**Human IGFBP-1 ELISA Kit – For the Quantitative In Vitro Determination of Human Insulin-like Growth Factor Binding Protein 1 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.**
Before using this kit, please read the entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used in clinical diagnostics. The principle of the assay is based on a sandwich ELISA format, where the presence of Human IGFBP-1 in the sample is detected through specific antibody interactions. The reaction is terminated by a stop solution, which changes the color from blue to yellow. The optical density (OD) is then measured at 450 nm using a spectrophotometer.
A set of calibration standards is included in the kit, allowing the user to create a standard curve by plotting OD values against known concentrations of IGFBP-1. The concentration of IGFBP-1 in unknown samples is determined by comparing their OD readings to this standard curve.
**Sample Collection and Storage:**
- **Serum:** Collect blood in a serum separator tube. Allow clotting 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 store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma:** Collect using heparin as an anticoagulant. Centrifuge within 30 minutes of collection at 2000×g for 30 minutes at 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 freezing and thawing.
**Important Notes:** Ensure proper centrifugation and avoid hemolysis or contamination with cellular debris.
**Materials Required but Not Supplied:**
- Incubator at 37°C
- 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 Strip Plate (12×8 or 12×4 strips)
- Standards (6 vials, 0.5 ml/vial)
- Sample Diluent (6.0 ml or 3.0 ml)
- HRP-Conjugated Reagent (10.0 ml or 5.0 ml)
- 20X Wash Solution (25 ml or 15 ml)
- Chromogen A (6.0 ml or 3.0 ml)
- Chromogen 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 per kit)
**Precautions:**
- Do not mix reagents from different kit lots.
- Allow all reagents to reach room temperature (20–25°C) before use.
- Do not use beyond the expiration date.
- Use only deionized or distilled water for dilution.
- Keep unused microtiter plate strips in sealed bags with desiccant.
- Wear gloves when handling biological samples due to potential infectious risk.
- Dispose of all waste properly, including inactivating viruses with 1% sodium hypochlorite.
- Avoid exposure to heat or flame when handling chromogen B, which contains 20% acetone.
**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 and ensure they are at room temperature.
2. Add 50 µl of standard or sample to appropriate wells (duplicate). Blank well receives no addition.
3. Add 100 µl of HRP-conjugate reagent to each well except the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times manually or automatically, ensuring complete removal of unbound conjugate.
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 change from blue to yellow.
7. Measure OD at 450 nm. Generate a standard curve using the mean OD values of standards. Calculate sample concentrations accordingly.
**Interpretation:**
- Subtract blank OD from all measurements.
- Each operator should generate their own standard curve.
- Intra-assay and inter-assay CVs are less than 15%.
- Assay range: 31.2 ng/ml – 1000 ng/ml.
- Sensitivity: <10 ng/ml.
- Cross-reactivity: No significant cross-reaction with other proteins.
**Storage:**
- Store at 2–8°C for frequent use; -20°C for long-term storage.
**Note:** Always follow good laboratory practices and handle all samples with care.
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