRII has a molecular mass of 78 to 80kDa and is restricted to hematopoietic cells. Both were expressed as homotrimers. TNF-α binds to TNFRI and TNFRII with similar affinity and promotes NFκB activation. However, only TNFRI with cell death domain structure, and can cause cell apoptosis. Both soluble receptors are released into human serum and urine and can neutralize TNF-α activity.

General Notes

1. Please use the kit within the validity period.
2. Components of different kits and different batch kits should not be mixed.
3. If the sample value is greater than the highest value of the standard curve, the sample should be diluted with (1×) The samples were diluted and retested. "If the cell culture supernatant sample needs to be diluted in a distributed manner, cell culture medium may be used for intermediate dilutions, except for the last step when diluent is used."
4. The difference of the test results can be caused by a variety of factors, including the operation of the experimenter, the use of the pipettor, the washing technique, the reaction time or temperature, the storage of the kit, etc.
5. The termination solution in the kit is acidic. Please protect your glasses, hands, face and clothes when using it.
6. For scientific research only, not for in vitro diagnosis.

Storage Temp.

Unopened kit, 2-8° C Storage

Test Range

31.3pg/mL-2000pg/mL