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Human IFN-α ELISA Kit
**Human IFN-α ELISA Kit – For the quantitative in vitro determination of Human Interferon α concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other biological fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.**
This kit is designed for research purposes only and should not be used for diagnostic or clinical applications. Before beginning the assay, please read and understand this entire user manual carefully.
The ELISA procedure involves a colorimetric reaction where the Stop Solution changes the color from blue to yellow. The optical density (OD) is measured at 450 nm using a microplate reader. A set of calibration standards is included in the kit, which allows the operator to generate a standard curve by plotting OD values against known IFN-α concentrations. Sample concentrations are then determined by comparing their OD readings to the standard curve.
**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. Centrifuge at 2000×g for 20 minutes. Remove 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 for 30 minutes at 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present in the sample.
**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):**
| Reagent Name | 96 Determinations | 48 Determinations |
|-----------------------------|-------------------|-------------------|
| MicroELISA Strip Plate | 12 x 8 strips | 12 x 4 strips |
| Standard (6 vials) | 0.5 ml/vial | 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 | 2 |
| User Manual | 1 | 1 |
| Sealed Bags | 1 | 1 |
**Note:**
- Standard concentrations: 160, 80, 40, 20, 10, 5 pg/mL
- If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
**Precautions:**
1. Do not mix reagents from different kit lots. Standards, conjugates, and plates are matched for optimal performance. Only use manufacturer-supplied reagents.
2. Allow all reagents to reach room temperature (20–25°C) before use. Do not use water baths to thaw samples or reagents.
3. Do not use reagents beyond their expiration date.
4. Use only deionized or distilled water for reagent dilution.
5. Keep microtiter plates in sealed bags until ready to use. Store unused strips at 2–8°C with desiccant.
6. Use fresh disposable pipette tips for each transfer to avoid contamination.
7. All blood-derived products should be treated as potentially infectious. Follow good laboratory practices.
8. Dispose of all samples properly. Allow waste to stand for at least 30 minutes to inactivate viruses before disposal.
9. Substrate solutions can be easily contaminated. Avoid exposure to heat or flame. Substrate B contains 20% acetone, so handle with care.
10. Bring all reagents to room temperature before starting the assay.
**Reagent Preparation and Storage:**
- **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. Add 50 µL of standard or sample to appropriate wells (excluding blank well). Cover with adhesive strip and incubate for 60 minutes at 37°C.
2. Wash the microtiter plate 4 times manually or automatically. For manual washing, fill each well with 1X Wash Solution, aspirate, and repeat four times. Dry the plate by blotting.
3. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution B to each well. Mix gently and incubate for 15 minutes at 37°C, protected from light.
4. Add 50 µL of Stop Solution to each well. Read OD at 450 nm using a microplate reader.
**Data Interpretation:**
1. Plot average OD values (450 nm) of standards on the Y-axis versus concentration on the X-axis.
2. Calculate mean OD for each standard and sample. Subtract blank OD value before interpretation.
3. Construct standard curve using graph paper or software.
4. To determine sample concentration, locate OD on Y-axis, draw a horizontal line to the curve, and then a vertical line to the X-axis.
5. Intra-assay and inter-assay CVs are less than 15%.
6. Assay range: 5 pg/mL – 160 pg/mL
7. Sensitivity: <1.0 pg/mL
8. Cross-reactivity: Recognizes recombinant and natural human IFN-α. No significant cross-reactivity observed.
9. Storage: 2–8°C (frequent use); 6 months at -20°C.
**Important Note:** Always follow proper safety protocols when handling biological materials. This kit is intended for research use only.