**Human CD14 ELISA Kit – For the Quantitative In Vitro Determination of Human Cluster of Differentiation 14 Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. NOT FOR USE IN DIAGNOSTIC PROCEDURES.**
**INTENDED USE AND TEST PRINCIPLE**
This CD14 ELISA Kit is intended for laboratory research use only and is not suitable for diagnostic or therapeutic purposes. The assay is based on a sandwich immunoassay format, where the color change from blue to yellow is induced by the Stop Solution, and the optical density (OD) is measured at 450 nm using a microplate reader. Calibration standards are included to generate a standard curve, allowing the quantification of CD14 concentrations in the samples.
**SAMPLE COLLECTION AND STORAGE**
- **Serum**: Use serum separator tubes. Allow samples to clot for 2 hours at room temperature or overnight at 4°C before centrifuging 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 and 2–8°C. Store at -20°C. Avoid repeated freeze-thaw.
- **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates, then assay immediately or store at -20°C. Ensure samples are properly centrifuged and free from hemolysis or granules.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. Incubator set 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**
All reagents should be stored at 2–8°C. Check expiration date on label.
| Reagent Name | 96 Determinations | 48 Determinations |
|---------------------------|-------------------|-------------------|
| MicroELISA Strip Plate | 12×8 strips | 12×4 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: 4000, 2000, 1000, 500, 250, 125 ng/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.
2. All reagents must reach room temperature (20–25°C) before use.
3. Do not use expired reagents.
4. Use only deionized or distilled water.
5. Keep microtiter plates in sealed bags until needed.
6. Use fresh pipette tips for each transfer to avoid contamination.
7. Wear disposable gloves and protective clothing during the procedure.
8. Dispose of waste in 1.0% hypochlorite solution for 30 minutes before disposal.
9. Substrate solutions are sensitive to contamination.
10. Substrate B contains 20% acetone—keep away from heat and flame.
11. Allow all reagents to reach 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 beginning. Add standards and samples in duplicate.
2. Add 50 µL of standard or sample to appropriate wells. Blank well has no addition.
3. Add 100 µL of HRP-conjugate reagent to all wells except blank. Cover with adhesive strip and incubate at 37°C for 60 minutes.
4. Wash the plate 4 times. Manual washing involves filling and aspirating; automated washing uses washer buffer.
5. 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 Stop Solution to each well. Color changes from blue to yellow. Gently tap if color is uneven.
7. Measure OD at 450 nm. Plot standard curve and calculate sample concentrations.
**QUALITY CONTROL**
- Intra-assay and inter-assay CV% < 15%.
- Assay range: 125 ng/mL – 4000 ng/mL.
- Sensitivity: < 100 ng/mL.
- Cross-reactivity: No significant cross-reaction with other proteins.
- Storage: 2–8°C (frequent use); 6 months at -20°C.
**NOTES**
- Always read the entire protocol before starting.
- Follow safety guidelines to prevent contamination and ensure accurate results.
- Each user should generate their own standard curve for best performance.
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