**Human IFN-α4 ELISA Kit – For the Quantitative In Vitro Determination of Human Interferon α 4 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
This ELISA kit is designed for the accurate quantification of Human Interferon alpha 4 (IFN-α4) in various biological samples. The assay is based on a sandwich ELISA principle, where specific antibodies are used to capture IFN-α4 from the sample. A horseradish peroxidase (HRP)-conjugated secondary antibody then binds to the captured protein, and the enzymatic reaction produces a color change that is measured at 450 nm using a microplate reader.
The kit includes a set of calibration standards to generate a standard curve, allowing the user to determine the concentration of IFN-α4 in unknown samples by comparing their optical density (OD) values to the standard curve. The Stop Solution changes the reaction from blue to yellow, which is critical for accurate measurement.
**Sample Collection and Storage:**
- **Serum:** Use a serum separator tube. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging at 2000×g for 20 minutes. Remove the serum and analyze 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. Store at -20°C.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and analyze immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
**Note:** Ensure proper centrifugation and avoid hemolysis or granules in the samples.
**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):**
- MicroELISA Strip Plate (12×8 strips / 12×4 strips)
- Standards (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml / 3.0 ml)
- HRP-Conjugate Reagent (10.0 ml / 5.0 ml)
- 20X Wash Solution (25 ml / 15 ml)
- Chromogen Solution A (6.0 ml / 3.0 ml)
- Chromogen Solution B (6.0 ml / 3.0 ml)
- Stop Solution (6.0 ml / 3.0 ml)
- Closure Plate Membrane (2 units)
- User Manual (1 copy)
- Sealed Bags (1 unit)
**Important Notes:**
- Standard concentrations: 160, 80, 40, 20, 10, 5 pg/mL
- If sample values exceed the highest standard, dilute with Sample Diluent
- Do not use water baths to thaw samples or reagents
- Do not use beyond expiration date
- Use only deionized or distilled water
- Keep microtiter plate in its sealed bag until needed
- Use fresh pipette tips to prevent contamination
- All blood-derived products should be treated as potentially infectious
- Dispose of all samples and waste according to local regulations
**Wash Solution Preparation (1X):**
Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized or distilled water. Store at 2–8°C for up to 1 month.
**Assay Procedure:**
1. Prepare all reagents and add standards and samples in duplicate.
2. Add 50 µL of standard or sample to each well.
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, protecting from light.
6. Add 50 µL of Stop Solution to each well. The color will turn yellow.
7. Read OD at 450 nm and plot a standard curve.
**Interpretation:**
- Calculate mean OD for each standard and sample. Subtract blank OD.
- Plot OD vs. concentration to generate the standard curve.
- Determine sample concentration by interpolating OD on the curve.
- Intra-assay and inter-assay CV < 15%
- Assay range: 5–160 pg/mL
- Sensitivity: <1.0 pg/mL
- Cross-reactivity: No significant cross-reaction with other IFN-α subtypes
**Storage:**
- 2–8°C for frequent use; 6 months at -20°C
**Safety & Disposal:**
- Liquid waste must be inactivated with 1.0% sodium hypochlorite for 30 minutes before disposal.
- Substrate B contains 20% acetone—keep away from heat and flame.
- Always follow good laboratory practices and treat all samples as potentially hazardous.
**Please read the entire user manual before performing the assay.**
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