**Mouse Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit – User Manual**
**Kit Specifications:**
This ELISA kit is available in 48-well or 96-well configurations. It includes:
- Standard Diluent: 1.5 mL × 1 vial
- Enzyme Standard Reagent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 vial, 2700 ng/L
- Sample Diluent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Developer A: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Chromogen B: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Stop Solution: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Concentrated Washing Solution: 20 mL × 20 times (48-well) / 20 mL × 30 times (96-well)
**Storage Conditions & Shelf Life:**
- Store the kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
**Principle of the Assay:**
The kit uses a double-antibody sandwich ELISA method to quantify TNF-α levels in samples. The microtiter plate is pre-coated with a specific monoclonal antibody against mouse TNF-α. After incubation with the sample, a biotinylated detection antibody is added, followed by HRP-conjugated secondary antibody. The reaction is developed using TMB substrate, which changes color in the presence of HRP. The absorbance is measured at 450 nm, and the concentration is calculated based on a standard curve.
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature, then centrifuge at 2000–3000 rpm for 10–20 minutes. Collect the supernatant.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing for 10–20 minutes.
3. **Urine:** 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 freeze-thaw cycles.
5. **Tissue Samples:** Homogenize in PBS, centrifuge, and collect the supernatant.
6. **Storage:** Process samples as soon as possible. If not used immediately, store at -20°C. Avoid repeated freezing and thawing.
**Important Notes:**
- Ensure all reagents are equilibrated to room temperature before use.
- Do not mix reagents from different batches.
- Avoid exposure of the substrate to light.
- Always prepare a standard curve and run samples in duplicate.
- Discard any unused enzyme reagent strips in sealed bags to prevent contamination.
- Handle all samples, washes, and waste as biohazardous materials.
**Performance Characteristics:**
- Linear regression correlation coefficient (R) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Usage Instructions:**
1. Prepare standards and dilute them according to the provided protocol.
2. Add 40 µL of sample diluent and 10 µL of sample to each well (final dilution 5×).
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted washing solution.
5. Add 50 µL of enzyme-labeled reagent to each well (except blank).
6. Incubate again at 37°C for 30 minutes.
7. Add 50 µL of TMB A and B, incubate at 37°C for 15 minutes.
8. Add 50 µL of stop solution and measure OD at 450 nm within 15 minutes.
**Service Commitment:**
- Free technical support during working hours.
- Sample testing services available upon request.
- Delivery time: From payment to delivery.
**Note:** This kit is for research use only. Results must be interpreted based on the microplate reader readings. Always refer to the latest version of the manual.
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