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Canine TIMP-1 ELISA Kit
**Canine TIMP-1 ELISA Kit – For the Quantitative In Vitro Determination of Canine Tissue Inhibitors of Metalloproteinase 1 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
This ELISA kit is designed for the quantitative measurement of Canine TIMP-1 concentrations in various biological samples. The test is based on the enzyme-linked immunosorbent assay (ELISA) principle, which allows for precise detection of TIMP-1 levels through a colorimetric reaction.
The procedure involves incubating the sample with specific antibodies, followed by the addition of a horseradish peroxidase (HRP)-conjugated secondary antibody. A chromogenic substrate is then added, producing a blue color that turns yellow upon the addition of the stop solution. The optical density (OD) is measured at 450 nm, and the concentration of TIMP-1 in the samples is determined by comparing the OD values to a standard curve generated from known concentrations.
**Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C. Centrifuge at ~2000×g for 20 minutes. Store at -20°C after aliquoting. Avoid repeated freeze-thaw cycles.
- **Plasma**: Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g (2–8°C). Store at -20°C.
- **Cell culture supernatants, tissue homogenates, and other fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination occurs.
**Materials Required but Not Supplied**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**Reagents Provided**
- Microtiter strip plates (12×8 or 12×4 strips)
- Standards (6 vials, 0.5 ml/vial)
- Sample diluent (6.0 ml or 3.0 ml)
- HRP-Conjugate reagent (10.0 ml or 5.0 ml)
- 20X Wash solution (25 ml or 15 ml)
- Chromogen solutions A & B (6.0 ml each)
- Stop solution (6.0 ml)
- Closure plate membranes (2 units)
- User manual and sealed bags
**Precautions**
- Do not mix reagents from different kits.
- Allow all reagents to reach room temperature before use.
- Never use expired reagents.
- Always use deionized or distilled water for dilution.
- Handle plasma as potentially infectious. Wear gloves during the procedure.
- Dispose of waste properly after inactivation for at least 30 minutes.
- Avoid contamination; use fresh pipette tips for each transfer.
- Keep substrate solutions away from heat and light.
**Reagent Preparation**
- Dilute 1 volume of 20X Wash Solution with 19 volumes of water to make 1X solution. Store at 2–8°C for up to one month.
**Assay Procedure**
1. Prepare all reagents before starting. Add standards and samples in duplicate.
2. Add 50 µl of standard or sample to the wells. Leave blank well without any addition.
3. Add 100 µl of HRP-conjugate reagent to all wells except the blank. Incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically.
5. Add 50 µl of chromogen A and B to each well. Incubate for 15 minutes at 37°C.
6. Add 50 µl of stop solution. The color should turn from blue to yellow. Gently mix if necessary.
7. Measure OD at 450 nm. Generate a standard curve and calculate sample concentrations accordingly.
**Performance Characteristics**
- Sensitivity: <1.0 ng/mL
- Dynamic range: 2.5–80 ng/mL
- Intra-assay and inter-assay CV <15%
- Cross-reactivity: No significant interference observed
**Storage**
- Store at 2–8°C for frequent use; at -20°C for long-term storage (up to 6 months).
**Note:** Always follow proper safety protocols and ensure accurate results by generating your own standard curve. This kit is intended for research purposes only.
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