**Mouse Tumor Necrosis Factor Alpha (TNF-α) ELISA Kit – Instructions for Use**
**Kit Specifications:**
Available in 48-well or 96-well configurations.
- **Standard Diluent:** 1.5 mL × 1 vial
- **Enzyme Standard Reagent:** 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
**Kit Composition:**
- Sealing Film: 2 pieces (for 48 wells) / 2 pieces (for 96 wells)
- Standard: 0.5 mL × 1 vial, concentration: 2700 ng/L
- Enzyme Standard: 1×48 / 1×96
- Sample Diluent: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Developer A: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Chromogen B: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Stop Solution: 3 mL × 1 vial (for 48 wells) / 6 mL × 1 vial (for 96 wells)
- Concentrated Washing Solution: 20 mL × 20 times (for 48 wells) / 20 mL × 30 times (for 96 wells)
**Storage Conditions & Shelf Life:**
- Store at 2–8°C
- Shelf life: 6 months from the date of receipt
**Principle of the Assay:**
This ELISA kit uses a double-antibody sandwich method to quantify mouse TNF-α levels in biological samples. The microplate is pre-coated with anti-TNF-α antibodies. After adding the sample and enzyme-labeled anti-TNF-α antibody, an immune complex is formed. After washing, TMB substrate is added, and the color changes from blue to yellow under the action of HRP. The intensity of the color is directly proportional to the TNF-α concentration in the sample. The OD value at 450 nm is measured, and the concentration is calculated using a standard curve.
**Objective:**
The kit is designed to measure TNF-α levels in serum, plasma, urine, cell culture supernatants, and tissue homogenates.
**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, mix, and centrifuge similarly.
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 by freezing and thawing before centrifugation.
5. **Tissue Samples:** Homogenize in PBS, centrifuge, and collect the supernatant.
6. **Storage:** Store samples at -20°C if not tested immediately. Avoid repeated freeze-thaw cycles.
7. **Note:** Avoid samples containing NaN₃, as it may inhibit HRP activity.
**Operation Steps:**
1. **Standard Dilution:** Prepare serial dilutions of the standard solution.
2. **Loading:** Add 40 μL of sample diluent and 10 μL of sample to each well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash 5 times with diluted washing solution.
5. **Add Enzyme Reagent:** Add 50 μL of enzyme-labeled reagent to each well.
6. **Incubation:** Repeat incubation at 37°C for 30 minutes.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to each well.
9. **Measurement:** Read OD450 within 15 minutes using a microplate reader.
**Notes:**
- Equilibrate the kit at room temperature for 15–30 minutes before use.
- Ensure accurate pipetting and avoid cross-contamination.
- Prepare a standard curve and run duplicates for better accuracy.
- Do not mix reagents from different batches.
- All waste should be handled as biohazardous material.
**Performance:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
**Technical Support:**
Free technical support is available during working hours. We also offer free sample testing to help ensure optimal results.
**Delivery:**
Orders are processed upon payment. Please contact us for any assistance.
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