Mouse TNF-α ELISA Kit

Catalog Number: abs520010 Application: ELISA Reactivity: Mouse Conjugation:

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$450 USD

Size: 96T

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Product Details

Product Specification

protein

TNF-α

Usage

Need to bring your own test equipment
1. Microplate reader (measurable 450nm Absorption value of detection wavelength and 540nm Or 570nm Absorption value of corrected wavelength)
2. High precision liquid dispenser and disposable tip
3. Distilled or deionized water
4. Bottle washer (spray bottle), multi-channel plate washer or automatic plate washer
5. 500mL Measuring cylinder

1. Preparation before the experiment
1. Sample collection and storage
① Cell culture supernatant: particulate matter should be removed by centrifugation; Test the sample immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the amount used once and store it frozen in -20℃ In the refrigerator, avoid repeated freezing and thawing. The sample may need to be used with a diluent ( 1× ) dilution.
② Serum: Use serum separation tubes ( SST ) Collect samples and place samples at room temperature 30 Minutes. Centrifugation 15 Minutes, with a rotation speed of 1000g 。 The serum was removed immediately and tested immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the amount used once and store it frozen in ≤-20℃ In the refrigerator, avoid repeated freezing and thawing. The sample may need to be used with a diluent ( 1× ) dilution.
③ Plasma: Use EDTA , heparin or citric acid as an anticoagulant to collect plasma, after collection 30 Centrifuge within minutes 15 Minutes, with a rotation speed of 1000g , and detect it immediately. If the sample is not tested in time after collection, it is recommended to pack it according to the amount used once and store it frozen in ≤-20℃ In the refrigerator, avoid repeated freezing and thawing. The sample may need to be used with a diluent ( 1× ) dilution.
2. Reagent Preparation ( Please place all reagents and samples at room temperature before use and let them stand 15 Minutes. All experimental samples and standards are recommended Do repeat hole detection ）
①1× Preparation of washing solution: The concentrated washing solution in the kit is 20× Mother liquor should be diluted with distilled water before use to 1× Working fluid. Example: Take 10mL Concentrated wash +190mL Distilled water to volume to 200mL In actual operation, the usage amount can be calculated first, and then prepared.
②1× Preparation of buffer for dilution: The concentration and dilution buffer in the kit is 10× Mother liquor, dilute with distilled water before use to 1× Working fluid. Example: Take 3mL Buffer for concentration and dilution +27mL Distilled water to volume to 30mL 。 In actual operation, the required amount of dilution buffer solution can be calculated according to the sample dilution factor, and then prepared.
③ Antibody detection: Centrifuge the dry powder to the bottom of the tube and use 110uL Buffer for dilution ( 1× ) Dissolve and let stand at room temperature 5 Get after minutes 100× Mother liquor; Before use, dilute to 1× Working fluid. According to the amount per well 100uL Calculate the required volume. Example: Used 10 Hole, then take 10uL Of 100 Double working concentration of detection antibody, using dilution buffer ( 1× ) Constant volume to 1mL , obtained 1mL Of 1× Detection antibody at working concentration.
④SA-HRP ： SA-HRP For 40× Mother liquor, use dilution buffer before use ( 1× ) Diluted and formulated 1× Working fluid, the required amount per hole is 100uL 。 Example: Used 10 Hole, then take 25uL Of 40× Mother liquor +975uL Buffer for dilution ( 1× ) Constant volume to 1mL , obtained 1mL Of 1× Detection antibody at working concentration.
⑤ Developer: per well 100uL Calculate the dosage required for the current test, take out the corresponding volume of color developer, and protect it from light; The developer removed is for the same day use only.
⑥ Standard: Dilution buffer for lyophilized standard ( 1× ) Re-dissolved, redissolved volume 1000uL , obtaining a concentration of 2000pg/mL Standard mother liquor. Gently shake at least 5 Minutes, it is fully dissolved. Add to each dilution tube 300uL Buffer for dilution ( 1× ）。 Make serial dilutions of the standard mother liquor according to the figure below, and each tube must be thoroughly mixed before pipetting to the next tube. Standard mother liquor without dilution can be used as the highest point of the standard curve ( 2000pg/mL ), buffer for dilution ( 1× ) can be used as a standard curve zero ( 0pg/mL ）。

