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**Rat Interleukin 23 (IL-23) Enzyme-Linked Immunosorbent Assay (ELISA) Kit – User Manual**
This reagent is intended for research purposes only and is designed to quantitatively measure Rat Interleukin 23 (IL-23) levels in serum, plasma, and other biological fluids. The assay is based on the double-antibody sandwich ELISA method. A microtiter plate pre-coated with a specific anti-IL-23 monoclonal antibody is used as the solid phase. After adding the sample, IL-23 binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following washing steps, TMB substrate is added, and the reaction is stopped with an acidic solution. The color intensity, measured at 450 nm, is directly proportional to the IL-23 concentration in the sample.
The kit includes all necessary reagents and components for performing the assay, such as standard solutions, enzyme conjugate, substrates, and wash buffers. It is available in 48-well and 96-well configurations. All components should be stored at 2–8°C, except for the enzyme-labeled plate, which must be kept sealed and protected from light.
**Sample Preparation Guidelines:**
- **Serum:** Collect blood at room temperature, allow it to clot 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:** 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:** Homogenize tissue in PBS, centrifuge, and collect the supernatant.
**Important Notes:**
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it may inhibit HRP activity.
- Always prepare a standard curve and perform duplicate measurements for accuracy.
- Ensure proper calibration of pipettes and use of dedicated tips to prevent cross-contamination.
- Store unused portions of the kit in a sealed bag to maintain stability.
**Assay Procedure Summary:**
1. Prepare serial dilutions of the IL-23 standards.
2. Add samples and standards to the appropriate wells.
3. Incubate the plate at 37°C for 30 minutes.
4. Wash the plate five times with diluted wash buffer.
5. Add the HRP-conjugated antibody and incubate again.
6. Add TMB substrate and develop the color.
7. Stop the reaction with the stop solution.
8. Measure absorbance at 450 nm using a microplate reader.
**Data Analysis:**
Plot the OD values of the standards against their concentrations to create a standard curve. Use linear regression or a four-parameter logistic (4PL) model to calculate the IL-23 concentration in unknown samples. Multiply by the dilution factor if applicable.
**Kit Performance:**
- Sensitivity: 4 ng/L
- Dynamic Range: 4 ng/L – 80 ng/L
- Correlation Coefficient (R²): ≥ 0.92
- Intra-assay CV < 9%, Inter-assay CV < 15%
**Storage and Shelf Life:**
- Store the unopened kit at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
**Safety and Disposal:**
All samples, reagents, and waste materials should be handled as biohazardous waste. Follow local regulations for proper disposal.
**Note:** This manual is provided in English. In case of any discrepancies, the English version takes precedence.
This detailed guide ensures accurate and reproducible results when measuring IL-23 levels in various biological matrices. Always follow the instructions carefully and validate the results with a microplate reader.