The human type III procollagen (PCIII) ELISA kit is based on a double-antibody one-step sandwich ELISA method. The process begins with microwells pre-coated with capture antibodies specific to PCIII. After adding the sample, standards, and HRP-labeled detection antibodies sequentially, the plate is incubated, washed thoroughly, and then developed using TMB substrate. Under the catalytic action of peroxidase, TMB turns blue and eventually becomes yellow when acid is added. The intensity of the color is directly proportional to the concentration of PCIII in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, allowing for accurate quantification of PCIII levels.
**Sample Collection and Handling**
1. **Serum**: Use endotoxin-free tubes to collect blood. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.
2. **Plasma**: Collect using EDTA, citrate, or heparin as anticoagulants. Centrifuge at 3000 rpm for 30 minutes and collect the supernatant.
3. **Cell Culture 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 collect the supernatant.
5. **Storage**: If not tested immediately, aliquot samples and store at -20°C. 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)
- 37°C incubator
**Precautions**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- The washing buffer may crystallize after refrigeration; warm it gently before use.
- Unused strips should be returned to the ziplock bag and stored at low temperature.
- No dilution is needed for pre-treated samples. Add 10 μL directly.
- Follow the protocol precisely, including timing, volume, and sequence.
- Shake all reagents well before use.
**Kit Components**
| Component | 96-well Configuration | 48-well Configuration | Notes |
|-------------------------|------------------------|------------------------|-------|
| Microporous plates | 12 wells × 8 strips | 12 wells × 4 strips | |
| Standards (30 U/ml) | 0.5 mL | 0.5 mL | |
| Standard Dilutions | 6 mL | 3 mL | As per instructions |
| Sample Diluent | 6 mL | 3 mL | |
| Detection Antibody (HRP) | 6 mL | 3 mL | |
| 20× Wash Buffer | 20 mL | 20 mL | |
| Substrate A | 6 mL | 3 mL | |
| Substrate B | 6 mL | 3 mL | |
| Stop Solution | 6 mL | 3 mL | |
| Sealing Membrane | 2 sheets | 2 sheets | |
| Ziplock Bag | 1 piece | 1 piece | |
**Reagent Preparation**
- **20× Wash Buffer**: Dilute 1 part of 20× wash buffer with 19 parts of distilled water.
**Washing Procedure**
- **Manual**: Drain each well, add washing solution, let stand for 1 minute, drain, and pat dry. Repeat 5 times.
- **Automatic**: Add 350 μL washing solution per well, soak for 1 minute, and repeat 5 times.
**Assay Procedure**
1. Remove the required strips from the foil pouch after equilibrating at room temperature for 20 minutes.
2. Set up standard, sample, and blank wells. Add 50 μL of standard solutions to standard wells.
3. Add 10 μL of sample and 40 μL of diluent to sample wells.
4. Add 50 μL of HRP-labeled antibody to each well. Seal with a membrane and incubate at 37°C for 60 minutes.
5. Discard liquid, wash 5 times with washing solution.
6. Add 50 μL of TMB A and B, incubate in the dark for 15 minutes.
7. Add 50 μL stop solution and measure OD at 450 nm within 15 minutes.
**Result Analysis**
Plot standard concentrations against their corresponding OD values in Excel. Generate a standard curve and calculate sample concentrations using the regression equation.
**Kit Performance**
- **Accuracy**: R ≥ 0.9900
- **Sensitivity**: <1.0 U/mL
- **Specificity**: No cross-reactivity with other similar proteins
- **Repeatability**: CV <15%
- **Storage**: 2–8°C, protected from light and moisture
- **Shelf Life**: 6 months
- **Dynamic Range**: 0.5–30 U/mL
**Disclaimer**
This kit is for research purposes only. Not suitable for clinical trials or human experimentation. The company is not responsible for misuse. Always follow the instructions carefully. Do not mix different batch numbers. Any deviation from the protocol is the responsibility of the user.
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