The human type III procollagen (PCIII) ELISA kit operates on the principle of a double-antibody one-step sandwich ELISA. The process begins by coating microwells with a capture antibody specific to PCIII. Afterward, samples, standards, and HRP-labeled detection antibodies are added sequentially, followed by incubation and thorough washing steps. A TMB substrate is then used to develop color; under the action of peroxidase, TMB turns blue and eventually yellow when an acid is introduced. 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 precise quantification of PCIII levels.
**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 such as EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes to obtain plasma.
3. **Cell Supernatant:** Centrifuge at 3000 rpm for 10 minutes to remove cellular debris.
4. **Tissue Homogenate:** Homogenize tissue in physiological saline, then centrifuge at 3000 rpm for 10 minutes to collect supernatant.
5. **Storage:** If not tested immediately, aliquot and store at -20°C. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use.
**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 During Operation:**
- Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use.
- If the washing buffer crystallizes after removal from the fridge, gently warm it in a water bath before use.
- Unused strips should be returned to the ziplock bag and stored in a dry, cool place.
- No dilution is required for pre-treated samples; add 10 μL directly.
- Follow the protocol precisely, including timing, volume, and sequence.
- Shake all reagents well before use to ensure homogeneity.
**Kit Components (96-well format):**
- Microwells: 12 × 8 strips
- Standards (30 U/mL): 0.5 mL
- Standard Dilutions: 6 mL
- Sample Diluent: 6 mL
- Detection Antibody (HRP-conjugated): 6 mL
- 20× Wash Buffer: 20 mL
- Substrate A & B: 6 mL each
- Stop Solution: 6 mL
- Sealing Membranes: 2 sheets
- Ziplock Bag: 1 piece
**Reagent Preparation:**
- Dilute the 20× wash buffer 1:20 with distilled water.
**Washing Procedure:**
- Manual: Fill each well with wash buffer, let stand for 1 minute, drain, and repeat 5 times.
- Automated: Add 350 μL wash buffer, soak for 1 minute, and repeat 5 times.
**Procedure Steps:**
1. Remove the required strips from the foil pouch and allow them to equilibrate at room temperature for 20 minutes.
2. Set up standard, sample, and blank wells.
3. Add 50 μL of standard solutions to standard wells.
4. Add 10 μL of sample and 40 μL of diluent to sample wells.
5. Add 50 μL of HRP-labeled detection antibody to each well, seal, and incubate at 37°C for 60 minutes.
6. Wash the plate 5 times with wash buffer.
7. Add 50 μL of TMB A and B, incubate in the dark for 15 minutes.
8. Stop the reaction by adding 50 μL stop solution, and measure OD at 450 nm within 15 minutes.
**Data Analysis:**
Plot the standard curve using Excel, with PCIII concentrations on the x-axis and OD values on the y-axis. Use the regression equation to calculate sample concentrations.
**Kit Performance:**
- **Accuracy:** R ≥ 0.9900
- **Sensitivity:** <1.0 U/mL
- **Specificity:** No cross-reactivity with other analogs
- **Repeatability:** CV <15% between plates
- **Storage:** 2–8°C, protected from light and moisture
- **Shelf Life:** 6 months
- **Detection Range:** 0.5–30 U/mL
**Disclaimer:**
This kit is for research use only. Not suitable for clinical trials or human experimentation. The company is not responsible for any misuse. Always follow the instructions carefully. Do not mix different batch numbers. Any deviation from the protocol is the responsibility of the user.
Body Wave Wig Glueless Wig,Glueless Wig Human Hair,Ready To Ship Wear 4x4 Hd Lace
Xu Chang Zhuo YunQing crafts Co., LTD , https://www.wowqueenhair.com