Human interleukin-15 (IL-15) ELISA test kit instruction manual

**Human Interleukin-15 (IL-15) ELISA Kit – Instructions for Use** This kit is intended for research purposes only and is designed to quantitatively determine the levels of Human Interleukin-15 (IL-15) in human serum, plasma, and other biological fluids. The assay is based on the sandwich ELISA method, which involves the use of two specific antibodies to capture and detect IL-15 in the sample. The procedure begins by coating a microtiter plate with a purified anti-IL-15 antibody. After incubation, the sample is added, allowing IL-15 to bind to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody then binds to the captured IL-15, forming a complex. Following a washing step, a TMB substrate is added, which changes color in the presence of HRP. The reaction is stopped, and the absorbance is measured at 450 nm using a microplate reader. The intensity of the color is directly proportional to the concentration of IL-15 in the sample. **Kit Components:** - Microtiter Plate (coated with anti-IL-15 antibody) - Standard (IL-15) - Enzyme-labeled antibody - Sample Diluent - TMB Substrate (A and B solutions) - Stop Solution - Washing Buffer (concentrated, 20x) - Sealing Film - Storage: 2–8°C **Sample Preparation Guidelines:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant. - **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix and centrifuge as above. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. - **Cell Culture Supernatant:** Centrifuge to remove debris. For intracellular components, lyse cells via freeze-thaw cycles and centrifuge again. - **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant. Store at -20°C if not used immediately. **Important Notes:** - Avoid repeated freezing and thawing of samples. - Do not use samples containing NaN₃, as it inhibits HRP activity. - Always prepare a standard curve with duplicate wells. If the sample OD exceeds the highest standard, dilute the sample before testing. - Ensure all reagents are equilibrated to room temperature before use. - Keep substrates away from light. - Follow all safety protocols when handling biological materials. **Procedure Summary:** 1. Prepare standards and samples. 2. Add samples and standards to the plate. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate 5 times with diluted 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 OD at 450 nm. 9. Calculate IL-15 concentration using the standard curve. **Performance Characteristics:** - Sensitivity: 0.5 ng/L - Dynamic Range: 0.5–10 ng/L - Intra- and inter-assay CV < 9% and < 11%, respectively - Correlation coefficient (R²) ≥ 0.95 **Storage and Shelf Life:** - Store all components at 2–8°C. - The kit is valid for 6 months from the date of manufacture. This ELISA kit provides a reliable and sensitive method for measuring IL-15 levels in various biological matrices. Proper execution of the protocol ensures accurate and reproducible results. Always refer to the latest version of the manual for any updates or additional instructions.

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