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-β 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-β 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-β in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.
Manual- Product Name
- Canine TNF-β (Tumor Necrosis Factor β) ELISA Kit
- Alias
- Lymphotoxin-alpha ELISA Kit, LT-alpha ELISA Kit, TNF-beta ELISA Kit, Tumor necrosis factor ligand superfamily member 1 ELISA Kit, LTA ELISA Kit, TNFB ELISA Kit, TNFSF1 ELISA Kit
- Catalogue No.
- ECA0040
- Size
- 48T/96T
- Species
- Canine
- UniProt ID
- Q5WR07
- 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-β , 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 0.101 0.103 0.102 15.625 0.203 0.209 0.206 31.25 0.277 0.285 0.281 62.5 0.43 0.442 0.436 125 0.651 0.669 0.66 250 0.862 0.888 0.875 500 1.429 1.471 1.45 1000 2.125 2.187 2.156 - Recovery
-
Add a certain amount of TNF-β into the sample. Calculate the recovery by comparing the measured value with the expected amount of TNF-β in the sample.
Sample Type Recovery Range(%) Average(%) serum(n=10) 93-102 96 EDTA plasma(n=10) 88-99 95 Heparin plasma(n=10) 89-101 95 - Linearity
-
Dilute the sample with a certain amount of TNF-β at 1:2, 1:4 and 1:8 to get the recovery range.
Sample Type 1:2 1:4 1:8 serum(n=10) 82-101% 81-102% 90-98% EDTA plasma(n=10) 83-96% 84-101% 84-101% Heparin plasma(n=10) 82-98% 87-101% 82-99% - 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) 31.03 125.9 512 31.56 129.3 498 Standard deviation 1.66 6.26 27.08 1.54 6.72 24.1 CV(%) 5.36 4.97 5.29 4.88 5.2 4.84 - 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