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

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This kit is based on Double antibody-Sandwich ELISA detection method and takes 4h assay time. The microplate provided in this kit has been precoated with anti TNF-α antibody. Add standard and properly diluted sample into relevant well respectively. After incubation, wash unbound components. Add biotinylated detection antibody. Then, it binds with TNF-α bound to precoated antibody. Wash unbound components and add HRP-Streptavidin Conjugate (SABC). Wash unbound components again and add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Calculate the concentration of TNF-α in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.

Catalogue No.: 
EH0302
Alias: 
Tumor necrosis factor ELISA Kit, Cachectin ELISA Kit, TNF-alpha ELISA Kit, Tumor necrosis factor ligand superfamily member 2 ELISA Kit, TNF-a ELISA Kit, Tumor necrosis factor, membrane form ELISA Kit, N-terminal fragment ELISA Kit, NTF ELISA Kit, Intracellular domain 1 ELISA Kit, ICD1 ELISA Kit, Intracellular domain 2 ELISA Kit, ICD2 ELISA Kit, C-domain 1 ELISA Kit, C-domain 2 ELISA Kit, Tumor necrosis factor, soluble form ELISA Kit, TNF ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit
Species: 
Human
Range: 
15.625-1000pg/ml
Sensitivity: 
9.375pg/ml
Manual MSDS
  • SPECIFICATIONS
  • CITATIONS
  • FIGURES
Product Name
Human TNF-α(Tumor Necrosis Factor Alpha) ELISA Kit
Alias
Tumor necrosis factor ELISA Kit, Cachectin ELISA Kit, TNF-alpha ELISA Kit, Tumor necrosis factor ligand superfamily member 2 ELISA Kit, TNF-a ELISA Kit, Tumor necrosis factor, membrane form ELISA Kit, N-terminal fragment ELISA Kit, NTF ELISA Kit, Intracellular domain 1 ELISA Kit, ICD1 ELISA Kit, Intracellular domain 2 ELISA Kit, ICD2 ELISA Kit, C-domain 1 ELISA Kit, C-domain 2 ELISA Kit, Tumor necrosis factor, soluble form ELISA Kit, TNF ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit
Catalogue No.
EH0302
Size
48T/96T
Species
Human
UniProt No.
P01375
Sample Type
Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples
Detection Method
Sandwich ELISA, Double Antibody
Detection Wavelength
OD450
Reaction Duration
4 hours
Range
15.625-1000pg/ml
Sensitivity
9.375pg/ml
Storage
2-8°C(Sealed), Don't cryopreserve.
Specificity
Specifically binds with TNF-α , no obvious cross reaction with other analogues.
ELISA Kit Components
Kit Components Item Size(48T) Size(96T) Storage Condition for Opened Kit
E001 ELISA Microplate(Dismountable) 8×6 8×12 Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
E002 Lyophilized Standard 1vial 2vial Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 month at -20°C
E003 Biotin-labeled Antibody(Concentrated, 100X) 60ul 120ul 2-8°C (Avoid Direct Light)
E034 HRP-Streptavidin Conjugate(SABC, 100X) 60ul 120ul
E024 TMB Substrate 5ml 10ml
E039 Sample Dilution Buffer 10ml 20ml 2-8°C
E040 Antibody Dilution Buffer 5ml 10ml
E049 SABC Dilution Buffer 5ml 10ml
E026 Stop Solution 5ml 10ml
E038 Wash Buffer(Concentrated, 25X) 15ml 30ml
E006 Plate Sealer 3 pieces 5 pieces  
E007 Product Description 1 copy 1 copy
Required Instruments and Reagents
  1. Microplate reader (wavelength: 450nm)
  2. 37°C incubator (CO2 incubator for cell culture is not recommenced.)
  3. Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
  4. Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
  5. Sterile tubes and Eppendorf tubes with disposable tips
  6. Absorbent paper and loading slot
  7. Deionized or distilled water
Assay Procedure Summary
Elisa实验原理图
  • Step 1: Add 100ul standard or sample into each well, seal the plate and statically incubate for 90 minutes at 37°C.
  • Washing: Wash the plate twice without immersing.
  • Step 2: Add 100ul biotin-antibody working solution, seal the plate and statically incubate for 60 minutes at 37°C.
  • Washing: Wash the plate three times and immerse for 1min each time.
  • Step 3: Add 100ul HRP-Streptavidin Conjugate (SABC) working solution, seal the plate and statically incubate for 30 minutes at 37°C.
  • Washing: Wash the plate five times and immerse for 1min each time.
  • Step 4: Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
  • Step 5: Add 50ul stop solution. Read at 450nm immediately and calculate.
Standard Curve

This product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.)

Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.

STD.(pg/ml) OD-1 OD-2 Average
0 0.142 0.149 0.145
15.625 0.187 0.197 0.191
31.25 0.246 0.258 0.251
62.5 0.302 0.317 0.308
125 0.422 0.444 0.431
250 0.746 0.784 0.761
500 1.256 1.32 1.281
1000 2.05 2.154 2.092
EH0302 Standard Curve Image
Recovery

Add a certain amount of TNF-α into the sample. Calculate the recovery by comparing the measured value with the expected amount of TNF-α in the sample.

Sample Type Recovery Range(%) Average(%)
serum(n=10) 92-103 95
EDTA plasma(n=10) 92-99 93
Heparin plasma(n=10) 86-99 94
Linearity

Dilute the sample with a certain amount of TNF-α at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type 1:2 1:4 1:8
serum(n=10) 81-93% 82-95% 86-95%
EDTA plasma(n=10) 86-103% 84-100% 83-94%
Heparin plasma(n=10) 85-96% 80-95% 81-99%
Precision(%)

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.

Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Item Intra-assay Precision Inter-assay Precision
Sample 1 2 3 1 2 3
n 20 20 20 20 20 20
Mean (pg/ml) 31.68 118.4 485 32.21 125.2 494
Standard deviation 1.76 7.09 28.71 1.78 7.32 27.12
CV(%) 5.56 5.99 5.92 5.53 5.85 5.49
Stability

Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.

ELISA kit(n=5) 37°C for 1 month 2-8°C for 6 months
Average(%) 80 95-100
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