**Rat Interleukin 5 (IL-5) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual**
This reagent is intended for research purposes only and not for human diagnostic use. The kit is designed to quantitatively measure the levels of Interleukin-5 (IL-5) in various biological samples, including rat serum, plasma, cell culture supernatants, and other similar liquid specimens.
**Principle of the Assay**
The IL-5 ELISA kit utilizes a sandwich immunoassay method. A microtiter plate is pre-coated with a specific monoclonal antibody against rat IL-5. After incubation with the sample, the IL-5 present in the sample 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 color development, which is proportional to the concentration of IL-5 in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the concentration is determined by comparing the OD values to a standard curve.
**Kit Components**
- Microtiter Plate: 1×48 or 1×96 wells
- Standard: 0.5 ml × 1 vial (1350 pg/ml)
- Standard Diluent: 1.5 ml × 1 vial
- Enzyme Conjugate: 3 ml × 1 vial
- TMB Substrate: 3 ml × 1 vial
- Stop Solution: 3 ml × 1 vial
- Wash Buffer (20×): 20 ml × 20 times or 20 ml × 30 times
- Sealing Film: 2 pieces
- Storage: 2–8°C
**Sample Preparation and Handling**
Proper sample handling is crucial for accurate results.
- **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 a sterile container and centrifuge.
- **Cell Culture Supernatant**: Centrifuge after collection. For intracellular components, lyse cells via repeated freeze-thaw cycles before centrifugation.
- **Tissue Homogenate**: Weigh the tissue, homogenize in PBS, and centrifuge. Store remaining samples at –20°C if not used immediately.
**Assay Procedure**
1. **Standard Dilution**: Prepare serial dilutions of the IL-5 standard on the microtiter plate.
2. **Sample Addition**: Add 40 μl of sample diluent and 10 μl of sample to each well.
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing**: Wash the plate five times with wash buffer.
5. **Enzyme Conjugate**: Add 50 μl of HRP-labeled antibody to each well.
6. **Second Incubation**: Incubate again at 37°C for 30 minutes.
7. **Color Development**: Add 50 μl of TMB A and B solutions, incubate for 15 minutes.
8. **Stop Reaction**: Add 50 μl of stop solution to each well.
9. **Reading**: Measure OD at 450 nm within 15 minutes of stopping the reaction.
**Notes and Recommendations**
- Allow the kit to equilibrate to room temperature before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Ensure all reagents are properly mixed and handled.
- Always prepare a standard curve and run samples in duplicate.
- If sample OD exceeds the highest standard, perform a dilution before testing.
- Do not use NaN3-containing samples, as it may inhibit HRP activity.
- All waste should be treated as biohazardous material.
**Data Analysis**
Plot the standard curve using the OD values vs. IL-5 concentrations. Calculate the sample concentration using linear regression or by interpolating from the standard curve. Multiply the result by the dilution factor if applicable.
**Performance Characteristics**
- Sensitivity: 1 pg/ml
- Dynamic Range: 1 pg/ml – 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.
- Shelf life: 6 months from the date of manufacture.
This manual provides detailed instructions for the accurate and reliable measurement of IL-5 in rat samples. Following these guidelines ensures consistent and reproducible results.
Microfiber dish drying mats are typically more absorbent than cotton and dry very quickly, making them a great option if you're looking for drying mats and palcemat to drying the water in any place. The microfiber fabric use for mats are super absorbent,thick and have a very short drying time.You can have many choices on different color and style.
Microfiber dish mat,microfiber drainer mats,microfiber mat for dish,microfiber dish drainer
jiangsu qiyun cleaning knitting product co.,ltd , https://www.jarfrry.com