**Rabbit Soluble Vascular Endothelial Cell Protein C Receptor (sEPCR) ELISA Kit – User Manual**
**Kit Specifications:**
This ELISA kit is available in 48-well or 96-well configurations. The standard dilution is 1.5 mL × 1 bottle. Enzyme standard reagent: 3 mL × 1 bottle (for 48 wells) or 6 mL × 1 bottle (for 96 wells). This product is for research use only.
**Standard Curve Preparation:**
Plot the standard concentration on the x-axis and OD values on the y-axis. Draw a standard curve, then determine the sample concentration based on its OD value. Multiply by the dilution factor, or calculate the linear regression equation using standard concentrations and OD values, then substitute the sample’s OD into the equation to obtain the actual concentration.
**Kit Components:**
- Sealing plate: 2 pieces (48) / 2 pieces (96)
- Standard: 0.5 mL × 1 bottle (2700 ng/L)
- Enzyme-labeled plate: 1 × 48 / 1 × 96
- Sample diluent: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- TMB A: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- TMB B: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Wash buffer: 3 mL × 1 bottle (48) / 6 mL × 1 bottle (96)
- Concentrated wash solution: 20 mL × 20 times (48) / 20 mL × 30 times (96)
**Storage Conditions & Shelf Life:**
Store at 2–8°C. Valid for 6 months from the date of manufacture.
**Purpose:**
The kit is designed to measure soluble vascular endothelial cell protein C receptor (sEPCR) levels in rabbit serum, plasma, and other biological fluids.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant, mix, centrifuge, and collect supernatant.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell culture supernatant:** Centrifuge, collect supernatant. For intracellular components, lyse cells via freezing/thawing and centrifuge again.
- **Tissue:** Homogenize in PBS, centrifuge, and collect supernatant.
- **Storage:** Avoid repeated freeze-thaw cycles. Store at -20°C if not used immediately. Do not use samples containing NaN3 due to HRP inhibition.
**Operating Procedures:**
1. **Standard Dilution:** Prepare a serial dilution series in 10 wells.
2. **Sample Loading:** Add 40 μL of sample diluent, then 10 μL of sample (final dilution 5×).
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute concentrated wash solution, wash 5 times.
5. **Enzyme Addition:** Add 50 μL of enzyme reagent to each well (except blank).
6. **Second Incubation:** Repeat incubation 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.
9. **Measurement:** Read OD at 450 nm within 15 minutes.
**Notes:**
- Equilibrate the kit at room temperature before use.
- If unsealed, store strips in a sealed bag.
- Wash solution may crystallize; dissolve in a water bath if needed.
- Use accurate pipettes, avoid cross-contamination.
- Prepare a standard curve with duplicates.
- Keep substrates away from light.
- Follow instructions strictly; results must be confirmed with a microplate reader.
- Treat all waste as biohazardous material.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Performance:**
- Linear correlation coefficient (R) ≥ 0.95.
- Intra-batch and inter-batch variation < 9% and < 11%, respectively.
- Detection range: 0.2 IU/L – 6 IU/L.
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