**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. The standard dilution is 1.5 mL × 1 vial. The enzyme standard reagent is 3 mL × 1 vial (for 48-well) or 6 mL × 1 vial (for 96-well).
**Kit Composition:**
- Sealing film: 2 pieces (48) / 2 pieces (96)
- Standard: 0.5 mL × 1 vial, concentration 2700 ng/L
- Enzyme standard: 3 mL × 1 vial (48) / 6 mL × 1 vial (96)
- Sample diluent: 3 mL × 1 vial (48) / 6 mL × 1 vial (96)
- Developer A: 3 mL × 1 vial (48) / 6 mL × 1 vial (96)
- Chromogen B: 3 mL × 1 vial (48) / 6 mL × 1 vial (96)
- Stop solution: 3 mL × 1 vial (48) / 6 mL × 1 vial (96)
- Concentrated washing solution: 20 mL × 20 times × 1 bottle (48) or 20 mL × 30 times × 1 bottle (96)
**Storage Conditions and Shelf Life:**
- Storage: 2–8°C
- Shelf life: 6 months from the date of manufacture
**Principle of the Assay:**
The kit employs a double-antibody sandwich ELISA method to quantify TNF-α levels in samples. Microtiter plates are pre-coated with purified mouse anti-TNF-α antibodies. After incubation with the sample, a biotinylated detection antibody is added, followed by HRP-conjugated secondary antibody. A TMB substrate is then used for color development, which changes from blue to yellow under acidic conditions. The absorbance at 450 nm is measured, and the concentration is calculated using a standard curve.
**Objective:**
This kit is designed for the quantitative determination of TNF-α in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Sample Preparation and Handling:**
- **Serum:** Allow blood to clot at room temperature, centrifuge at 2000–3000 rpm for 10–20 minutes, and collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix well, and centrifuge similarly.
- **Urine:** Collect in sterile tubes, centrifuge, and take the supernatant.
- **Cell Culture Supernatant:** Centrifuge after collection; for intracellular components, lyse cells via freezing/thawing and centrifuge again.
- **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
- **General Notes:** Avoid repeated freeze-thaw cycles. Store samples at -20°C if not tested immediately. Do not use samples containing NaN3.
**Operation Steps:**
1. **Standard Dilution:** Prepare a serial dilution of the standard, starting from 1800 ng/L down to 150 ng/L.
2. **Sample 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 buffer.
5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well.
6. **Second Incubation:** Repeat incubation at 37°C for 30 minutes.
7. **Color Development:** Add 50 µL of TMB A and B, incubate for 15 minutes at 37°C.
8. **Stop Reaction:** Add 50 µL of stop solution to each well.
9. **Measurement:** Read OD450 nm within 15 minutes.
**Notes and Precautions:**
- Allow the kit to equilibrate to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Handle all reagents carefully, especially TMB, which is light-sensitive.
- Always run a standard curve, preferably in duplicate.
- If the sample OD exceeds that of the highest standard, perform a preliminary dilution.
- Follow the manufacturer’s instructions strictly.
**Performance:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²): ≥ 0.95
- Intra-batch and inter-batch variability: < 9% and < 11%, respectively
**Service Commitment:**
We offer free technical support during working hours. Sample testing services are available upon request to ensure optimal results.
**Delivery:**
Orders are processed and shipped after payment confirmation.
This manual provides detailed guidance for accurate and reliable TNF-α quantification. For any questions or assistance, please contact our support team.
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