**Human Platelet Factor 4 (PF-4/CXCL4) ELISA Kit Instruction Manual**
**Kit Specifications:**
The Human PF-4/CXCL4 ELISA Kit is available in either a 48-well or 96-well configuration. The standard dilution is 1.5 mL × 1 vial, and the enzyme standard reagent is 3 mL × 1 vial (for 48-well) or 6 mL × 1 vial (for 96-well). This kit is intended for **research use only**.
**Standard Curve Preparation:**
To determine the concentration of PF-4/CXCL4 in the sample, prepare a standard curve by plotting the standard concentrations (on the x-axis) against the corresponding OD values (on the y-axis). Alternatively, calculate the linear regression equation using the standard concentrations and their OD values. Then, input the OD value of the sample into this equation to obtain the calculated concentration, which should be multiplied by the dilution factor to get the actual sample concentration.
**Kit Components:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 2700 ng/L, 0.5 mL × 1 vial
- Enzyme standard: 1×48 / 1×96
- Sample diluent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
- Developer A: 3 mL × 1 vial / 6 mL × 1 vial
- Chromogen B: 3 mL × 1 vial / 6 mL × 1 vial
- Wash buffer: 3 mL × 1 vial / 6 mL × 1 vial
- Concentrated wash solution: 20 mL × 20 times / 20 mL × 30 times
**Storage Conditions:**
All components should be stored at 2–8°C. The shelf life of the kit is 6 months from the date of receipt.
**Principle of the Assay:**
This ELISA kit uses the **double-antibody sandwich method** to detect human PF-4/CXCL4. The microplate is pre-coated with a specific antibody against PF-4/CXCL4. After adding the sample, the target antigen binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, leading to a color change that correlates with the amount of PF-4/CXCL4 present. The reaction is stopped, and the absorbance is measured at 450 nm.
**Purpose:**
This kit is designed for the quantitative determination of PF-4/CXCL4 in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature, centrifuge at 2000–3000 rpm for 10–20 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 after collection; for intracellular components, lyse cells via freeze-thaw cycles.
- **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
**Important Notes:**
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it may inhibit HRP activity.
- Always prepare a standard curve and run samples in duplicate.
- Keep all reagents away from light and handle with care.
- Follow the manual strictly to ensure accurate results.
- Discard all waste materials as biohazardous.
**Performance Characteristics:**
- Linear range: 0.2 IU/L – 6 IU/L
- Correlation coefficient (R²): ≥ 0.95
- Intra-assay and inter-assay variability: < 9% and < 11%, respectively
**Service Commitment:**
- Free technical support during working hours.
- Sample testing services available upon request.
- Delivery within the agreed period.
**Operation Steps:**
1. Dilute standards and load them into the plate.
2. Add sample diluent and test samples.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash solution.
5. Add enzyme-labeled reagent and incubate again.
6. Add TMB substrate and incubate for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure OD at 450 nm within 15 minutes.
**Additional Tips:**
- Allow the kit to equilibrate to room temperature before use.
- Ensure proper pipetting accuracy and avoid cross-contamination.
- Use fresh solutions and follow the instructions carefully.
This kit provides a reliable and efficient method for measuring PF-4/CXCL4 levels, supporting research in immunology, inflammation, and related fields.
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