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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 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Biological Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.**
This kit is designed for the quantitative measurement of Canine TIMP-1 levels in various biological samples. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, using a competitive binding format. The Stop Solution changes the color from blue to yellow, allowing the operator to generate a standard curve by plotting Optical Density (OD) values against known TIMP-1 concentrations. The sample concentration is then determined by comparing the OD readings to this standard curve.
**Sample Collection and Storage:**
- **Serum:** Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifugation at ~2000×g for 20 minutes. Remove serum and analyze immediately or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and analyze immediately or store at -20°C. Ensure no hemolysis or granules are present.
**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 (Stored at 2–8°C):**
- 96-well microplate (12×8 strips)
- 6 standards (0.5 ml/vial)
- Sample diluent (6.0 ml)
- HRP-conjugated reagent (10.0 ml)
- 20X Wash solution (25 ml)
- Chromogen A (6.0 ml)
- Chromogen B (6.0 ml)
- Stop solution (6.0 ml)
- Closure plate membrane (2 pieces)
- User manual and sealed bags
**Note:** Standard concentrations are 80, 40, 20, 10, 5, and 2.5 ng/mL. If sample values exceed the highest standard, dilute with sample diluent and repeat the test.
**Precautions:**
- Do not mix reagents from different kit lots.
- All reagents must be brought to room temperature (20–25°C) before use.
- Do not use reagents past their expiration date.
- Use only deionized or distilled water for dilution.
- Keep unused strips in the storage bag with desiccant.
- Use fresh pipette tips for each transfer to prevent contamination.
- Handle all blood-derived samples as potentially infectious.
- Dispose of waste after 30 minutes of inactivation.
**Reagent Preparation:**
- Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents before starting the assay. Run standards and samples in duplicate.
2. Add 50 μL of standard or sample to appropriate wells. Blank well receives no addition.
3. Add 100 μL of HRP-conjugate reagent to all wells except the blank. Cover and incubate at 37°C for 60 minutes.
4. Wash the plate 4 times manually or automatically. Ensure complete removal of liquid.
5. Add 50 μL of Chromogen A and 50 μL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 μL of Stop Solution to each well. The color should turn from blue to yellow. If green or uneven, gently tap the plate.
7. Measure OD at 450 nm. Generate a standard curve by plotting average OD values against concentrations.
**Interpretation:**
- Calculate mean OD for each standard and sample. Subtract blank OD before interpretation.
- Locate the sample OD on the Y-axis and draw a line to the standard curve. Read the corresponding concentration on the X-axis.
- Each user should create their own standard curve. Intra-assay and inter-assay CV% are <15%.
**Performance Characteristics:**
- Assay range: 2.5 ng/mL – 80 ng/mL
- Sensitivity: <1.0 ng/mL
- Cross-reactivity: No significant cross-reactivity observed
- Storage: 2–8°C (frequent use); 6 months at -20°C
**Important:** This kit is intended for research purposes only. Do not use for diagnostic procedures. Always follow safety protocols when handling biological samples.