**Human IFN-α4 ELISA Kit – For Quantitative In Vitro Determination of Human Interferon α 4 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Procedures.**
This ELISA kit is designed for the quantitative measurement of Human Interferon alpha-4 (IFN-α4) in various biological samples. The method is based on a sandwich ELISA format, where the colorimetric reaction is detected at 450 nm using a spectrophotometer. The intensity of the color change is directly proportional to the concentration of IFN-α4 in the sample.
The kit includes a set of calibration standards that allow the operator to generate a standard curve by plotting optical density (OD) values against known concentrations. Once the standard curve is established, the IFN-α4 levels in unknown samples can be accurately determined by comparing their OD values to the curve.
**Sample Collection and Storage:**
- **Serum:** Use a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Remove the serum and assay immediately, or aliquot and store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes at 2000×g, 2–8°C. Avoid repeated freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates, then assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
*Note: Ensure proper centrifugation and avoid hemolysis or granulation.*
**Materials Required but Not Supplied:**
1. Incubator at 37°C
2. Microplate reader capable of measuring absorbance at 450 nm
3. Precision pipettes, disposable tips, and absorbent paper
4. Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- 96-well microplate (12 x 8 strips)
- 6 standard vials (0.5 ml/vial)
- Sample diluent (6.0 ml)
- HRP-conjugated reagent (10.0 ml)
- 20X Wash solution (25 ml)
- Chromogen A (6.0 ml)
- Chromogen B (6.0 ml)
- Stop solution (6.0 ml)
- Closure plate membrane (2 pieces)
- User manual and sealed bags
**Standard Concentrations:** 160, 80, 40, 20, 10, 5 pg/mL
**Important Notes:**
- Do not use water baths to thaw samples or reagents.
- Do not use any components past their expiration date.
- Only use distilled or deionized water.
- Keep microtiter plates in their sealed bags until needed. Store unused strips at 2–8°C with desiccant.
- Always use fresh pipette tips to prevent contamination.
**Safety and Waste Disposal:**
- All samples and reagents should be handled with care, as they may contain infectious agents.
- Dispose of liquid waste by adding sodium hypochlorite to a final concentration of 1.0%, allowing it to stand for at least 30 minutes before disposal.
- Substrate B contains 20% acetone; keep away from heat or flame.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to one month.
**Assay Procedure:**
1. Prepare all reagents before starting.
2. Add 50 µL of standard or sample to each well (duplicate).
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution. The color changes from blue to yellow. If uneven, gently tap the plate.
7. Measure OD at 450 nm. Generate a standard curve and calculate sample concentrations.
**Performance Characteristics:**
- Intra-assay CV <15%, Inter-assay CV <15%.
- Assay range: 5–160 pg/mL.
- Sensitivity: <1.0 pg/mL.
- Cross-reactivity: No significant cross-reactivity with other IFN subtypes.
- Storage: 2–8°C (frequent use), -20°C (up to 6 months).
Always read the complete user manual before performing the assay. This kit is intended for research purposes only.
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