2. Operation steps
1. Prepare all required reagents and standards;
2. Take out the microplate from the sealed bag that has been equilibrated to room temperature. Please put the unused slats back into the aluminum foil bag and re-seal;
3. Add to the microplate 300uL Washing liquid, let stand and soak 30 Seconds, discard the lotion and pat the microplate dry on absorbent paper. Please use it immediately and do not let the microplate dry;
4. Add different concentration standards, experimental samples or quality control articles into the corresponding wells, and each well 100uL 。 Seal the reaction wells with plate sealing tape and incubate at room temperature 2 hour ;
5. The liquid in the plate is sucked off and the plate is washed using a washing bottle, a multi-channel plate washer or an automatic plate washer. Washing solution per well 300uL Then the intra-plate wash liquid is aspirated. Repeat Operation 3 Times. Trying to absorb the residual liquid as much as possible every time you wash the plate will help to get good experimental results. At the end of the last plate washing, please suck all the liquid in the plate or turn the plate upside down, and pat all the residual liquid dry on absorbent paper;
6. Within each well added 100uL Detect antibodies. Seal the reaction wells with plate sealing tape and incubate at room temperature 2 Hour;
7. Repeat th 5 Step washing operation;
8. Within each well added 100uLSA-HRP , room temperature incubation 20 Minutes. Be careful to avoid light;
9. Repeat th 5 Step washing operation;
10. Within each well added 100uL Chromogenic solution, incubate at room temperature 5-30 Minutes, pay attention to avoiding light;
11. Within each well added 50uL Stop solution, the color of the solution in the well will change from blue to yellow. If the color of the solution changes to green or the color changes are inconsistent, pat the microplate gently to mix the solution evenly;
12. After addition of stop solution 30 Within minutes, measured using a plate reader 450nm Absorbance value, set 540nm Or 570nm As a correction wavelength. If dual-wavelength correction is not used, the accuracy of the results may be affected;
13. Calculation results: Add the corrected absorbance values of each standard and sample (OD450-OD540 Or OD570) , average of repeated well readings and then subtract the average zero standard OD Value. Using computer software for four-parameter logic (4-PL) Curve fitting creates a standard curve. Another way is to plot the standard concentration and make the logarithm with the corresponding OD The values were logarithmic to generate a curve, and the best fit line was determined by regression analysis. This process can generate a data fit that is sufficiently useful but less accurate. If the sample is diluted, the concentration should be calculated by multiplying the dilution factor.

Note: The standard curve data provided is for reference only, and the sample content should be calculated according to the standard curve drawn in the same test.

3. Kit parameters
1. Recovery: Different levels of mice were spiked in cell culture medium samples TNF-α The recovery rate was determined. The recoveries range from 88-107% , the average recovery was in 94% 。
2. Sensitivity: Mouse TNF-α The lowest measurable dose ( MDD ) is generally less than 5.5pg/mL 。 The lowest measurable value is determined according to 20 The corresponding concentration is calculated by adding two standard deviations to the mean value of the zero-point absorbance values of each standard curve.
3. Correction: This ELISA High purity recombinant mice expressed by Escherichia coli TNF-α Corrected by protein.
4. Linearity: 4 High concentrations of mice were spiked into different samples TNF-α , followed by a diluent ( 1× ) Dilute the sample to the detection range and determine its linearity.

Dilution factor	Average / Expected value ( % ）	Range ( % ）
1:2	97	94-103
1:4	105	99-111
1:8	106	97-114
1:16	107	104-111

5. Specificity: This ELISA Method can detect natural and recombinant mice TNF-α Egg whites. The following factors were mixed with diluent ( 1× ) formulated into 50ng/mL Concentration to detect with mice TNF-α Cross-reactivity of. Will 50ng/mL Interfering factors incorporated into the intermediate range of recombinant mice TNF-α In the control article, to detect the effect on mice TNF-α Of interference. No significant cross-reactivity or interference was observed.

Recombinant human protein	Recombinant mouse protein
Lymphotoxin α1/β2	TNF RII
Lymphotoxin α2/β1
TNF-α
TNF RI
TNF RI

4. Analysis of frequently asked questions
1. Whiteboard (no color appears after color rendering is complete)

Serial number	Possible cause	Solutions
1	Improper storage of kits; Mixing reagents of different kits	Purchase new kits and pay attention to storage conditions; Not mixable
2	Low incubation temperature and short time	If the temperature is low, extend the incubation time and color development time
3	Wrong addition or missed addition of reagents	Add the correct reagent strictly according to the instructions
4	The container used to prepare the solution is not clean or there is a problem with the water	Use clean containers and qualified distilled water
5	In the process of plate washing, the soaking time is long, the times of plate washing are too many, and the impact force of plate washing is large	Follow the instructions strictly
6	Heterogeneity of reagent temperature	All reagents should be equilibrated at room temperature 30 minute
7	Detecting antibodies and / Or HRP Concentration too low	Refer to the instructions, do not dilute at will

2. Flower plate (blank, negative positive control normal, but specimen well OD Values are significantly higher)

Serial number	Possible cause	Solutions
1	Less washing times, insufficient	Wash as per instructions
2	Substrate 3,3',5,5'- Tetramethylbenzidine ( TMB ) contaminated or exposed by metal ions or oxidants	Use clean containers and qualified distilled water during preparation; Store protected from light
3	Incubation temperature is high and / Or too long	Controlling the temperature and time of incubation and final enzymatic reaction
4	The gun tip was not changed during sample loading, resulting in cross-contamination	Change the gun head for each sample
5	Cross contamination of nearby holes	Vertical clapper, use suitable clapper paper to avoid paper scraps in the hole
6	Presence of endogenous interfering substances in sample	Estimate possible infectious substances and perform corresponding treatment
7	Samples were hemolyzed, stored for too long, incomplete agglutination, contaminated by bacteria, influence of additives in blood collection tube	Avoid hemolysis, contamination, long storage and other phenomena

