This Human TNF-α ELISA Kit is designed for precise quantification of human tumor necrosis factor alpha (TNF-α) in serum, plasma, cell culture supernatants, or other biological fluids. With a detection range from 7.8 to 500 pg/mL, it’s ideal for research in immunology, inflammation, cytokine profiling, and disease biomarkers.
Kit Contents and Storage
Component | Quanitity/Form |
96-well Pre-coated ELISA microplate | 1 plate |
Lyophilized TNF- α Standrad Protein | 2 Vials |
Lyophilized Biotinylated Detection Antibody | 2 Vials |
Streptavidin-HRP Conjugate (Ready to use) | 0.6 mL |
Assay Diluent (1% BSA in PBS) | 50 mL |
TMB Substrate | 10 mL |
Stop solution | 10 mL |
Wash buffer concentrate (20 ×) | 50 mL |
Plat Sealers | 3 pieces |
Storage and Stability
Store the kit at 4 °C immediately upon receipt. The reagents are stable for up to one year under proper storage conditions. Reconstituted or diluted components should be used fresh or stored as indicated.
Sample Preparation
- For serum: collect, clot, centrifuge at 3,000 rpm for 10 min at 4 °C.
- For plasma: collect with anticoagulants, centrifuge similarly.
- For culture medium: remove debris by centrifugation.
- Aliquot and store samples at –80 °C until use. Avoid repeated freeze-thaw cycles.
Reagent Preparation
- Reconstitute the standard; perform serial dilutions to generate a standard curve (500 → 7.8 pg/mL).
- Prepare detection antibody, streptavidin-HRP, wash buffer, etc., as per instructions.
ELISA Procedure
- Add standards / blanks / samples to wells, incubate ~2 hours.
- Wash; add detection antibody, incubate; wash again.
- Add streptavidin-HRP; incubate; wash.
- Add TMB substrate, allow color development; add stop solution.
- Read absorbance at 450 nm.
Calculation
- Generate standard curve.
- Determine sample TNF-α concentrations by interpolation within the linear range.
Troubleshooting Tips
Problem | Possible cause | Solution |
Low or no signal | Reagents degraded; incubation too short; temperature too low | Use fresh reagents; ensure room temp; extend incubation if necessary. |
High background | Inadequate washing; contaminated or old substrate; improper dilution | Increase wash steps; use fresh substrate; correct dilution. |
Poor reproducibility | Pipetting errors; plate rows unevenly filled; bubbles | Use calibrated pipettes; fill plate carefully; remove bubbles. |
Applications
Cytokine profiling in clinical and research studies
Monitoring inflammation in disease models (autoimmune, metabolic disorders)
Biomarker discovery for diagnostics