**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 method to detect TIMP-2 levels in the sample. A microplate is pre-coated with a specific anti-TIMP-2 antibody. After adding the sample, TIMP-2 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex. Following a wash step, the substrate TMB is introduced. Under the catalytic action of HRP, TMB changes from blue to yellow, with the color intensity directly proportional to the concentration of TIMP-2 in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the concentration is determined by comparing the sample OD to a standard curve.
**Kit Components**
- 48-well or 96-well configuration
- Coated microplate (1×48 or 1×96)
- 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 & B (3 ml × 1 bottle each)
- Wash Buffer (20 ml × 20/30 times)
- Sealing Plate (2 pieces)
- Sealing Bag (1 piece)
**Sample Preparation**
- **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. Mix and centrifuge similarly.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freezing and thawing.
- **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C if not used immediately.
**Storage Conditions**
All components should be stored at 2–8°C. Avoid repeated freeze-thaw cycles. Do not store samples containing NaN3, as it may inhibit HRP activity.
**Procedure Summary**
1. Prepare standards by serial dilution.
2. Add samples and blanks to the microplate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted washing buffer.
5. Add enzyme-labeled reagent and incubate again.
6. Add TMB substrate and develop color for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure OD at 450 nm within 15 minutes.
**Important Notes**
- Allow the kit to equilibrate to room temperature before use.
- Ensure accurate pipetting and avoid cross-contamination.
- Always prepare a standard curve and perform duplicate measurements.
- Discard all waste as biohazardous material.
- Follow the manufacturer’s instructions carefully.
**Calculation**
Plot the standard concentrations against their corresponding OD values. Determine the sample concentration from the standard curve, adjusting for any dilution factor. The linear regression equation can also be used for more precise calculations.
**Performance**
- Correlation coefficient (R) ≥ 0.92
- Intra-assay CV < 9%, Inter-assay CV < 15%
- Detection range: 26 pg/ml – 1300 pg/ml
This kit provides a reliable and sensitive method for measuring TIMP-2 levels in biological samples, making it suitable for various research applications. Always refer to the original manual for detailed information.
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