**Mouse Glial Cell Line-Derived Neurotrophic Factor (GDNF) ELISA Kit – Instructions for Use**
This reagent is intended for research use only. It is designed to quantitatively measure the levels of Glial Cell Line-Derived Neurotrophic Factor (GDNF) in mouse serum, plasma, and related biological fluids.
**Principle of the Assay**
The kit employs a sandwich ELISA technique. A microtiter plate is pre-coated with a specific anti-GDNF antibody. After incubation with the sample, GDNF binds to the immobilized antibody. An HRP-conjugated secondary antibody then recognizes the captured GDNF, forming an antibody-antigen-enzyme complex. Following washing steps, TMB substrate is added, which changes color under the catalytic action of HRP. The reaction is stopped with an acidic solution, causing the color to shift from blue to yellow. The intensity of the color is directly proportional to the GDNF concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the GDNF concentration is determined by comparing the sample OD values to a standard curve.
**Kit Components**
- Microtiter Plate: 1 × 48 or 1 × 96 wells
- Standard: 0.5 mL × 1 bottle (90 ng/L)
- 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 Films: 2 pieces per configuration
- Storage: 2–8°C
**Sample Preparation**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant, mix well, and centrifuge similarly.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm; for intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
- **Tissue**: Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C after thawing.
**Precautions**
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it inhibits HRP activity.
- Always prepare a standard curve and run duplicates for accuracy.
- Ensure all reagents are brought to room temperature before use.
- Discard any unsealed enzyme-labeled plate parts immediately after opening.
**Procedure Summary**
1. Prepare standards and samples according to dilution instructions.
2. Add 100 μL of standard or sample to respective wells.
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted wash buffer.
5. Add 50 μL of enzyme conjugate and incubate again.
6. Add TMB substrate, incubate for 15 minutes, then stop the reaction.
7. Measure absorbance at 450 nm within 15 minutes.
**Data Analysis**
Plot a standard curve using OD values vs. GDNF concentrations. Calculate sample concentrations using linear regression. Adjust for dilution if necessary.
**Performance**
- Intra-batch CV < 9%, Inter-batch CV < 15%
- Linear range: 2 ng/mL – 80 ng/mL
- Correlation coefficient (R²) ≥ 0.92
**Storage**
- Store at 2–8°C.
- Shelf life: 6 months from date of manufacture.
**Safety and Disposal**
All samples, reagents, and waste should be treated as biohazardous materials. Do not mix components from different batches. Follow all safety guidelines when handling chemicals.
**Note**
For optimal results, follow the protocol precisely. The English manual takes precedence in case of discrepancies.
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