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Duck interleukin 4 (IL-4) elisa kit instruction manual
**Duck Interleukin 4 (IL-4) ELISA Kit Instruction Manual**
**Kit Specifications:**
This ELISA kit is available in either a 48-well or 96-well configuration. The standard reagent is provided as 1.5 mL × 1 vial, while the enzyme standard is 3 mL × 1 vial (for 48-well) or 6 mL × 1 vial (for 96-well). The kit is intended for research use only.
**Standard Curve Preparation:**
To determine the concentration of IL-4 in the sample, plot the standard concentrations on the x-axis and the corresponding OD values on the y-axis. Draw a standard curve and calculate the sample concentration based on its OD value. Alternatively, use linear regression to derive an equation from the standard curve and input the sample's OD into the equation. Multiply the result by the dilution factor to obtain the actual sample concentration.
**Kit Components:**
- Sealing film: 2 pieces (48-well) / 2 pieces (96-well)
- Standard: 0.5 mL × 1 vial, 2700 ng/L
- Enzyme standard: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Sample diluent: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Developer A: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Chromogen B: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Wash buffer: 3 mL × 1 vial (48-well), 6 mL × 1 vial (96-well)
- Concentrated wash solution: 20 mL × 20 times (48-well), 20 mL × 30 times (96-well)
**Storage Conditions:**
Store all components at 2–8°C. The shelf life is 6 months from the date of receipt.
**Principle of the Assay:**
The Duck IL-4 ELISA kit uses a double-antibody sandwich method. Microtiter plates are pre-coated with anti-IL-4 antibodies. After adding the sample, IL-4 binds to the coated antibody. HRP-conjugated secondary antibody is then added, forming a complex. After washing, TMB substrate is added, and the reaction is stopped with an acidic solution. The color intensity is directly proportional to the IL-4 concentration, which is measured at 450 nm using a microplate reader.
**Purpose:**
This kit is designed to quantitatively measure IL-4 levels in duck serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids.
**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. Mix well and centrifuge as above.
- **Urine:** Collect in sterile tubes and centrifuge similarly.
- **Cell Culture Supernatant:** Centrifuge after collection; for intracellular components, lyse cells by repeated freeze-thaw cycles before centrifugation.
- **Tissue:** Homogenize in PBS, centrifuge, and collect the supernatant.
- **Storage:** Avoid repeated freezing and thawing. Store samples at -20°C if not tested immediately.
**Operation Steps:**
1. **Standard Dilution:** Prepare a serial dilution of the standard solution across the plate.
2. **Sample Loading:** Add 40 μL of sample diluent and 10 μL of sample to each test well.
3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes.
4. **Washing:** Rinse the plate 5 times with diluted wash buffer.
5. **Enzyme Addition:** Add 50 μL of enzyme-labeled reagent to each well.
6. **Second Incubation:** Repeat step 3.
7. **Color Development:** Add 50 μL each 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 values at 450 nm within 15 minutes.
**Notes:**
- Equilibrate the kit at room temperature for 15–30 minutes before use.
- Avoid cross-contamination by using a new sealing film for each experiment.
- Ensure accurate pipetting and avoid light exposure during the color development phase.
- Always prepare a standard curve and run duplicates for better accuracy.
- Do not mix reagents from different batches.
- Follow the manual strictly for reliable results.
**Performance:**
- Correlation coefficient (R) ≥ 0.95
- Intra-batch and inter-batch variation < 9% and < 11%, respectively
- Detection range: 0.2 IU/L – 6 IU/L
**Technical Support:**
Free guidance and assistance are available during working hours. We also offer sample testing services to help maximize your experimental outcomes.
**Safety Note:**
All samples, washes, and waste should be treated as biohazardous materials. Avoid contact with NaN3-containing samples, as it may inhibit HRP activity.