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**Human Interleukin-15 (IL-15) ELISA Kit – Instructions for Use**
This ELISA kit is intended for research use only and is designed to quantitatively determine the levels of human interleukin-15 (IL-15) in various biological samples, including serum, plasma, urine, cell culture supernatants, and other body fluids. The method employed is a sandwich ELISA technique, ensuring high specificity and sensitivity.
**Principle of the Assay:**
The assay utilizes a two-step binding process involving a solid-phase antibody and a detection antibody labeled with horseradish peroxidase (HRP). The microplate is pre-coated with a specific anti-IL-15 monoclonal antibody. After incubation with the sample, IL-15 binds to the immobilized antibody. A second HRP-conjugated antibody then binds to the captured IL-15, forming a complex. Following washing steps, a TMB substrate is added, producing a color change that is proportional to the IL-15 concentration. The reaction is stopped with an acidic solution, and the absorbance is measured at 450 nm using a microplate reader.
**Kit Components (96-well format):**
- 1 × 96-well plate (pre-coated with anti-IL-15 antibody)
- 2 sealing films
- 1 standard (13.5 ng/L)
- 1 standard diluent (1.5 mL)
- 1 enzyme-labeled reagent (3 mL)
- 1 sample diluent (3 mL)
- 1 developer A (3 mL)
- 1 developer B (3 mL)
- 1 stop solution (3 mL)
- 1 concentrated wash buffer (20×, 20 mL × 30)
- 1 manual (English)
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of manufacture when stored properly.
**Sample Preparation Guidelines:**
- **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 as above.
- **Urine:** Collect in a sterile container and centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes after collection.
- **Tissue Homogenate:** Weigh tissue, homogenize in PBS, centrifuge, and collect supernatant.
**Important Notes:**
- Avoid repeated freeze-thaw cycles.
- Do not use samples containing NaN₃, as it may inhibit HRP activity.
- Always prepare a standard curve and run samples in duplicate.
- Keep all reagents away from light and handle with care.
- Follow all safety procedures when handling biological materials.
**Procedure Summary:**
1. Prepare serial dilutions of the standard.
2. Add samples and standards to the microplate.
3. Incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted 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.
9. Calculate concentrations using the standard curve.
**Data Interpretation:**
Plot the standard curve with IL-15 concentrations on the x-axis and OD values on the y-axis. Use linear regression to calculate unknown sample concentrations, adjusting for any dilution factors. Ensure accuracy by validating results with multiple replicates.
**Quality Control:**
The kit provides a linear range between 0.5 ng/L and 10 ng/L, with a correlation coefficient (R²) of ≥0.95. Intra- and inter-assay variations are less than 9% and 11%, respectively.
Always refer to the most recent version of the manual for detailed instructions and troubleshooting tips.