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    Rat TNF-α ELISA Kit

    Rat TNF-α ELISA Kit

    Catalog Number: abs530007 Reactivity: Rat Conjugation: Brand: Absin
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    $450 USD
    $450 USD
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    Product Details

    Product Specification

    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 Color 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 color developer removed is for use on the same day only.

    ⑥ Standard: Dilution buffer for lyophilized standard ( 1× ) Re-dissolved, redissolved volume 1000uL , obtaining a concentration of 4000pg/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 ( 4000pg/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 wash liquid in the plate is aspirated off. 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 were spiked into cell culture medium samples Rat TNF-α The recovery rate was determined. The recoveries range from 75-112% , the average recovery was in 90% 。

    2. Sensitivity: Rat TNF-α The lowest measurable dose ( MDD ) is generally less than 7.8pg/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 kit expressed by Escherichia coli Rat TNF-α Corrected by protein.

    4. Linearity: 4 High concentrations of different samples were incorporated into Rat TNF-α , followed by a diluent ( 1× ) Dilute the sample to the detection range and determine its linearity.
    Dilution factor Average / Expected value ( % ） Range ( % ）
    1:2 102 96-105
    1:4 103 98-108
    1:8 109 103-116
    1:16 102 98-105

    5. Specificity: This ELISA Method can detect natural and recombinant Rat TNF-α Egg whites.

    4. Analysis of frequently asked questions

    1. Whiteboard (no color appears after color rendering is complete)
    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 application 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)
    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
    Component Specifications Shelf life of diluted or redissolved reagent after unpacking
    Rat TNF-α Microplate 1 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
    Rat TNF-α Standard 2 After dissolution, calculate the dosage and divide it into packages, which can be used in -20°C store 14 Day
    Rat TNF-α Detection antibodies 1 After the concentrated volume is dissolved, it can be in 2-8°C store 14 Day
    40×SA-HRP 1 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 After opening, it can be found in 2-8°C store 30 Day
    Chromogenic liquid 1
    Stop Solution 1
    20× Concentrated Wash Buffer 1
    Sealing film 3 Stored at room temperature, to avoid contamination, not reusable
    Background Tumor necrosis factor alpha (TNF-alpha), also known as cachectin and TNFSF1A, is the prototype ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. TNF-α is also implicated in a number of pathological conditions, including asthma, Crohn's disease, rheumatoid arthritis, neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmune diseases, and cancer. Rat TNF-alpha is a 26kDa type II transmembrane protein, which consists of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane fragment, and a 156 aa extracellular domain (ECD). In ECD, rat TNF-alpha has 95% amino acid sequence identity with mouse and 73%-79% amino acid sequence identity with bovine, canine, cotton rat, horse, cat, human, macaque, and pig TNF-alpha. It is produced by a variety of immune cells, epithelial cells, endothelial cells and tumor cells. TNF-α assembles intracellularly to form non-covalently linked homotrimers expressed on the cell surface. Cell surface TNF-α can not only induce lysis of tumor cells and virus-infected cells, but also produce its own downstream cell signaling after ligation through soluble TNFRI. TACE/ADAM17 releases membrane-bound TNF-α to release a bioactive cytokine, which is a 55 kDa soluble trimer of the extracellular domain of TNF-α. TNF-α binds to the ubiquitous 55-60 kDa TNFRI and the hematopoietic cell-restricted 78-80 kDa TNFRII, both of which are also expressed as homotrimers. Both type I and type II receptors bind TNF-α with comparable affinity and can promote NFκB activation. However, only TNF RI contains a cytoplasmic death domain, which triggers the activation of apoptosis. Soluble forms of both types of receptors are released into human serum and urine and can neutralize the biological 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 (1 ×) 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 62.5pg/mL-4000pg/mL