Human TNFalpha ELISA Kit

SKU: orb561883

Description

Human TNFalpha(Tumor Necrosis Factor Alpha) ELISA Kit

Research Area

Immunology & Inflammation

Images & Validation

• Tested Applications: KO/KD Validated
• Application Notes:
  Capture antibody and detection antibody by WB KO validation (Detect TNF-α KO THP-1 cells).

Key Properties

• Reactivity: Human
• Sample Types: 9.375pg/ml
• Assay Type: Sandwich
• Assay Time: 4 hours
• Range: 15.6-1000pg/ml
• Sensitivity: 1pg/ml

Procedure & Performance

• Assay Principle:
  The kit is based on a sandwich enzyme immunoassay principle. The microtiter plate is pre-coated with a capture antibody specific to the target analyte. Standards or samples are added to the wells, followed by a biotin-conjugated detection antibody specific for the analyte. Avidin conjugated to horseradish peroxidase (HRP) is then added and incubated. After addition of the TMB substrate, color develops only in wells containing the analyte bound to the detection antibody and HRP–avidin complex. The reaction is stopped with an acidic solution, and absorbance is measured at 450 nm ± 10 nm. The analyte concentration in the samples is determined by comparison with a standard curve.
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• Kit Components:
  1. ELISA Microplate; 2. Standards; 3. Detection Antibody ; 4. HRP-Streptavidin Conjugate; 5. TMB Substrate; 6. Dilution buffers; 7. Stop Solution; 8. Wash Buffer; 9. Plate Sealers; 10. Manual
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• Reagent Preparation:
  1. Wash Buffer: Prepare the 1X Wash Buffer using distilled water according to the manual.; 2. Standard: Perform gradient dilution according to the instructions in the manual. ; 3. Other Concentrated Reagents: Dilute the concentrated reagents using the Dilution Buffers provided in the kit to 1 X working solutions as instructed in the manual. Always use a clean pipette tip for each different solution.
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• Assay Procedure:
  After the kit equilibrates to room temperature, add standards or samples to each well and incubate.; Discard liquid, add wash buffer to each well, wash the plate twice, and blot dry on clean absorbent paper.; Add biotinylated antibody working solution to each well and incubate.; Discard liquid, add wash buffer to each well, wash the plate three times, and blot dry on clean absorbent paper.; Add streptavidin-HRP working solution to each well and incubate.; Discard liquid, add wash buffer to each well, wash the plate five times, and blot dry on clean absorbent paper.; Add TMB substrate solution to each well and incubate in the dark.; Add stop solution to each well, mix thoroughly, and immediately read OD at 450 nm.
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• Materials Required:
  1. Microplate readers; 2. Centrifuge; 3. Incubator; 4. Automated plate washer; 5. Single-channel or multi-channel high-precision pipettes; 6. Disposable pipette tips; 7. Sterile tubes; 8. Eppendorf tubes; 9. Absorbent paper; 10. Loading slots
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• Precision:
  Intra-assay Precision (Precision within an assay): CV% < 8% ; Intra-assay precision was evaluated by testing multiple replicates of samples within the same plate.; ; Inter-assay Precision (precision between assays): CV% < 10% ; Inter-assay precision was evaluated by testing samples across different plates.
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• Calculation of Results:
  1. Average the duplicate readings for each Standard, Control, and Sample, and subtract the mean optical density of the zero Standard. ; 2. Construct a standard curve by plotting the target concentration on the y-axis against absorbance on the x-axis and draw a curve through the data points. ; 3. Determine the sample concentration by substituting the OD450 value into the standard curve. For diluted samples, multiply the calculated value by the corresponding dilution factor.
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• Curve Fitting Softwares:
  1. Curve Expert; 2. Thermo SkanIt RE; 3. SciDAVis; 4. LabPlot; 5. ……
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Storage & Handling

• Storage: 2 - 8 °C for 12 months
• Expiration Date: 6 months from date of receipt.
• Disclaimer: For research use only

Alternative Names

TNFα|Tumor Necrosis Factor Alpha|TNF-α|DIF|TNF-alpha|TNFA|TNFSF2

Quick Database Links

Gene Symbol

TNF-α

UniProt

P01375