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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)
This reagent is intended solely for research purposes. The standard curve is plotted by plotting the concentration of the standard on the x-axis and the OD value on the y-axis. The sample concentration can be determined from the standard curve, multiplied by the dilution factor. Alternatively, a linear regression equation can be calculated using the standard concentrations and corresponding OD values. The sample OD is then substituted into the equation to calculate the actual concentration, which is also multiplied by the dilution factor.
**Kit Components:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 bottle (2700 ng/L)
- Enzyme Standard: 1×48 / 1×96
- Sample Diluent: 3 mL × 1 bottle / 6 mL × 1 bottle
- Developer A: 3 mL × 1 bottle / 6 mL × 1 bottle
- Chromogen B: 3 mL × 1 bottle / 6 mL × 1 bottle
- Stop Solution: 3 mL × 1 bottle / 6 mL × 1 bottle
- Concentrated Washing Solution: (20 mL × 20 times) × 1 bottle / (20 mL × 30 times) × 1 bottle
**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.
**Principle of the Assay:**
The kit uses the double-antibody sandwich method to detect endotoxin (ET) levels in samples. Microtiter plates are coated with purified rat anti-ET antibodies. After adding the sample, ET binds to the immobilized antibody. HRP-labeled anti-ET antibody is then added, forming an antibody-antigen-enzyme complex. After washing, TMB substrate is added, producing a blue color that turns yellow in the presence of acid. The intensity of the color is directly proportional to the ET concentration in the sample. Absorbance is measured at 450 nm, and the ET concentration is determined using a standard curve.
**Purpose:**
This kit is used to quantify endotoxin (ET) in serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**Sample Preparation and Handling:**
- **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant.
- **Plasma:** Use EDTA or sodium citrate as anticoagulant. Centrifuge after mixing.
- **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes.
- **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm. For intracellular components, lyse cells by freezing/thawing, then centrifuge.
- **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant.
- **General Note:** Avoid repeated freeze-thaw cycles. Store at -20°C if not tested immediately. Do not use samples containing NaN₃, as it inhibits HRP activity.
**Procedure:**
1. **Standard Dilution and Loading:** Prepare a 10-point standard curve (1800, 1200, 600, 300, 150 ng/L). Add 100 μL of each standard to designated wells.
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample (final dilution 5×) to each test well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Dilute the concentrated washing solution (30×) and wash 5 times.
5. **Enzyme Addition:** Add 50 μL of enzyme conjugate to each well (except blank).
6. **Second Incubation:** Repeat step 3.
7. **Color Development:** Add 50 μL of TMB A and B, incubate at 37°C for 15 minutes.
8. **Stop Reaction:** Add 50 μL of stop solution to each well.
9. **Measurement:** Read OD at 450 nm within 15 minutes.
**Notes:**
- Equilibrate the kit at room temperature for 15–30 minutes before use.
- Avoid cross-contamination; use a new sealing film for each experiment.
- Keep all reagents away from light.
- Always run a standard curve in duplicate. If the sample OD exceeds the first standard well, dilute the sample before testing.
- Follow the manual strictly; results must be based on microplate reader readings.
- Dispose of all waste as biohazardous material.
- Do not mix reagents from different batches.
- In case of discrepancy, the English manual takes precedence.
**Performance Characteristics:**
- Linear correlation coefficient (R²) ≥ 0.95
- Intra-batch and inter-batch variation < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
**Service Commitment:**
We offer free technical support during working hours. Sample testing services are available upon request to ensure optimal experimental results.
**Delivery Period:** From payment to delivery.
**Note:** This kit is for research use only. Not for diagnostic or therapeutic purposes.