**Rat Monocyte Chemoattractant Protein 1 (MCP-1/CCL2/MCAF) ELISA Kit – Instructions for Use**
**Kit Specifications:**
This kit is available in 48-well or 96-well configurations. It includes:
- Standard dilution: 1.5 ml × 1 bottle
- Enzyme standard reagent: 3 ml × 1 bottle (for 48 wells) / 6 ml × 1 bottle (for 96 wells)
- The kit is intended for research use only.
**Principle of the Assay:**
The Rat MCP-1 ELISA Kit uses a double-antibody sandwich method to quantify MCP-1 levels in biological samples. A monoclonal antibody specific to MCP-1 is pre-coated on a microplate. After incubation with the sample, a biotinylated detection antibody binds to the captured MCP-1, followed by HRP-conjugated streptavidin. TMB substrate is added, and the reaction is stopped with an acidic solution. The color intensity, measured at 450 nm, correlates with the concentration of MCP-1 in the sample.
**Standard Curve Preparation:**
Plot the OD values (y-axis) against the known concentrations of the standard (x-axis). Determine the sample concentration using either the standard curve or linear regression equation. Multiply the calculated value by the dilution factor to obtain the actual sample concentration.
**Kit Components:**
- Sealing film: 2 pieces (48 wells) / 2 pieces (96 wells)
- Standard: 0.5 ml × 1 bottle (2700 ng/L)
- Enzyme standard: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Sample diluent: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Developer A: 3 ml × 1 bottle (48) / 6 ml × 1 bottle (96)
- Developer 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:**
- Store all components at 2–8°C.
- Shelf life: 6 months from the date of receipt.
**Sample Preparation Guidelines:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing for 10–20 minutes.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell culture supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freezing-thawing.
- **Tissue:** Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
**Notes for Optimal Performance:**
- Allow the kit to equilibrate to room temperature for 15–30 minutes before use.
- Avoid repeated freeze-thaw cycles.
- Do not mix reagents from different batches.
- Keep the substrate away from light.
- Always prepare a standard curve and perform duplicate measurements for accuracy.
- If sample OD exceeds the highest standard, dilute the sample and recalculate accordingly.
- Discard all waste as biohazardous material.
**Testing Procedure:**
1. Prepare standards and samples according to instructions.
2. Add 100 μL of standard or sample to each well.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add 50 μL of enzyme-labeled reagent to each well.
6. Incubate again for 30 minutes.
7. Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. Stop the reaction with 50 μL stop solution.
9. Measure absorbance at 450 nm within 15 minutes.
**Performance Characteristics:**
- Sensitivity: 0.2 IU/L – 6 IU/L
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Correlation coefficient (R²) ≥ 0.95
**Technical Support:**
Free technical support is available during working hours. We also offer free sample testing services to help you achieve optimal results.
**Important:** This kit is for research purposes only and should not be used for diagnostic or clinical applications.
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