The mouse type IV collagen (Col IV) ELISA kit operates on the principle of a double-antibody one-step sandwich ELISA. This method involves pre-coated microplates with capture antibodies specific to Col IV. Following sample or standard addition, a horseradish peroxidase (HRP)-labeled detection antibody is introduced. After thorough washing, the substrate TMB is added, which changes color in the presence of peroxidase activity—first turning blue and then yellow when an acidic stop solution is applied. The intensity of the color is directly proportional to the amount of Col IV in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the concentration is determined by comparing it to a standard curve.
**Sample Collection and Handling**
1. **Serum**: Collect blood in pyrogen-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Use anticoagulants like EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.
3. **Cell Supernatant**: Centrifuge at 3000 rpm for 10 minutes to remove debris before use.
4. **Tissue Homogenate**: Homogenize tissue in physiological saline, centrifuge at 3000 rpm for 10 minutes, and take the supernatant.
5. **Storage**: Store samples at -20°C in single-use aliquots. Avoid repeated freeze-thaw cycles. Thaw at room temperature before testing.
**Required Equipment**
- Microplate reader (450 nm)
- Precision pipettes: 0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL
- Incubator at 37°C
**Precautions During Operation**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- If the wash buffer crystallizes after refrigeration, gently warm it in a water bath before use.
- Seal unused plates in a ziplock bag and store them at low temperature.
- Do not dilute samples; add 10 μL directly.
- Follow the incubation time and sequence precisely.
- Shake all reagents well before use.
**Kit Components**
- Microporous plates (96-well or 48-well configurations)
- Standard solutions (1600 pg/mL)
- Diluted standards (1600, 800, 400, 200, 100, 0 pg/mL)
- Sample diluent
- HRP-labeled detection antibody
- 20× Wash buffer
- Substrate A and B
- Stop solution
- Seal film
- Ziplock bags and instructions
**Reagent Preparation**
- Dilute the 20× wash buffer 1:20 with distilled water.
**Washing Procedure**
- Manual: Fill each well with wash buffer, let sit for 1 minute, drain, and repeat 5 times.
- Automatic: Use 350 μL per well, soak for 1 minute, and wash 5 times.
**Procedure Steps**
1. Remove the required microplate from the foil pouch after 20 minutes at room temperature.
2. Set up standard, sample, and blank wells.
3. Add 50 μL of standard solutions and 10 μL of sample plus 40 μL diluent.
4. Add 50 μL of HRP-labeled antibody to each well. Seal and incubate at 37°C for 60 minutes.
5. Wash 5 times with wash buffer.
6. Add 50 μL of TMB A and B, incubate in the dark for 15 minutes.
7. Add 50 μL of stop solution and measure OD at 450 nm within 15 minutes.
**Data Analysis**
Plot the standard curve in Excel, using standard concentrations on the x-axis and OD values on the y-axis. Calculate sample concentrations based on the regression equation.
**Kit Performance**
- Accuracy: R² ≥ 0.9900
- Sensitivity: <1.0 pg/mL
- Specificity: No cross-reactivity with other proteins
- Repeatability: CV <15% between plates
- Storage: 2–8°C, protected from light and moisture
- Shelf Life: 6 months
- Detection Range: 25 pg/mL – 1600 pg/mL
**Disclaimer**
This kit is for research purposes only. Not intended for clinical use or animal experimentation. The user assumes all risks. Follow the instructions strictly. Do not mix different batch numbers. Any deviation may lead to inaccurate results.
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