**Rat Interleukin-6 (IL-6) ELISA Kit – Instructions for Use**
*This kit is for research use only. It is designed to quantify interleukin-6 (IL-6) in rat serum, plasma, urine, cell culture supernatants, and tissue homogenates.*
### **Principle of the Assay**
The Rat IL-6 ELISA Kit employs a double-antibody sandwich immunoassay. A microtiter plate is pre-coated with a specific monoclonal antibody against IL-6. The sample or standard is added, allowing IL-6 to bind to the immobilized antibody. After washing, horseradish peroxidase (HRP)-labeled secondary antibody is added, forming an antibody-antigen-enzyme complex. A TMB substrate is then introduced, producing a color change that is proportional to the IL-6 concentration. The reaction is stopped with an acidic solution, and the absorbance is measured at 450 nm using a microplate reader. The IL-6 concentration is determined by comparing the sample OD value to a standard curve.
### **Kit Components**
- **Microtiter Plate**: 1 × 48 or 1 × 96 wells
- **Standard**: 180 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
- **Stop Solution**: 3 mL × 1 bottle
- **Washing Solution (20×)**: 20 mL × 20 times or 20 mL × 30 times
- **Sealing Film**: 2 pieces (for 48-well), 2 pieces (for 96-well)
- **Storage Bag**: 1 piece
### **Sample Preparation**
- **Serum/Plasma**: Centrifuge at 2000–3000 rpm for 20 minutes after clotting or anticoagulant addition.
- **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.
- **Tissue Homogenate**: Weigh the tissue, add PBS (pH 7.4), homogenize, centrifuge, and collect the supernatant. Store at 2–8°C if not used immediately.
### **Procedure**
1. **Standard Dilution**: Prepare a serial dilution of the IL-6 standard (120 ng/L to 10 ng/L).
2. **Sample Addition**: Add 40 μL sample diluent and 10 μL sample to each well.
3. **Incubation**: Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing**: Wash 5 times with diluted washing buffer.
5. **Enzyme Addition**: Add 50 μL enzyme conjugate to each well (except blank).
6. **Second Incubation**: Incubate at 37°C for 30 minutes.
7. **Color Development**: Add 50 μL TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction**: Add 50 μL stop solution to each well.
9. **Measurement**: Read OD 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 inhibits HRP activity.
- Always prepare a standard curve and run duplicates for accuracy.
- Discard unused reagents and follow biosafety protocols.
- Do not mix reagents from different batches.
### **Calculation**
Plot the standard curve using concentration vs. OD values. Calculate the sample concentration using linear regression and multiply by the dilution factor.
### **Performance**
- Sensitivity: 8 ng/L
- Range: 8–150 ng/L
- Correlation coefficient (R²): ≥ 0.92
### **Storage and Shelf Life**
- Store all components at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
Always refer to the English manual for the most accurate and up-to-date instructions.
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