**Rat Interleukin 5 (IL-5) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Manual**
This reagent is intended for research use only. The IL-5 ELISA Kit is designed to quantitatively determine the concentration of interleukin-5 (IL-5) in rat serum, plasma, cell culture supernatants, and other biological fluids.
**Principle of the Assay**
The kit utilizes a sandwich ELISA method. A microplate pre-coated with a specific monoclonal antibody against rat IL-5 serves as the solid-phase capture antibody. After incubation with the sample, IL-5 binds to the immobilized antibody. A second biotinylated detection antibody is then added, followed by horseradish peroxidase (HRP)-conjugated streptavidin. The enzyme-substrate reaction leads to a color change, which is measured spectrophotometrically at 450 nm. The intensity of the color is directly proportional to the amount of IL-5 present in the sample.
**Kit Components**
- 48-well or 96-well plate format
- Standard: 1350 pg/ml, 0.5 ml × 1 bottle
- Standard Diluent: 1.5 ml × 1 bottle
- Enzyme-labeled Antibody: 3 ml × 1 bottle
- Sample Diluent: 3 ml × 1 bottle
- TMB Substrate: 3 ml × 1 bottle
- Stop Solution: 3 ml × 1 bottle
- Washing Buffer: 20× concentrated, 20 ml × 1 bottle
- Sealing Film: 2 pieces (for 48-well), 2 pieces (for 96-well)
- Storage: 2–8°C
**Sample Preparation**
- **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes.
- **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge similarly.
- **Urine**: Collect in sterile tubes and centrifuge.
- **Cell Culture Supernatant**: Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
- **Tissue Homogenate**: Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
- **Storage**: All samples should be processed immediately if possible. If not, store at -20°C and avoid repeated freeze-thaw cycles.
**Important Notes**
- Avoid using samples containing NaN₃, as it may inhibit HRP activity.
- Always prepare a standard curve with duplicate wells.
- Ensure proper dilution if sample OD exceeds the standard range.
- Discard any unused reagents after opening.
- Keep all reagents away from light and moisture.
- Follow the manual strictly; results must be read on a microplate reader.
**Procedure Summary**
1. Prepare standards 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 washing buffer.
5. Add enzyme-labeled antibody and incubate again.
6. Add TMB substrate and develop color for 15 minutes.
7. Stop the reaction with stop solution.
8. Measure absorbance at 450 nm.
9. Calculate concentrations using the standard curve.
**Data Analysis**
Plot the standard curve using OD values vs. known concentrations. Use linear regression or software to calculate the unknown sample concentrations. Multiply by the dilution factor to obtain the actual value.
**Performance Characteristics**
- Sensitivity: 1 pg/ml
- Dynamic Range: 1–1000 pg/ml
- Intra-assay CV < 9%, Inter-assay CV < 11%
- Correlation coefficient (R²) ≥ 0.95
**Storage and Shelf Life**
- Store the kit at 2–8°C.
- Valid for 6 months from the date of manufacture.
**Safety and Disposal**
All reagents and waste materials should be handled as biohazardous waste. Do not mix reagents from different batches. This manual is written in English, and in case of discrepancies, the English version shall prevail.
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