Human Tumor Necrosis Factor Alpha (TNF-α) High Sensitivity ELISA Kit

SKU: orb1945913

Description

Human Tumor Necrosis Factor Alpha (TNF-α) High Sensitivity ELISA Kit

Research Area

Cancer Biology; Immunology & Inflammation

Images & Validation

• Application Notes:
  This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human TNF-α. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human TNF-α and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human TNF-α, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human TNF-α. You can calculate the concentration of Human TNF-α in the samples by comparing the OD of the samples to the standard curve.

Key Properties

• Reactivity: Human
• Sample Types: Serum, Plasma and Other biological fluids,Sample Volume= 100μL
• Assay Type: Sandwich
• Assay Time: 3.5H
• Range: 0.78-50pg/mL
• Sensitivity: 0.47 pg/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.
• 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
• 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.
• Assay Procedure:
  This procedure is for reference only.; ; After the kit equilibrates to room temperature, add standard or sample to the wells and incubate.; Aspirate and wash the plate for three times.; Immediately add Biotinylated Detection Antibody Working Solution to each well and incubate.; Aspirate and wash the plate for three times ; Add Streptavidin-HRP Working Solution to each well and incubate.; Aspirate and wash the plate for three times ; Add TMB Substrate Reagent to each well and incubate.; Add Stop Reagent to each well, read the plate at OD 450 nm immediately, and calculate the results.
• 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
• Precision:
  Intra-assay Precision (Precision within an assay): CV% ≤ 10% ; 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.
• 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.
• Curve Fitting Softwares:
  1. Curve Expert; 2. Thermo SkanIt RE; 3. SciDAVis; 4. LabPlot; 5. ……

Storage & Handling

• Storage: Refer to the Storage Guidelines in the Manual
• Expiration Date: 6 months from date of receipt.
• Disclaimer: For research use only

Alternative Names

DIF, TNF-alpha, TNFA, TNFSF2, TNF-A, TNFα

Standard curve for orb1945913 ELISA kit.

Quick Database Links

UniProt

P01375