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.
Manual
- Product Name
- Sheep TNF-α(Tumor Necrosis Factor Alpha) ELISA Kit
- Alias
- TNFα ELISA Kit, Tumor Necrosis Factor Alpha ELISA Kit, TNF-α ELISA Kit, DIF ELISA Kit, TNF-alpha ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit
- Catalogue No.
- ESH0025
- Size
- 48T/96T
- Species
- Sheep
- UniProt ID
- P23383
- 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
-
- Microplate reader (wavelength: 450nm)
- 37°C incubator (CO2 incubator for cell culture is not recommenced.)
- Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
- Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
- Sterile tubes and Eppendorf tubes with disposable tips
- Absorbent paper and loading slot
- Deionized or distilled water
- Assay Procedure Summary
-
- 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 0.08 0.084 0.082 15.625 0.1 0.104 0.102 31.25 0.123 0.129 0.126 62.5 0.129 0.135 0.132 125 0.221 0.231 0.226 250 0.429 0.447 0.438 500 1.01 1.052 1.031 1000 2.181 2.27 2.225 
- 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) 89-105 97 EDTA plasma(n=10) 89-100 96 Heparin plasma(n=10) 88-101 93 - 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) 85-98% 95-99% 82-95% EDTA plasma(n=10) 87-102% 87-99% 81-99% Heparin plasma(n=10) 86-102% 89-94% 80-100% - 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) 30.45 119.2 503 31.87 121.9 491 Standard deviation 1.53 7.39 26.26 2.05 5.88 30.44 CV(%) 5.02 6.2 5.22 6.43 4.82 6.2 - 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