Human pentameric 3 (PTX3) ELISA kit instruction manual
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**Human Pentameric 3 (PTX3) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Manual**
This reagent is intended for research use only. The Human Pentameric 3 (PTX3) ELISA Kit is designed to quantitatively measure PTX3 levels in human serum, plasma, and other biological fluids.
**Principle of the Assay**
The kit employs a double-antibody sandwich ELISA method. A microtiter plate is pre-coated with a specific monoclonal antibody against PTX3. After adding the sample, PTX3 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following washing steps, TMB substrate is introduced, which turns blue under HRP activity and then yellow upon acid termination. The intensity of the color is directly proportional to the PTX3 concentration in the sample. Absorbance is measured at 450 nm using a microplate reader, and the concentration is determined by comparing the sample OD values to a standard curve.
**Kit Components**
- Microtiter Plate: 1 × 48 or 1 × 96 wells
- Standard: 9 μg/L, 0.5 mL × 1 bottle
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Conjugate: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate A & B: 3 mL × 1 bottle each
- Wash Buffer (20×): 20 mL × 20 or 30 times, as per configuration
- Sealing Film: 2 pieces
- Storage: 2–8°C
**Sample Preparation Guidelines**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant, mix, and centrifuge similarly.
- **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant**: Centrifuge after collection; for intracellular components, lyse cells via freeze-thaw cycles before centrifugation.
- **Tissue Homogenate**: Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
**Procedure Summary**
1. Prepare standards by serial dilution.
2. Add samples and blanks to the plate.
3. Incubate at 37°C for 30 minutes.
4. Wash 5 times with diluted wash buffer.
5. Add HRP-conjugated antibody and incubate again.
6. Add TMB substrate, incubate for 15 minutes.
7. Stop reaction with stop solution.
8. Measure absorbance at 450 nm.
**Notes**
- Allow all reagents to equilibrate to room temperature before use.
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it inhibits HRP activity.
- Always run standards in duplicate.
- Discard sealing film after single use to prevent cross-contamination.
**Data Analysis**
Plot a standard curve using standard concentrations vs. OD values. Calculate the unknown sample concentration by interpolation or linear regression. Multiply by the dilution factor if applicable.
**Performance**
- Intra-assay CV < 9%, Inter-assay CV < 11%.
- Linear range: 0.3–7 μg/L.
- Correlation coefficient (R²) ≥ 0.95.
**Storage and Shelf Life**
- Store kit at 2–8°C.
- Shelf life: 6 months from date of manufacture.
This manual provides detailed instructions to ensure accurate and reproducible results. Always follow the protocol carefully and validate results with a microplate reader. All waste should be handled as biohazardous material.