**Human Cyclin-D1 ELISA Kit – For Quantitative In Vitro Determination of Human Cyclin-D1 in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Purposes.**
This ELISA kit is designed for the quantitative measurement of Human Cyclin-D1 in various biological samples. The assay is based on the enzyme-linked immunosorbent assay (ELISA) principle, where the colorimetric reaction is used to determine the concentration of the target protein.
The test works by using a set of calibrated standards to generate a standard curve. The optical density (OD) of each sample is measured at 450 nm, and the concentration of Cyclin-D1 is determined by comparing the OD values with the standard curve.
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### **Intended Use**
This kit is intended for laboratory research purposes only and should not be used for diagnostic or clinical testing. It is suitable for measuring Cyclin-D1 levels in serum, plasma, cerebrospinal fluid, tissue homogenates, and other body fluids.
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### **Test Principle**
The Human Cyclin-D1 ELISA Kit uses a sandwich immunoassay format. A monoclonal antibody specific to Cyclin-D1 is pre-coated on the microtiter plate. After incubation with the sample, a secondary HRP-conjugated antibody binds to the captured antigen. The addition of a chromogenic substrate leads to a color change, which is stopped by the Stop Solution. The intensity of the color is directly proportional to the amount of Cyclin-D1 present in the sample.
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### **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 (20 minutes at ~2000×g). Store at -20°C after aliquoting. 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.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or contamination.
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### **Materials Required (Not Included)**
1. Incubator 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
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### **Reagents Provided**
| Reagent | 96 Tests | 48 Tests |
|--------|----------|----------|
| Microplate (12×8 strips) | 1 | 1 |
| Standards (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 A | 6.0 ml | 3.0 ml |
| Chromogen 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 are 40, 20, 10, 5, 2.5, 1.25 μg/L. If sample values exceed the highest standard, dilute with Sample Diluent and repeat.*
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### **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 expiration date.
- Use only deionized or distilled water for dilutions.
- Keep unused strips in sealed bags with desiccant.
- Wear gloves when handling biological materials.
- Discard waste after 30 minutes of inactivation.
- Avoid contamination; use fresh pipette tips for each step.
- Substrate solutions must be protected from light and heat.
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### **Reagent Preparation**
- **Wash Solution (1X)**: Dilute 1 volume of 20X Wash Solution with 19 volumes of deionized water. Store at 2–8°C for 1 month.
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### **Assay Procedure**
1. Prepare all reagents and standards.
2. Add 50 µL of standard or sample to appropriate wells (duplicate).
3. Add 100 µL of HRP-Conjugate to all wells except blank. Incubate for 60 minutes at 37°C.
4. Wash 4 times manually or automatically.
5. Add 50 µL of Chromogen A and B, incubate for 15 minutes at 37°C.
6. Add 50 µL Stop Solution. Color changes from blue to yellow.
7. Read OD at 450 nm. Plot standard curve and calculate sample concentrations.
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### **Interpretation of Results**
- Generate a standard curve using average OD values vs. standard concentrations.
- Subtract blank OD from all readings.
- Use graph paper or software to plot and interpret results.
- Intra-assay and inter-assay CV% < 15%.
- Sensitivity: <1.0 µg/L.
- Assay range: 1.25–40 µg/L.
- No significant cross-reactivity with other proteins.
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### **Storage**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C.
- Shelf life: 6 months at -20°C.
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**Please read the entire user manual before use.**
**For Research Use Only.**
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