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Rat endotoxin (ET) elisa kit instruction manual
**Rat Endotoxin (ET) ELISA Kit – Instructions for Use**
**Kit Specifications:**
Available in 48-well or 96-well configurations.
- Standard Dilution: 1.5 mL × 1 bottle
- Enzyme Standard Reagent: 3 mL × 1 bottle (for 48-well) / 6 mL × 1 bottle (for 96-well)
**Usage Note:** This reagent is for research purposes only. The standard curve should be plotted with the concentration on the x-axis and OD value on the y-axis. Using this curve, the sample’s OD value can be converted to its corresponding concentration by interpolation or by calculating the linear regression equation of the standard curve. Once the sample OD is input into the equation, the actual sample concentration can be determined by multiplying it by the dilution factor.
**Kit Composition:**
- Sealing Film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 bottle (2700 ng/L)
- Sample Diluent: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Developer A: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Chromogen B: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Wash Solution: 3 mL × 1 bottle (48-well) / 6 mL × 1 bottle (96-well)
- Concentrated Wash Solution: 20 mL × 20 times (48-well) / 20 mL × 30 times (96-well)
**Storage Conditions:**
All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of receipt.
**Principle of the Assay:**
This ELISA kit uses the sandwich method to detect endotoxin (ET) levels in biological samples. The microplate is pre-coated with purified rat ET-specific antibodies. After adding the sample and HRP-labeled secondary antibody, an antigen-antibody-enzyme complex is formed. After washing, TMB substrate is added, producing a blue color that turns yellow upon acid termination. The intensity of the color correlates with the ET concentration in the sample. Absorbance is measured at 450 nm, and the concentration is calculated using a standard curve.
**Purpose:**
The kit is designed for quantitative determination of endotoxin (ET) in serum, plasma, urine, cell culture supernatants, and tissue homogenates.
**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. Centrifuge after mixing.
- **Urine:** Collect in sterile tubes and centrifuge.
- **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells by repeated freezing and thawing before centrifugation.
- **Tissue Specimens:** Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C if not used immediately.
**Important Notes:**
- Avoid repeated freeze-thaw cycles.
- Do not use samples containing NaN₃, as it may inhibit HRP activity.
- All samples, washes, and waste should be treated as biohazardous materials.
- Ensure all reagents are properly mixed and equilibrated before use.
- Perform the standard curve in duplicate for accuracy. If the sample OD exceeds the first standard well, dilute the sample and adjust the calculation accordingly.
- Always use a new sealing film for each experiment to prevent cross-contamination.
- Keep the substrate away from light during incubation.
**Operation Steps:**
1. Prepare standard dilutions in a serial manner.
2. Add 40 μL of sample diluent and 10 μL of sample to each well (final 5x dilution).
3. Incubate the plate at 37°C for 30 minutes.
4. Wash the plate 5 times with diluted wash solution.
5. Add 50 μL of enzyme-labeled reagent to each well (except blank).
6. Incubate again for 30 minutes.
7. Add 50 μL of chromogen A and B, incubate for 15 minutes at 37°C.
8. Stop the reaction by adding 50 μL of stop solution.
9. Measure OD450 within 15 minutes.
**Performance:**
- Linear correlation coefficient (R²) ≥ 0.95
- Intra-assay and inter-assay variability < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
- Free technical support during working hours.
- Sample testing services available upon request.
- Delivery time: From payment to shipment.
**Note:** Always refer to the English manual for official instructions. Any discrepancies should be resolved using the English version.