**Human Platelet Factor 4 (PF-4/CXCL4) ELISA Kit Instruction Manual**
**Kit Specifications:**
The Human Platelet Factor 4 (PF-4/CXCL4) ELISA Kit is available in either a 48-well or 96-well configuration. The kit includes:
- Standard Diluent: 1.5 mL × 1 vial
- Enzyme Standard Reagent: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Sealing Film: 2 pieces (48-well), 2 pieces (96-well)
- Standard: 0.5 mL × 1 vial, 2700 ng/L
- Sample Diluent: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Developer A: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Chromogen B: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Wash Solution: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Concentrated Wash Solution: 20 mL × 20 times (48-well), 20 mL × 30 times (96-well)
**Storage Conditions & Expiration:**
- Kit storage: 2–8°C
- Shelf life: 6 months from the date of manufacture
**Purpose:**
This ELISA kit is designed for the quantitative determination of human platelet factor 4 (PF-4/CXCL4) in serum, plasma, urine, cell culture supernatants, and other biological fluids. It is intended for research use only.
**Principle of Operation:**
The kit uses a double-antibody sandwich ELISA method. Purified anti-PF-4 antibodies are coated on the microplate wells. After incubation with the sample, PF-4 binds to the immobilized antibody. An HRP-conjugated secondary antibody then binds to the captured PF-4, forming an immune complex. TMB substrate is added, and the color develops under the catalytic action of HRP. The reaction is stopped, and the absorbance is measured at 450 nm. A standard curve is plotted using known concentrations, and the sample concentration is determined accordingly.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature, centrifuge at 2000–3000 rpm for 10–20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well, centrifuge, and collect supernatant.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Cell Culture Supernatant:** Centrifuge to remove debris. For intracellular components, lyse cells by freezing/thawing and centrifuge again.
- **Tissue:** Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C after thawing.
**Operation Steps:**
1. **Standard Dilution:** Prepare a serial dilution of the standard solution.
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample into each well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Rinse the plate 5 times with wash buffer.
5. **Enzyme Addition:** Add 50 μL of enzyme-labeled reagent to each well.
6. **Second Incubation:** Incubate at 37°C for 30 minutes.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to each well.
9. **Measurement:** Read OD values at 450 nm within 15 minutes.
**Notes for Use:**
- Equilibrate the kit to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each test.
- Ensure accurate pipetting and avoid excessive shaking during mixing.
- Always run a standard curve and consider sample dilution if OD values exceed the standard range.
- Do not mix reagents from different batches.
- Keep substrates away from light.
**Performance Characteristics:**
- Linear regression correlation coefficient (R²) ≥ 0.95
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
**Technical Support:**
Free technical support is available during working hours. We also offer free sample testing services to help ensure optimal experimental results.
**Safety & Disposal:**
All samples, washes, and waste materials should be treated as biohazardous. Follow local regulations for disposal.
This manual provides detailed instructions for accurate and reliable detection of PF-4/CXCL4 levels in various biological samples. Proper handling and adherence to protocol are essential for obtaining consistent and reproducible results.
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