Multi-effect growth factor (PTN) elisa technical specification
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**Multi-Effect Growth Factor (PTN) ELISA Kit Technical Specification**
**Kit Composition:**
- **48-well configuration:**
- Sealing film: 2 pieces
- Enzyme-labeled plate: 1×48
- Standard: 1 bottle of 0.5 ml (1350 pg/mL)
- Standard dilution: 1 bottle of 1.5 ml
- Enzyme standard reagent: 1 bottle of 3 ml
- Sample dilution: 1 bottle of 3 ml
- Developer A: 1 bottle of 3 ml
- Developer B: 1 bottle of 3 ml
- Stop solution: 1 bottle of 3 ml
- Concentrated washing solution (20x): 1 bottle (20 ml × 20 times)
- **96-well configuration:**
- Sealing film: 2 pieces
- Enzyme-labeled plate: 1×96
- Standard: 1 bottle of 0.5 ml (1350 pg/mL)
- Standard dilution: 1 bottle of 1.5 ml
- Enzyme standard reagent: 1 bottle of 6 ml
- Sample dilution: 1 bottle of 6 ml
- Developer A: 1 bottle of 6 ml
- Developer B: 1 bottle of 6 ml
- Stop solution: 1 bottle of 6 ml
- Concentrated washing solution (20x): 1 bottle (20 ml × 30 times)
**Storage Instructions:**
All components should be stored at 2–8°C, and the kit is stable for up to 12 months when stored properly.
**Sample Preparation and Requirements:**
1. **Serum:**
Collect blood at room temperature, allow it to clot for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant. If precipitation occurs during storage, centrifuge again before use.
2. **Plasma:**
Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. If precipitate forms, re-centrifuge.
3. **Urine:**
Collect using a sterile tube, centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant. Re-centrifuge if precipitate appears.
4. **Cell Culture Supernatant:**
For secreted components: 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, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant carefully.
5. **Tissue Specimens:**
Weigh the tissue, add PBS (pH 7.4), freeze rapidly in liquid nitrogen. After thawing, homogenize with a manual or mechanical homogenizer, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant and store the rest at 2–8°C.
6. **General Notes:**
Extract samples as soon as possible after collection. Perform the test promptly. If not used immediately, store at –20°C. Avoid repeated freezing and thawing. Do not use samples containing NaN₃, as it inhibits HRP activity.
**Operating Procedure:**
1. **Standard Dilution and Loading:**
Prepare a serial dilution of standards in 10 wells. Start with 100 μL of standard in the first two wells, mix 50 μL with diluent, then transfer 100 μL to next wells, repeating until final concentrations of 900, 600, 300, 150, and 75 pg/mL are achieved.
2. **Sample Addition:**
Add 40 μL of sample diluent to each sample well, then 10 μL of the sample (final dilution 5×). Avoid touching the well walls and gently mix.
3. **Incubation:**
Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:**
Remove the sealing film, discard liquid, wash each well 5 times with washing solution, and pat dry.
5. **Enzyme Addition:**
Add 50 μL of enzyme-labeled reagent to each well except blank wells.
6. **Second Incubation:**
Incubate at 37°C for 30 minutes.
7. **Second Washing:**
Repeat the washing procedure.
8. **Color Development:**
Add 50 μL of developer A and 50 μL of developer B to each well. Incubate at 37°C for 15 minutes in the dark.
9. **Stop Reaction:**
Add 50 μL of stop solution to each well to terminate the reaction. The color will turn yellow.
10. **Measurement:**
Measure OD values at 450 nm within 15 minutes of adding the stop solution. Use a blank well for zero adjustment.
This detailed protocol ensures accurate and reproducible results for PTN detection using the ELISA method. Always follow the manufacturer’s instructions and handle all reagents with care.