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**Human Interleukin-15 (IL-15) ELISA Kit – Instructions for Use**
This kit is intended for research use only and is designed to quantitatively determine the concentration of Human Interleukin-15 (IL-15) in human serum, plasma, urine, cell culture supernatants, and other biological fluids.
**Principle of the Assay**
The IL-15 ELISA uses a sandwich immunoassay format. A microtiter plate is pre-coated with a specific monoclonal antibody against IL-15. After adding the sample, IL-15 binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. The plate is washed to remove unbound reagents, followed by incubation with a TMB substrate. The color develops in proportion to the amount of IL-15 present. The reaction is stopped with a stop solution, and the absorbance is measured at 450 nm using a microplate reader. The concentration of IL-15 in the sample is determined by comparing the OD value to a standard curve.
**Kit Components**
- Microtiter Plate: 1 × 48 or 1 × 96 wells
- Standard: 13.5 ng/L (0.5 mL × 1 bottle)
- Standard Diluent: 1.5 mL × 1 bottle
- Enzyme Conjugate: 3 mL × 1 bottle
- Sample Diluent: 3 mL × 1 bottle
- TMB Substrate A & B: 3 mL × 1 bottle each
- Wash Buffer (20x): 20 mL × 20 or 30 times
- Sealing Film: 2 pieces (for 48-well), 2 pieces (for 96-well)
- Storage: 2–8°C
**Sample Preparation**
- **Serum**: Collect blood, allow clotting at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
- **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 at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles before centrifuging.
- **Tissue**: Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
**Procedure Summary**
1. Prepare standard dilutions in a serial manner.
2. Add 40 μL of sample diluent and 10 μL of sample to each well.
3. Seal the plate and incubate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash buffer.
5. Add 50 μL of enzyme conjugate to each well (except blank).
6. Incubate again at 37°C for 30 minutes.
7. Wash the plate again.
8. Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
9. Stop the reaction with 50 μL of stop solution.
10. Measure absorbance at 450 nm within 15 minutes.
**Notes and Precautions**
- Allow the kit to equilibrate to room temperature before use.
- Avoid repeated freezing and thawing of samples.
- Do not use samples containing NaN3, as it may inhibit HRP activity.
- Always run a standard curve and use duplicate wells for accuracy.
- Keep all reagents away from light and store properly.
- Discard all waste as biohazardous material.
**Data Analysis**
Plot the standard curve using concentration vs. OD values. Calculate the sample concentration using linear regression and multiply by the dilution factor.
**Performance**
- Sensitivity: 0.5 ng/L
- Range: 0.5–10 ng/L
- Intra-assay CV <9%, Inter-assay CV <11%
- Correlation coefficient (R²) ≥ 0.95
**Storage and Shelf Life**
- Store at 2–8°C.
- Valid for 6 months from the date of manufacture.
This kit provides a reliable and accurate method for detecting IL-15 levels in various biological samples, ensuring consistent and reproducible results when used correctly.