**BDNF ELISA Kit: Experimental Principle and Usage Instructions**
The Brain-Derived Neurotrophic Factor (BDNF) Enzyme-Linked Immunosorbent Assay (ELISA) is a highly sensitive and specific method used to quantify BDNF levels in biological samples. This kit utilizes the double-antibody sandwich ELISA technique, which ensures accurate detection of human BDNF. The process begins with a microplate pre-coated with a specific monoclonal antibody against BDNF. After adding the sample, BDNF binds to the immobilized antibody. A secondary HRP-conjugated antibody is then introduced, forming an antibody-antigen-enzyme complex. Following thorough washing, the substrate TMB is added, leading to a color change from blue to yellow under the action of HRP and an acidic stop solution. The intensity of the color is directly proportional to the concentration of BDNF in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the BDNF concentration is determined by comparing the results to a standard curve.
**Kit Components (48-well and 96-well configurations):**
- Coated Microplate: 1 × 48 or 1 × 96
- Standard: 13.5 μg/L, 0.5 mL × 1 bottle
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Conjugate: 3 mL × 1 bottle (48-well), 6 mL × 1 bottle (96-well)
- Sample Diluent: 3 mL × 1 bottle (48-well), 6 mL × 1 bottle (96-well)
- TMB Substrate: 3 mL × 1 bottle (48-well), 6 mL × 1 bottle (96-well)
- Stop Solution: 3 mL × 1 bottle (48-well), 6 mL × 1 bottle (96-well)
- Wash Buffer (20×): 20 mL × 20 times (48-well), 20 mL × 30 times (96-well)
- Sealing Film: 2 pieces (48-well), 2 pieces (96-well)
- Storage: All components should be stored at 2–8°C.
**Sample Preparation and Handling Guidelines:**
1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant; if precipitate forms, re-centrifuge.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge similarly. Store and handle with care.
3. **Urine:** Collect in sterile tubes, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant and re-centrifuge if needed.
4. **Cell Culture Supernatant:** For secreted proteins, collect in sterile tubes and centrifuge. For intracellular components, lyse cells via freeze-thaw cycles, then centrifuge again.
5. **Tissue Samples:** Weigh the tissue, add PBS (pH 7.4), homogenize, and centrifuge. Store remaining samples at 2–8°C or freeze for later use.
6. **General Notes:** Process samples as soon as possible after collection. If not tested immediately, store at -20°C and avoid repeated freeze-thaw cycles.
7. **Note:** Avoid using samples containing NaN3, as it may inhibit HRP activity and affect assay accuracy.
This detailed procedure ensures reliable and reproducible BDNF quantification, making it ideal for research and diagnostic applications.
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