**Human Rheumatoid Factor (RF) ELISA Kit – For Quantitative In Vitro Determination of Human RF Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
This package insert must be read in its entirety before using the product. The Human RF ELISA Kit is designed for research purposes only and is not intended for use in diagnostic or clinical procedures. The kit provides a reliable method for quantifying rheumatoid factor (RF) levels in various biological samples.
The principle of the assay is based on a competitive immunoassay. A set of calibration standards is included to generate a standard curve, which is essential for determining RF concentrations in unknown samples. The optical density (OD) values obtained from the samples are compared to this standard curve to calculate RF levels accurately.
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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 before centrifugation at 2000×g for 20 minutes. 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 of collection at 2000×g for 30 minutes at 2–8°C. Store at -20°C. Avoid freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure no hemolysis or granules are present in the sample.
Note: Proper centrifugation is crucial. Avoid any contamination or degradation of samples.
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### **Materials Required but Not Supplied**
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 (Stored at 2–8°C)**
| 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 |
**Standard Concentration:** 48, 24, 12, 6, 3, 1.5 IU/mL
**Note:** If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay. Do not substitute reagents between different kit lots. All components are matched for optimal performance. Use only manufacturer-supplied reagents.
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### **Important Safety and Handling Instructions**
1. Allow all reagents and materials to reach room temperature (20–25°C) before use.
2. Do not use water baths for thawing samples or reagents.
3. Do not use reagents beyond their expiration date.
4. Always use deionized or distilled water for dilutions.
5. Keep microtiter plates in sealed bags until needed. Unused strips should be stored with desiccant at 2–8°C.
6. Use fresh, disposable pipette tips for each transfer to prevent cross-contamination.
7. Do not mix disposable knives or other instruments that may come into contact with blood or body fluids.
8. Treat all biological samples as potentially infectious. Follow good laboratory practices.
9. Dispose of waste properly: Add 1% sodium hypochlorite to liquid waste and allow it to stand for 30 minutes before disposal.
10. Substrate Solution A is sensitive to contamination; discard if it appears bluish before use.
11. Substrate B contains 20% acetone—keep away from heat and open flames.
12. Avoid exposure to light during incubation steps.
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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 the assay. It is recommended to run standards and samples in duplicate.
2. Add 50 μL of standard or sample to appropriate wells. The blank well receives no addition.
3. Add 100 μL of HRP-conjugate reagent to all wells except the blank.
4. Incubate for 60 minutes at 37°C.
5. Wash the plate 4 times with 1X Wash Solution. Manual washing involves aspirating and refilling each well.
6. After the final wash, invert the plate and blot dry.
7. Add 50 μL of Chromogen Solution A and 50 μL of Chromogen Solution B to each well. Incubate for 15 minutes at 37°C, protected from light.
8. Add 50 μL of Stop Solution to each well. The color will change from blue to yellow.
9. Read OD at 450 nm within 15 minutes using a microplate reader.
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### **Data Analysis and Interpretation**
1. Generate a standard curve by plotting average OD values (450 nm) against corresponding RF concentrations.
2. Subtract the blank OD value from all measurements before interpretation.
3. Construct the standard curve using graph paper or software.
4. At the point of intersection, draw a vertical line to the X-axis and read the RF concentration.
5. Variations in technique, incubation time, temperature, or kit age may affect results. Each user should establish their own standard curve.
6. Intra-assay CV: <15%, Inter-assay range: 1.5–48 IU/mL.
7. Sensitivity: <1.0 IU/mL.
8. Cross-reactivity: No significant cross-reactivity observed with recombinant or natural human RF.
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### **Storage and Shelf Life**
- Store at 2–8°C for frequent use.
- For long-term storage, keep at -20°C for up to 6 months.
- Do not freeze-thaw repeatedly.
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**Note:** This kit is intended solely for research purposes. It is not approved for diagnostic use. Always follow proper safety protocols when handling biological samples. Consult the manufacturer's guidelines for further details.
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