**Human HIF-1α ELISA Kit – For the quantitative in vitro determination of Human hypoxia-inducible factor 1α concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. Intended for laboratory research use only. NOT FOR DIAGNOSTIC OR THERAPEUTIC USE.**
This ELISA kit is designed to measure the levels of Human HIF-1α in various biological samples. The principle of the assay is based on a competitive enzyme-linked immunosorbent assay (ELISA). A stop solution is added to terminate the reaction, causing the color to change from blue to yellow. The optical density (OD) is measured at 450 nm using a spectrophotometer.
To determine the concentration of HIF-1α in your sample, this kit includes a set of calibration standards. These standards are run alongside the samples to generate a standard curve, which allows for accurate quantification of HIF-1α levels by comparing the OD values of the samples to the 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 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 freezing and thawing multiple times.
- **Cell culture supernatants, tissue homogenates, and other biological fluids:** Centrifuge to remove particulates and assay immediately, or store at -20°C. Ensure no hemolysis or granules are present.
**Materials Required (Not Included):**
- 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 (Store at 2–8°C):**
- Microtiter plate (12×8 strips or 12×4 strips)
- Standard (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml or 3.0 ml)
- Enzyme Conjugate (10.0 ml or 5.0 ml)
- Wash Solution (25 ml or 15 ml)
- Substrate A (6.0 ml or 3.0 ml)
- Substrate B (6.0 ml or 3.0 ml)
- Stop Solution (6.0 ml or 3.0 ml)
- Closure plate membrane (2 units)
- User manual (1 copy)
- Sealed bags (1 unit)
**Notes:**
- 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.
- Only use reagents provided by the manufacturer.
- Allow all reagents and samples to reach room temperature (20–25°C) before use.
- Do not use water baths to thaw samples or reagents.
- Do not use reagents beyond their expiration date.
- Always use deionized or distilled water for dilutions.
- Store unused microtiter plate strips in their pouch with desiccant at 2–8°C.
- Use fresh pipette tips for each transfer to prevent contamination.
- Wear gloves throughout the procedure to ensure safety.
**Waste Disposal:**
- Liquid waste should be treated with sodium hypochlorite at a final concentration of 1.0% and left 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 or distilled water. Stable for 1 month at 2–8°C.
**Assay Procedure:**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to the appropriate wells. Leave the blank well empty.
3. Add 100 µL of Enzyme Conjugate to all wells except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically. After washing, invert and blot dry.
5. Add 50 µL of Substrate A and 50 µL of Substrate B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. The color will turn yellow. If the color is green or inconsistent, gently mix the plate.
7. Measure OD at 450 nm within 15 minutes.
**Calculation of Results:**
- Plot the average OD values of the standards against their concentrations to create a standard curve.
- Subtract the blank OD from all readings before interpreting results.
- Determine the HIF-1α concentration by matching the sample OD to the 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
- No significant cross-reactivity observed
- Storage: 2–8°C (frequent use); 6 months at -20°C
**Important Notes:**
- This kit is intended for research purposes only.
- Handle all reagents with care and follow proper safety protocols.
- Always refer to the user manual for detailed instructions and troubleshooting.
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