**Human CD14 ELISA Kit – For the Quantitative In Vitro Determination of Human Cluster of Differentiation 14 (CD14) Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Use in Diagnostic Procedures.**
This kit is intended for research purposes only and is not suitable for diagnostic or therapeutic use. The CD14 ELISA Kit utilizes a sandwich immunoassay technique to quantitatively measure CD14 levels in biological samples. The reaction involves binding of CD14 to immobilized antibodies on microtiter strips, followed by detection using a horseradish peroxidase (HRP)-conjugated secondary antibody. A chromogenic substrate is then added, producing a color change that is measured at 450 nm using a spectrophotometer. The intensity of the color is directly proportional to the concentration of CD14 in the sample.
To ensure accurate results, calibration standards are included in the kit. These standards are run alongside the test samples to generate a standard curve, which allows for the precise determination of CD14 concentrations in unknown samples.
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**Sample Collection and Storage**
- **Serum**: Collect using 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 store at -20°C. Avoid repeated freeze-thaw cycles.
- **Plasma**: Use 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. Assay immediately or store at -20°C. Ensure no hemolysis or granulation occurs.
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**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
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**Reagents Provided**
All reagents are stored at 2–8°C. Refer to the expiration date on the label.
- MicroELISA Strip Plate: 12×8 strips (96 determinations), 12×4 strips (48 determinations)
- Standard (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 unit
- Sealed Bags: 1 unit
**Standard Concentrations**: 4000, 2000, 1000, 500, 250, 125 ng/mL
If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
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**Precautions**
- Do not substitute reagents from different kit lots.
- All reagents must reach room temperature (20–25°C) before use.
- Do not use water baths for thawing.
- Do not use reagents beyond their expiration date.
- Use only deionized or distilled water.
- Keep microtiter plates in storage bags until needed.
- Use fresh pipette tips for each transfer to avoid contamination.
- Wear disposable gloves and lab coats during the procedure.
- Dispose of waste in 1.0% hypochlorite solution for 30 minutes before disposal.
- Avoid contamination of Substrate Solutions.
- Substrate B contains 20% acetone; keep away from heat or flame.
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**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 1 month.
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**Assay Procedure**
1. Prepare all reagents before starting. Run standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well receives no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times.
- **Manual Washing**: Fill each well with 1X Wash Solution, aspirate, and repeat 4 times.
- **Automated Washing**: Aspirate, wash 4 times with 1X Wash Buffer, adjust brush to remove maximum liquid, set fill volume to 350 µL/well.
5. After final wash, add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. Color changes from blue to yellow. If uneven, gently tap the plate.
7. Measure OD at 450 nm using a microplate reader.
**Data Interpretation**
- Plot average OD values of standards vs. concentrations.
- Subtract blank OD from all readings.
- Locate sample OD on Y-axis, draw horizontal line to standard curve, and read X-axis for CD14 concentration.
- Each user should generate their own standard curve.
- Intra-assay CV <15%, Inter-assay CV <15%.
- Assay range: 125–4000 ng/mL
- Sensitivity: <100 ng/mL
- Cross-reactivity: No significant cross-reactivity with other proteins.
**Storage**
- 2–8°C for frequent use
- -20°C for long-term storage (up to 6 months)
**Note**: This product is not intended for diagnostic use. Always follow safety protocols and proper disposal procedures.
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