5. Experimental flow chart

Species Reactivity

Mouse

Theory

This kit adopts double antibody sandwich enzyme-linked immunosorbent detection technology. Specific anti-mouse TNF-α antibodies were pre-coated on high affinity enzyme labeled plates. The standard substance, the sample to be tested and the biotinylated detection antibody are added to the well of the enzyme label plate, and after incubation, TNF-α present in the sample binds to the solid phase antibody and the detection antibody to form an immune complex. After washing to remove unbound material, horseradish peroxidase-labeled Streptavidin-HRP was added. After washing, a chromogenic substrate is added to protect the color from light. A stop solution was added to stop the reaction, and the absorbance value was measured at a wavelength of 450 nm (reference calibration wavelength of 540 nm or 570 nm).

Source

Mouse

Synonym

APC1 protein, Cachectin, Cachetin, DIF, TNF, TNF, monocyte-derived, TNFA, TNF-A, TNFalpha, TNF-alpha, TNF-alphacachectin, TNFATNF, macrophage-derived, TNFG1F, TNFSF1A, TNFSF2, TNFSF2TNF superfamily, member 2, tumor necrosis factor ligand superfamily member 2, tumor necrosis factor, tumor necrosis factor-alpha, Mouse tumor necrosis factor alpha ELisa kit

Wells

Composition

Please use it within the validity period of the kit (new and old products are shipped randomly)

form	Specifications	Shelf life of diluted or redissolved reagent after unpacking
Mouse TNF-α Microplate	1 Block	After the unused slats are placed back in the aluminum foil bag with desiccant and sealed, they can be used in 2-8℃ store 30 Day
Mouse TNF-α Standard	2 branch	After dissolution, calculate the dosage and divide it into packages, which can be used in -20°C store 14 Day
Mouse TNF-α Detection antibodies	1 branch	After the concentrated volume is dissolved, it can be in 2-8°C store 14 Day
40×SA-HRP	1 branch	40× The concentration can be at 2-8°C Storage; 1× Working concentration is not recommended for storage
10× Buffer for concentration and dilution	1 Bottle	After opening, it can be found in 2-8°C store 30 Day
Chromogenic liquid	1 Bottle
Stop liquid	1 Bottle
20× Concentrated Wash Buffer	1 Bottle
Sealing film	3 Zhang	Stored at room temperature, to avoid contamination, not reusable

Background

Tumor necrosis factor alpha (TNF-α), also known as cachectin and TNFSF1A, is the prototype ligand of tumor necrosis factor superfamily. It is a pleiotropic factor that plays a central role in inflammatory response, 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 26kDa type II transmembrane protein consisting of a 35 amino acid (aa) intracellular domain, a 21aa transmembrane segment, and a 177aa extracellular domain (ECD). In the ECD region, human TNF-α has 97% amino acid sequence homology with macaque monkey, and 71-92% amino acid sequence homology with cattle, dog, cotton rat, horse, cat, mouse, pig and rat. It can be expressed by a variety of different cells such as immune cells, epithelial cells, endothelial cells, tumor cells. TNF-α can induce lysis of tumor cells and virus-infected cells, and binds to soluble TNFRI to generate transmembrane transfer signals of downstream cells. TACE/ADAM17 can cause the shedding of TNF-α-containing cell membrane to release active TNF-α cytokine. It is a trimer composed of TNF-α extracellular soluble structure with a molecular weight of 55 kDa.
TNF-α has two receptors: TNFRI and TNFRII. TNFRI has a molecular weight of 55-60kDa and is widely expressed; TNFRII has a molecular weight of 78-80 kDa and is limited to hematopoietic cell expression. Both are expressed as homotrimers; TNF-α and TNFRI and TNFRII bind with similar affinities and can promote the activation of NFκB. However, only TNFRI has a cell death domain, which can trigger apoptosis. Both soluble receptors are released into human serum and urine and can neutralize the activity of TNF-α.

General Notes

1. Please use the kit within the validity period.
2. The components of different kits and different batch kits cannot be mixed.
3. If the sample value is greater than the highest value of the standard curve, the sample should be diluted with diluent (1 ×) and re-tested; If the cell culture supernatant sample needs to be distributed and diluted, cell culture medium can be used for other intermediate dilutions except dilution with diluent in the last step.
4. Differences in test results can be caused by a variety of factors, including the operation of the experimenter, the use of the pipette, the plate washing technique, the reaction time or temperature, the storage of the kit, etc.
5. The terminating solution in the kit is an acidic solution. Please protect your glasses, hands, face and clothes when using it.
6. For scientific research only, not for in vitro diagnosis.

Storage Temp.

Kit unopened, stored at 2-8 °C.

Test Range

31.3pg/mL-2000pg/mL

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Mouse TNF-α ELISA Kit