**Human Matrix Metalloproteinase-2 (MMP-2) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Instructions**
This ELISA kit is intended for **research use only**. It is designed to quantitatively measure **Matrix Metalloproteinase-2 (MMP-2)** in **human serum, plasma, and related body fluids**. The assay employs a **double-antibody sandwich method**, ensuring high specificity and sensitivity.
**Principle of the Assay:**
The microtiter plate is pre-coated with a specific antibody against human MMP-2. After adding the sample, the target MMP-2 binds to the immobilized antibody. A secondary HRP-conjugated antibody then recognizes the captured MMP-2, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, which turns blue under HRP catalysis and changes to yellow when an acid stop solution is introduced. The intensity of the color is directly proportional to the concentration of MMP-2 in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the concentration is determined from a standard curve.
**Kit Components (48-well & 96-well configurations):**
- Microtiter plate (coated with anti-MMP-2 antibody)
- Standard: 13.5 μg/L, 0.5 mL × 1 bottle
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Reagent: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate A & B: 3 mL × 1 bottle each
- Wash Buffer (20×): 20 mL × 20/30 times × 1 bottle
- Sealing Film: 2 pieces
- Storage: 2–8°C
**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 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 before centrifugation.
- **Tissue Homogenate**: Weigh the tissue, homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
- **Storage**: Samples should be processed promptly. If not tested immediately, store at -20°C. Avoid repeated freezing and thawing. Note: Samples containing NaN3 are not suitable due to HRP inhibition.
**Procedure Summary:**
1. Prepare standards by serial dilution (final concentrations: 9, 6, 3, 1.5, 0.75 μg/L).
2. Add 40 μL sample diluent and 10 μL sample to each test well.
3. Seal and incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted wash buffer.
5. Add 50 μL enzyme reagent to all wells except blanks.
6. Incubate again at 37°C for 30 minutes.
7. Wash again and add 50 μL each of TMB A and B. Incubate for 15 minutes at 37°C.
8. Stop the reaction with 50 μL stop solution.
9. Measure OD at 450 nm within 15 minutes.
**Important Notes:**
- Allow the kit to equilibrate at room temperature before use.
- Store unopened strips in a sealed bag to prevent moisture.
- Use separate pipettes for each step to avoid cross-contamination.
- Always include a blank control and run duplicates for accuracy.
- If sample OD exceeds the highest standard, perform a preliminary dilution.
- Do not mix reagents from different batches.
- Keep TMB away from light.
- Follow the manual strictly; results must be confirmed with a microplate reader.
- All waste should be treated as biohazardous material.
**Calculation:**
Plot the standard curve using standard concentrations vs. OD values. Determine the unknown sample concentration by interpolation or linear regression. Multiply by the dilution factor if applicable.
**Performance Characteristics:**
- Linear range: 0.3–10 μg/L
- Correlation coefficient (R²): ≥ 0.92
- Intra-batch CV < 9%, Inter-batch CV < 15%
**Storage & Shelf Life:**
- Store at 2–8°C.
- Valid for 6 months from the date of manufacture.
**Note:** This guide is intended for reference only. Always refer to the official instruction manual provided with the kit for full details.
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