Human pentameric 3 (PTX3) ELISA kit instruction manual
Custom Pattern Handkerchief,Affordable Mens Handkerchief,Scalloped Edge Handkerchief,Personalized Wedding Handkerchief Fashiontex Industries Ltd , https://www.fashiontexjs.com
**Human Pentameric 3 (PTX3) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual**
This ELISA kit is intended for research use only and is designed to quantitatively determine the levels of human pentameric 3 (PTX3) in serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Principle of Operation**
The kit utilizes a double-antibody sandwich ELISA method. A microtiter plate is pre-coated with a purified anti-PTX3 antibody. After adding the sample, PTX3 binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an antibody-antigen-enzyme complex. Following washing steps, TMB substrate is introduced, producing a blue color that turns yellow in the presence of an acidic stop solution. 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 results are calculated using a standard curve.
**Kit Components**
The kit includes:
- Microtiter plate (48 or 96 wells)
- Standard (6 μg/L to 0.5 μg/L)
- Enzyme-labeled reagent
- Sample diluent
- TMB substrate (A and B solutions)
- Wash buffer (concentrated, 20× or 30×)
- Sealing film and bag
- Instructions and reference data
**Storage Conditions**
- Store all components at 2–8°C.
- Unopened kit has a shelf life of 6 months.
**Sample Preparation**
1. **Serum/Plasma**: Centrifuge blood samples at 2000–3000 rpm for 20 minutes.
2. **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes.
3. **Cell Culture Supernatant**: Centrifuge after collection. For intracellular proteins, lyse cells via repeated freeze-thaw cycles.
4. **Tissue Homogenate**: Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
**Procedure Summary**
1. Prepare standards and samples by serial dilution.
2. Add samples and standards to the plate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with wash buffer.
5. Add HRP-conjugated antibody and incubate again.
6. Add TMB substrate and incubate for 15 minutes.
7. Stop the reaction with stop solution.
8. Read absorbance at 450 nm.
**Notes and Precautions**
- Allow the kit to reach room temperature before use.
- Avoid repeated freezing and thawing of samples.
- Do not use NaN3-containing samples as it inhibits HRP activity.
- Always run a standard curve and perform duplicate measurements.
- Discard any unsealed strips immediately after use.
- Handle all reagents as potential biohazards.
**Data Analysis**
Plot the standard curve using concentration vs. OD values. Use linear regression to calculate the unknown sample concentrations. Multiply by the dilution factor if applicable.
**Performance Characteristics**
- Intra- and inter-assay coefficients of variation <9% and <11%, respectively.
- Linear range: 0.3 μg/L to 7 μg/L.
- Correlation coefficient (R²) ≥ 0.95.
This manual provides detailed guidance for accurate and reliable detection of PTX3 in various biological matrices. Ensure all steps are followed precisely to obtain consistent results.