**BDNF ELISA Kit: Experimental Principle and Usage Guide**
The Brain-Derived Neurotrophic Factor (BDNF) Enzyme-Linked Immunosorbent Assay (ELISA) kit is designed to quantify BDNF levels in various biological samples using a double-antibody sandwich method. This technique involves immobilizing a specific anti-BDNF antibody on the surface of microtiter plate wells. Once the sample is added, BDNF binds to the immobilized antibody. A second, HRP-conjugated anti-BDNF antibody then recognizes and binds to the captured BDNF, forming an immune complex. After thorough washing to remove unbound components, a TMB substrate is introduced. The HRP enzyme catalyzes the conversion of TMB into a blue color, which changes to yellow upon acid termination. The intensity of the color is directly proportional to the BDNF concentration in the sample.
The optical density (OD) at 450 nm is measured using a microplate reader, and the BDNF concentration is determined by comparing the sample OD to a standard curve generated from known concentrations of BDNF.
**Kit Components:**
- Coated Plate: 1 × 48 or 1 × 96 wells
- Standard: 13.5 μg/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 A & B: 3 mL × 1 bottle each
- Stop Solution: 3 mL × 1 bottle
- Wash Buffer (20×): 20 mL × 20 times or 20 mL × 30 times
- Sealing Film: 2 pieces (for 48-well), 2 pieces (for 96-well)
- Sealed Bag: 1 piece
**Storage Instructions:**
All components should be stored at 2–8°C. Avoid freezing 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 supernatant carefully; if precipitate forms, re-centrifuge before use.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge similarly.
3. **Urine:** Collect in sterile tubes and centrifuge for 20 minutes.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm. For intracellular components, lyse cells by repeated freeze-thaw cycles and centrifuge again.
5. **Tissue Samples:** Weigh and homogenize in PBS (pH 7.4). Centrifuge after homogenization. Store remaining portions at -20°C if not used immediately.
6. **General Notes:** Process samples as soon as possible after collection. If delayed, store at -20°C, avoiding repeated freeze-thaw cycles. Do not use samples containing NaN3, as it inhibits HRP activity.
This ELISA kit provides a reliable and sensitive method for measuring BDNF levels, making it suitable for research in neuroscience, clinical diagnostics, and drug development. Always follow the manufacturer’s instructions for optimal performance.
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