**Rat Interleukin-1 (IL-1) Enzyme-Free ELISA Kit – Technical Specification**
*Supplier: Guangrui Bio, China – High-Quality ELISA Kit Provider for European Markets*
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### **Product Overview**
This Rat Interleukin-1 (IL-1) Enzyme-Free ELISA Kit is designed for the quantitative determination of IL-1 levels in rat serum, cell culture supernatants, and other biological fluids. The kit utilizes a double-antibody sandwich immunoassay method, ensuring high specificity and sensitivity. It is intended for research purposes only and not for diagnostic or therapeutic use.
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### **Principle of Operation**
The assay is based on the enzyme-linked immunosorbent assay (ELISA) technique. A microtiter plate is pre-coated with a purified monoclonal anti-rat IL-1 antibody. After incubation with the sample, IL-1 binds to the immobilized antibody. An HRP-conjugated secondary antibody is then added, forming a complex. TMB substrate is used for color development, and the reaction is stopped with sulfuric acid. The absorbance is measured at 450 nm, and the concentration of IL-1 is determined by comparing the OD values to a standard curve.
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### **Kit Composition**
- **48-well configuration:**
- 1×48 coated microplate
- 1×48 sealed bags
- 1×48 standard dilution tubes
- 1×48 HRP-conjugate reagent
- 1×48 sample diluent
- 1×48 chromogen solutions (A & B)
- 1×48 stop solution
- 1×48 washing buffer (20× diluted)
- **96-well configuration:**
- 1×96 coated microplate
- 1×96 sealed bags
- 1×96 standard dilution tubes
- 1×96 HRP-conjugate reagent
- 1×96 sample diluent
- 1×96 chromogen solutions (A & B)
- 1×96 stop solution
- 1×96 washing buffer (20× diluted)
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### **Storage Conditions**
- Store all components at 2–8°C.
- Shelf life: 6 months from the date of manufacture.
- Avoid repeated freezing and thawing.
- Keep away from light and moisture.
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### **Sample Preparation Guidelines**
1. **Serum:** Collect blood at room temperature, allow clotting for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes.
2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, centrifuge for 20 minutes.
3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
4. **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles.
5. **Tissue Samples:** Homogenize in PBS (pH 7.4), centrifuge, and collect supernatant.
**Note:** Avoid samples containing NaN₃, as it may inhibit HRP activity.
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### **Assay Procedure**
1. **Standard Dilution:** Prepare a serial dilution of the IL-1 standard.
2. **Sample Addition:** Add 40 μL of sample diluent and 10 μL of sample to each well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Wash the plate 5 times with wash buffer.
5. **Enzyme Conjugate Addition:** Add 50 μL of HRP-labeled antibody to each well.
6. **Second Incubation:** Incubate at 37°C for 30 minutes.
7. **Color Development:** Add 50 μL of TMB solution A and B. Incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to each well.
9. **Reading:** Measure absorbance at 450 nm within 15 minutes using a microplate reader.
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### **Important Notes**
- Allow the kit to equilibrate to room temperature before use.
- Do not reuse the sealing film to prevent cross-contamination.
- Ensure accurate pipetting and maintain consistent timing during each step.
- Always include blank wells and perform duplicate measurements for better accuracy.
- If sample OD exceeds the highest standard, dilute the sample before testing.
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### **Calculation**
Plot the standard curve using OD values vs. IL-1 concentrations. Calculate the sample concentration using linear regression or interpolation. Multiply by the dilution factor if applicable.
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### **Performance Characteristics**
- Sensitivity: 8 ng/L
- Dynamic Range: 8 ng/L – 150 ng/L
- Intra- and inter-assay CV < 9% and < 11%, respectively.
- Correlation coefficient (R²) ≥ 0.95.
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### **Safety and Disposal**
All samples, reagents, and waste should be treated as biohazardous materials. Follow local regulations for disposal.
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This kit provides a reliable and efficient method for measuring IL-1 levels in rat samples, ideal for researchers studying inflammatory responses, immune regulation, and related biological processes.
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