**Human Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed for the quantitative determination of human tissue inhibitor of metalloproteinase-2 (TIMP-2) in human serum, plasma, and other body fluids in vitro. The shelf life of the kit is 6 months when stored properly at 2–8°C.
**Principle of the Assay**
The TIMP-2 ELISA Kit utilizes a double-antibody sandwich immunoassay method. A microtiter plate is pre-coated with a specific monoclonal antibody against TIMP-2. After adding the sample, TIMP-2 binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following washing steps, a TMB substrate is introduced, which changes color under the action of HRP. The reaction is stopped by adding an acidic solution, turning the blue color to yellow. The intensity of the color is directly proportional to the concentration of TIMP-2 in the sample. The absorbance is measured at 450 nm using a microplate reader, and the TIMP-2 concentration is determined from a standard curve.
**Kit Components**
- 48-well or 96-well configuration
- Microtiter plate coated with anti-TIMP-2 antibody
- Standard: 1800 pg/ml, 0.5 ml × 1 bottle
- Standard Diluent: 1.5 ml × 1 bottle
- Enzyme-labeled Reagent: 3 ml × 1 bottle
- Sample Diluent: 3 ml × 1 bottle
- TMB Substrate A and B: 3 ml × 1 bottle each
- Wash Buffer (20×): 20 ml × 20 times or 20 ml × 30 times
- Sealing Film: 2 pieces (for 48-well), 2 pieces (for 96-well)
- Storage: All components should be kept at 2–8°C unless otherwise stated.
**Sample Preparation and Handling**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix and centrifuge as above.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant**: Centrifuge and collect the supernatant. For intracellular components, lyse cells via freeze-thaw cycles before centrifugation.
- **Tissue Samples**: Homogenize in PBS (pH 7.4), centrifuge, and collect the supernatant. Avoid repeated freezing and thawing.
- **Storage**: If not tested immediately, store samples at -20°C. Avoid multiple freeze-thaw cycles. Note: NaN3-containing samples are not suitable due to HRP inhibition.
**Procedure Summary**
1. Prepare serial dilutions of the TIMP-2 standard.
2. Add 40 μL of sample diluent and 10 μL of 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 (except blank).
6. Incubate again at 37°C for 30 minutes.
7. Wash the plate.
8. Add 50 μL of TMB A and B, incubate for 15 minutes at 37°C.
9. Stop the reaction with 50 μL of stop solution.
10. Measure OD at 450 nm within 15 minutes.
**Notes and Precautions**
- Allow the kit to equilibrate to room temperature before use.
- Do not reuse sealing films to prevent cross-contamination.
- Avoid light exposure during TMB development.
- Always prepare a standard curve and run duplicates.
- Do not mix reagents from different batches.
- Discard all waste as biohazardous material.
- Follow instructions carefully; results must be read using a microplate reader.
**Performance Characteristics**
- Sensitivity: 26 pg/mL
- Dynamic Range: 26 pg/mL – 1300 pg/mL
- Correlation Coefficient (R²): ≥ 0.92
- Intra-assay CV < 9%, Inter-assay CV < 15%
**Data Analysis**
Plot the standard concentrations on the x-axis and corresponding OD values on the y-axis. Determine the sample concentration from the standard curve or use linear regression analysis. Multiply the result by the dilution factor to obtain the actual concentration.
**Note:** This manual is provided for reference. Always refer to the official English version for critical procedures.
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