**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 (for 48-well) / 6 mL × 1 vial (for 96-well)
- Coating Antibody, Detection Antibody, HRP Conjugate, Substrate, and Wash Solution
**Principle of Operation:**
The kit employs a sandwich ELISA method to quantify TNF-α levels in biological samples. The microtiter plate is pre-coated with anti-TNF-α antibodies. After incubation with the sample, the captured TNF-α is detected using an HRP-labeled secondary antibody. A colorimetric reaction occurs upon substrate addition, with the intensity directly proportional to the TNF-α concentration. The optical density (OD) is measured at 450 nm, and the concentration is determined by comparing the sample OD to a standard curve.
**Storage Conditions & Shelf Life:**
- Store all components at 2–8°C.
- Shelf life: 6 months from the date of receipt.
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature, then centrifuge at 2000–3000 rpm for 10–20 minutes. Collect supernatant carefully.
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 after cell lysis by repeated freezing/thawing.
5. **Tissue Homogenate:** Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
6. Avoid repeated freeze-thaw cycles. Store samples at -20°C if not tested immediately.
**Standard Curve Preparation:**
Dilute the standard solution in serial dilutions (1800 ng/L, 1200 ng/L, 600 ng/L, 300 ng/L, 150 ng/L). Plot the OD values against concentrations on a logarithmic scale. Calculate the sample concentration using linear regression or by referencing the standard curve. Multiply by the dilution factor to obtain the actual concentration.
**Procedure Summary:**
1. Prepare standards and samples.
2. Add 40 µL of sample diluent and 10 µL of sample to each well (final 5× dilution).
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add 50 µL of enzyme conjugate to each well (except blank wells).
6. Incubate again at 37°C for 30 minutes.
7. Wash 5 times.
8. Add 50 µL of TMB A and B, incubate at 37°C for 15 minutes.
9. Stop the reaction with 50 µL of stop solution.
10. Measure OD at 450 nm within 15 minutes.
**Important Notes:**
- Allow the kit to equilibrate to room temperature before use.
- Do not reuse sealing films.
- Avoid light exposure during substrate incubation.
- Always prepare a standard curve and run duplicates for accuracy.
- Do not mix reagents from different batches.
- All waste should be treated as biohazardous material.
**Performance Characteristics:**
- Correlation coefficient (R²) ≥ 0.95.
- Intra-batch CV < 9%, Inter-batch CV < 11%.
- Detection range: 0.2 IU/L – 6 IU/L.
**Technical Support:**
Free technical assistance is available during working hours. Sample testing services are also offered to ensure optimal results.
**Warranty & Guarantee:**
The kit is intended for research use only. If you have any questions or need further guidance, please contact our support team.
**Delivery & Payment:**
Order processed and shipped after payment confirmation.
This manual provides detailed instructions for accurate and reliable TNF-α detection in various biological matrices. Follow all steps carefully to ensure consistent and reproducible results.
Jiangsu Raymeel Home Decoration Co., Ltd. , https://www.raymeelhome.com