**Rabbit Tumor Necrosis Factor-α (TNF-α) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – Instructions for Use**
This reagent is intended for research purposes only. The kit is designed to quantitatively measure tumor necrosis factor alpha (TNF-α) in rabbit serum, plasma, and other biological fluids.
**Principle of the Assay**
The assay is based on the double-antibody sandwich ELISA method. A microplate is pre-coated with a specific monoclonal antibody against rabbit TNF-α. After incubation with the sample, TNF-α binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex of antibody-TNF-α-enzyme. Following washing steps, the substrate TMB is introduced. Under the catalytic action of HRP, TMB turns blue and then changes to yellow upon addition of an acidic stop solution. The intensity of the color is directly proportional to the concentration of TNF-α in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the concentration is determined by comparing it to a standard curve.
**Kit Components**
- Coated Microplate: 1 × 48 wells / 1 × 96 wells
- Standard: 22.5 ng/L, 0.5 mL × 1 bottle
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Conjugate: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate: 3 mL × 1 bottle
- Stop Solution: 3 mL × 1 bottle
- Wash Buffer (20×): 20 mL × 20 times × 1 bottle
- Sealing Film: 2 pieces (for 48-well) / 2 pieces (for 96-well)
- Sealed Bag: 1 piece
**Storage Conditions**
All components should be stored at 2–8°C. Do not freeze unless specified.
**Sample Preparation and Handling**
1. **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. If precipitate forms during storage, re-centrifuge.
2. **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix well, then centrifuge as above.
3. **Urine**: Collect in a sterile container and centrifuge at 2000–3000 rpm for 20 minutes.
4. **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via repeated freezing and thawing, then centrifuge again.
5. **Tissue Samples**: Weigh the tissue, add PBS (pH 7.4), homogenize, and centrifuge. Store at 2–8°C after thawing.
6. **General Notes**: Process samples immediately after collection. If not used right away, store at -20°C. Avoid repeated freeze-thaw cycles.
7. **Note**: Samples containing NaN3 cannot be tested, as it inhibits HRP activity.
This kit provides a reliable and sensitive method for measuring TNF-α levels in various biological matrices. Follow all instructions carefully to ensure accurate results.
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