Multi-effect growth factor (PTN) elisa technical specification
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**Multi-Effect Growth Factor (PTN) ELISA Technical Specification Kit**
**Kit Composition:**
- **48-well configuration:**
- 1×48 coated plate
- 2 pieces of sealing film (for 48 wells)
- 1 sealed bag
- 1×1 enzyme-labeled plate
- 0.5 ml × 1 bottle of standard solution
- 1.5 ml × 1 bottle of standard dilution
- 3 ml × 1 bottle of sample dilution
- 3 ml × 1 bottle of developer A
- 3 ml × 1 bottle of developer B
- 3 ml × 1 bottle of stop solution
- 20 ml × 20 times concentrated wash solution
- **96-well configuration:**
- 1×96 coated plate
- 2 pieces of sealing film (for 96 wells)
- 1 sealed bag
- 1×1 enzyme-labeled plate
- 0.5 ml × 1 bottle of standard solution
- 1.5 ml × 1 bottle of standard dilution
- 3 ml × 1 bottle of sample dilution
- 3 ml × 1 bottle of developer A
- 3 ml × 1 bottle of developer B
- 3 ml × 1 bottle of stop solution
- 20 ml × 30 times concentrated wash solution
**Storage Instructions:**
All components should be stored at 2–8°C. The standard solution and reagents must be protected from light and kept in a cool, dry place.
**Sample Preparation and Requirements:**
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 precipitation occurs during storage, re-centrifuge before use.
2. **Plasma:**
Use EDTA or sodium citrate as anticoagulant. Mix thoroughly for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. Re-centrifuge if precipitate forms.
3. **Urine:**
Collect using a sterile tube, then centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant. Re-centrifuge if needed.
4. **Cell Culture Supernatant:**
For secreted components, collect in a sterile tube and centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, dilute cell suspension with PBS (pH 7.2–7.4) to ~1 million cells/ml. Freeze-thaw repeatedly to release components, then centrifuge again. Collect the supernatant carefully.
5. **Tissue Specimen:**
Weigh the tissue, add PBS (pH 7.4), freeze rapidly in liquid nitrogen, and store at 2–8°C after thawing. Homogenize with a homogenizer, centrifuge at 2000–3000 rpm for 20 minutes, and collect the supernatant. Only test one portion; store the rest at -20°C.
6. **General Sample Handling:**
Process samples as soon as possible after collection. If not tested immediately, store at -20°C. Avoid repeated freezing and thawing. Do not use samples containing NaN3, as it may inhibit HRP activity.
**Procedure:**
1. **Standard Dilution and Loading:**
Prepare a serial dilution of the standard solution in 10 wells. Start with 100 µL of standard in the first two wells, mix 50 µL with 50 µL diluent, and continue diluting across the remaining wells. Final concentrations: 900 pg/mL, 600 pg/mL, 300 pg/mL, 150 pg/mL, 75 pg/mL.
2. **Sample Addition:**
Add 40 µL of sample diluent and 10 µL of sample to each test well. Avoid touching the walls of the microplate. Gently mix.
3. **Incubation:**
Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:**
Remove the seal, discard the liquid, and wash each well 5 times with washing solution. Pat dry.
5. **Enzyme Addition:**
Add 50 µL of enzyme-labeled reagent to each well except blank controls.
6. **Second Incubation:**
Incubate at 37°C for another 30 minutes.
7. **Second Washing:**
Repeat the washing steps as above.
8. **Color Development:**
Add 50 µL of developer A and 50 µL of developer B to each well. Mix gently and incubate at 37°C for 15 minutes, away from direct light.
9. **Stop Reaction:**
Add 50 µL of stop solution to each well to terminate the reaction. The color will turn yellow.
10. **Measurement:**
Measure absorbance at 450 nm within 15 minutes of adding the stop solution. Use a blank well for zero adjustment.
This kit is designed for high sensitivity and specificity in detecting PTN levels. Follow all instructions carefully to ensure accurate results.