**Human HIF-1α ELISA Kit – For Quantitative In Vitro Determination of Human Hypoxia-Inducible Factor 1α in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not Intended for Diagnostic or Therapeutic Purposes.**
This ELISA kit is designed for the quantitative determination of Human HIF-1α concentrations in various biological samples. The assay is based on the principle of a competitive enzyme-linked immunosorbent assay (ELISA). The color change from blue to yellow, induced by the Stop Solution, is measured at 450 nm using a microplate reader. A standard curve is generated using a set of calibration standards, allowing accurate quantification of HIF-1α levels in unknown samples.
**Intended Use:**
The Human HIF-1α ELISA Kit is intended for research use only and should not be used in diagnostic or therapeutic procedures. It provides a reliable method for measuring HIF-1α in serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids.
**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. Store aliquots 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 freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids:** Centrifuge to remove particulates and store at -20°C. Ensure no hemolysis or granules are present.
**Materials Required but Not Supplied:**
- 37°C incubator
- Microplate reader capable of measuring absorbance at 450 nm
- Precision pipettes, disposable tips, and absorbent paper
- Distilled or deionized water
**Reagents Provided (Stored at 2–8°C):**
- Microtiter Plate (12×8 strips / 12×4 strips)
- Standard (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml / 3.0 ml)
- Enzyme Conjugate (10.0 ml / 5.0 ml)
- Wash Solution (25 ml / 15 ml)
- Substrate A (6.0 ml / 3.0 ml)
- Substrate 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)
**Standard Concentrations:** 200, 100, 50, 25, 12.5, 6.25 pg/mL. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the test.
**Important Notes:**
- Always allow reagents and samples to reach room temperature (20–25°C) before use.
- Do not use water baths for thawing.
- Do not use reagents past their expiration date.
- Use only deionized or distilled water for dilution.
- Keep unused microtiter plate strips in the sealed bag with desiccant.
- Use fresh pipette tips for each transfer to avoid contamination.
- Wear gloves during the procedure to prevent cross-contamination.
**Waste Disposal:**
Dispose of liquid waste by adding sodium hypochlorite to achieve a final concentration of 1.0%. Allow it to stand for at least 30 minutes to inactivate viruses before disposal.
**Reagent Preparation:**
- Wash Solution (1X): Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized water. Store at 2–8°C for up to one 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 (blank well receives nothing).
3. Add 100 µL of Enzyme Conjugate to all wells except blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate four times (manual or automated).
5. Add 50 µL of Substrate A and 50 µL of Substrate B. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. Mix gently if color change is uneven.
7. Read OD at 450 nm within 15 minutes.
**Calculation of Results:**
- Plot average OD values of standards against their concentrations.
- Subtract blank OD from all readings.
- Determine sample concentration by comparing OD to the standard curve.
- Each user should generate their own standard curve.
**Performance Characteristics:**
- Intra-assay and Inter-assay CV (%) < 15%
- Assay Range: 6.25 pg/mL – 200 pg/mL
- Sensitivity: <1.0 pg/mL
- Cross-reactivity: No significant interference observed
- Storage: 2–8°C (frequent use); -20°C for long-term storage (up to 6 months)
**Note:** This kit is not intended for clinical use. Follow all safety guidelines and dispose of waste properly.
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