TNF alpha ELISA Kit

Catalog No. ABIN7672436

Human TNF alpha ELISA Kit, Colorimetric assay for quantification of Human TNF alpha.

Quick Overview for TNF alpha ELISA Kit (ABIN7672436)

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 15.625 pg/mL - 1000 pg/mL

Application: ELISA

Sample Type: Biological Fluids, Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Lysate

Grade: Quick

Product Details TNF alpha ELISA Kit

Minimum Detection Limit: 15.625 pg/mL

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

Analytical Method: Quantitative

Specificity: Specifically binds with TNF-α , no obvious cross reaction with other analogues. Recommended Sample Dilution Ratio The following table shows the recommended dilution ratios for this kit for a limited number of samples for your reference only. (When the sample content is very low, the original solution can be added without dilution, but it is necessary to ensure that the pH is between 6.8 and 8.0, and it does not contain more than 10 % organic solvents or high-concentration protein denaturants.) 1. Common sample validation: Sample Type Recommended Dilution Ratio Content Healthy human serum/plasma (EDTA, Citrate , heparin) (n=10) undiluted ND-21.6pg/mL Serum of patients with bacterial infection (CRP >7 μg/mL) (n=3) undiluted or 1/2 dilution 117-680pg/mL Plasma of patients with bacterial infection (CRP >7 μg/mL) (n=3) undiluted or 1/2 dilution 135-775pg/mL THP-1 cells were treated with 80nM TPA and cultured overnight. Take the cell culture supernatant for detection. undiluted ND THP-1 cells were treated with 80nM TPA and cultured overnight. Then, stimulate with 100 ng/mL LPS for 6 hours and take the cell culture supernatant for detection. 1/10 dilution 2.1 ng/mL THP-1 cells were treated with 80nM TPA and cultured overnight. Then, they were stimulated with 100 ng/mL LPS for 6 hours. After that, 300 ng/mL Brefeldin A (BFA) was added and cultured for 3 hours. The cell lysate (Cat No: E050) was collected, and the total protein concentration of 1.2 mg/mL was detected by BCA. 1/2 dilution 683pg/mg(total protein) 2. KO sample validation (Detect TNF-α KO THP-1 cells): Sample Type Dilution Ratio Content Wild THP-1 cells were treated with 80nM TPA and cultured overnight, and then stimulated with 200 ng/mL LPS for 6 hours. The cell culture supernatant was taken for detection. 1/5 dilution 1.6 ng/mL KO THP-1 cells were treated with 80nM TPA and cultured overnight, and then stimulated with 200 ng/mL LPS for 6 hours. The cell culture supernatant was taken for detection. undiluted ND KO THP-1 cells were treated with 80nM TPA and cultured overnight. Then they were stimulated with 200 ng/mL LPS for 6 hours. After that, 300 ng/mL Brefeldin A (BFA) was added and cultured for 3 hours. The cell lysate was collected (Cat No: E050), and the total protein concentration of 0.92 mg/mL was detected by BCA. undiluted ND Note:ND is lower than the sensitivity of the kit and was not detected 3. Capture antibody and detection antibody by WB KO validation (Detect TNF-α KO THP-1 cells):

Sensitivity: 9.375 pg/mL

Components:

• ELISA Microplate(Dismountable)
• Lyophilized Standard
• Cap/Det Ab (Ready to use
• blue)
• TMB Substrate
• Sample Dilution Buffer
• Stop Solution
• Plate Sealer
• Product Description

Material not included:

• Microplate reader (wavelength: 450nm)
• 37 °C incubator (CO2 incubator for cell culture is not recommenced.)
• Automated plate washer or multi-channel pipette/5 mL pipettor
• 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

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

Sandwich ELISA, Double Antibody

Sample Volume: 50 μL

Assay Time: 2 h

Plate: Pre-coated

Protocol: Step 1: Take out the required plate wells, add 50 µL Cap/Det Ab into each well, then add 50 µL Standard or Sample into individual well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure thorough mixing then static incubate for 60 minutes at 37 °C. Washing: Wash the plate twice without immersion. Step 2: Add 100 µL HRP-Streptavidin (orange) into each well, seal the plate and static incubate for 30 minutes at 37 °C. Washing: Wash the plate five times without immersion. Step 3: Add 90 µL TMB substrate solution, seal the plate and static incubate for 10-20 minutes at 37 °C. (Accurate TMB visualization control is required.) Step 4: Add 50 µL stop solution. Read at 450nm immediately and calculate.

Assay Procedure:

1. Take out the required strips, add 50 µL Cap/Det Ab into each well, then add 50 µL Standard or Sample into relevant well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure complete mixing then statically incubate for 60 minutes at 37 °C.
2. Wash the plate twice without immersion.
3. Add 100 µL HRP-Streptavidin (orange) into each well, seal the plate and statically incubate for 30 minutes at 37 °C.
4. Wash the plate five times without immersion.
5. Add 90 µL TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37 °C (Accurate TMB visualization control is required.).
6. Add 50 µL stop solution. Read at 450nm immediately and calculate.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid direct light exposure to antibody solutions

Storage: 4 °C

Storage Comment: Store sealed Kit at 2-8° C. Do Not Cryopreserve. Store ELISA Microplate for 1 month at 2-8°C, Store for 12 month at -20°C. Put the leftover strips into a sealed foil bag with the desiccant

Expiry Date: 12 months

Target Details for TNF alpha

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Alternative Name: TNF-alpha

Background: Tumor necrosis factor, Cachectin, TNF-alpha, Tumor necrosis factor ligand superfamily member 2, TNF-a, Tumor necrosis factor, membrane form, N-terminal fragment, NTF, Intracellular domain 1, ICD1, Intracellular domain 2, ICD2, C-domain 1, C-domain 2, Tumor necrosis factor, soluble form, TNF, TNFA, TNFSF2

UniProt: P01375

Pathways: NF-kappaB Signaling, Apoptosis, Caspase Cascade in Apoptosis, TLR Signaling, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome