**Mouse Matrix Metalloproteinase 1 (MMP-1) ELISA Kit – Instructions for Use**
**Kit Specifications:**
Available in 48-well or 96-well configurations.
Standard Dilution: 1.5 mL × 1 vial
Enzyme Standard Reagent: 3 mL × 1 vial (48-well) / 6 mL × 1 vial (96-well)
Storage Conditions: 2–8°C
**Calculation Method:**
Plot the standard concentration on the x-axis and OD values on the y-axis to generate a standard curve. Determine the sample concentration based on the corresponding OD value from the curve, then multiply by the dilution factor. Alternatively, use linear regression analysis of the standard curve to calculate the sample concentration using its OD value, then apply the dilution factor to obtain the actual concentration.
**Kit Components:**
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 vial (2700 ng/L)
- 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 (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)
**Principle of Operation:**
The kit uses a double-antibody sandwich ELISA method to detect MMP-1 levels in samples. A microplate is coated with purified anti-MMP-1 antibodies. After adding the sample, the target antigen binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. After washing, TMB substrate is added, and the color changes from blue to yellow under HRP catalysis. The intensity of the color is directly proportional to the MMP-1 concentration in the sample. The absorbance is measured at 450 nm, and the concentration is determined via a standard curve.
**Objective:**
This kit is designed for the quantitative determination of mouse MMP-1 in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Service Commitment:**
- Delivery period: From payment to delivery
- Free technical support during working hours
- Free sample testing services available upon request to ensure optimal results
**Storage and Expiration:**
- Storage: 2–8°C
- Shelf Life: 6 months from the date of receipt
**Sample Preparation Guidelines:**
1. **Serum:** Allow blood to clot at room temperature, centrifuge at 2000–3000 rpm for 10–20 minutes. Collect the supernatant; if precipitate forms, re-centrifuge.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge for 10–20 minutes. Collect supernatant.
3. **Urine:** Collect in a sterile tube, centrifuge at 2000–3000 rpm for 20 minutes. Carefully collect the supernatant.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells by repeated freeze-thaw cycles, then centrifuge again.
5. **Tissue Samples:** Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge, and collect the supernatant. Store at 2–8°C after thawing.
6. **General:** Process samples immediately after collection. If not tested right away, store at -20°C. Avoid repeated freezing and thawing.
7. **Note:** Avoid using samples containing NaN₃, as it inhibits HRP activity.
**Procedure Steps:**
1. **Standard Dilution and Loading:** Prepare serial dilutions of the standard, loading 100 µL into the first two wells, followed by 50 µL of diluent and subsequent dilutions.
2. **Sample Loading:** Add 40 µL of sample diluent and 10 µL of sample into each test well. Ensure proper mixing.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute concentrated wash solution, then wash 5 times.
5. **Enzyme Addition:** Add 50 µL of enzyme-labeled reagent to each well except blank.
6. **Second Incubation:** Repeat the incubation step.
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 at 450 nm within 15 minutes.
**Notes:**
- Allow the kit to equilibrate at room temperature for 15–30 minutes before use.
- If the enzyme reagent is exposed, store remaining strips in a sealed bag.
- Concentrated wash solution may crystallize; warm gently if needed.
- Use a pipette for accuracy. Avoid cross-contamination by using separate sealing films.
- Always prepare a standard curve and perform duplicates for better accuracy.
- Keep all materials away from light and follow the manual strictly.
- Treat all waste as biohazardous material.
- Do not mix reagents from different batches.
- In case of conflict, the English manual takes precedence.
**Kit Performance:**
- Linear regression correlation coefficient (R²) ≥ 0.95
- Intra-batch CV < 9%, Inter-batch CV < 11%
- Detection range: 0.2 IU/L – 6 IU/L
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