**Human Rheumatoid Factor (RF) ELISA Kit – For the Quantitative In Vitro Determination of Human RF in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Body Fluids**
*For Laboratory Research Use Only. Not for Diagnostic or Therapeutic Procedures.*
Before using this product, please read this entire package insert carefully. This ELISA kit is designed for research purposes only and is not intended for clinical diagnostic use. The kit allows for the accurate quantification of rheumatoid factor (RF) in various biological samples through an enzyme-linked immunosorbent assay (ELISA) method.
The test works by measuring the optical density (OD) at 450 nm, which correlates with the concentration of RF in the sample. A set of calibration standards is included to generate a standard curve, enabling the determination of RF levels in unknown samples.
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**Sample Collection and Storage**
- **Serum**: Collect using a serum separator tube. Allow clotting for 2 hours at room temperature or overnight at 4°C before centrifugation at 2000×g for 20 minutes. Assay 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 assay immediately or store at -20°C. Avoid repeated freeze-thaw cycles.
- *Note: Ensure proper centrifugation, and avoid hemolysis or granules in the 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**
| 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: The standard concentrations are 48, 24, 12, 6, 3, and 1.5 IU/mL. If sample values exceed the highest standard, dilute with sample diluent and repeat the assay. Do not substitute reagents between different kit lots. All reagents must be used as provided.*
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**Preparation and Storage of Reagents**
- **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.
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**Assay Procedure**
1. Prepare all reagents before starting. Add standards and samples in duplicate to the microtiter plate.
2. Add 50 μL of standard or sample to each well (blank well receives nothing).
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 four times with 1X Wash Solution. Manual washing: aspirate, fill, and repeat. Automated washing: follow manufacturer instructions.
6. 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.
7. Add 50 μL of Stop Solution to each well. The color will change from blue to yellow.
8. Read OD at 450 nm within 15 minutes using a microplate reader.
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**Data Interpretation**
- Plot average OD (450 nm) against standard concentrations on a graph.
- Subtract blank OD from all readings before interpreting results.
- Construct the standard curve using graph paper or software.
- Determine RF concentration by interpolating the sample OD on the standard curve.
- Each user should generate their own standard curve due to possible variations in technique.
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**Performance Characteristics**
- **Sensitivity**: <1.0 IU/mL
- **Intra-assay CV**: <10%
- **Inter-assay CV**: <15%
- **Cross-reactivity**: No significant cross-reaction with other proteins
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**Storage**
- Store unused reagents at 2–8°C if frequently used.
- Store at -20°C for long-term storage (up to 6 months).
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**Safety and Disposal**
- Handle all samples with care, assuming potential infectious risk.
- Dispose of liquid waste by adding 1% sodium hypochlorite and allowing it to sit for 30 minutes before disposal.
- Avoid using water baths to thaw samples or reagents.
- Do not use reagents beyond their expiration date.
- Always use fresh pipette tips to prevent contamination.
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**Important Notes**
- The substrate solution may become contaminated; do not use if it appears bluish.
- Chromogen B contains 20% acetone—keep away from heat and flame.
- Allow all reagents to reach room temperature (20–25°C) before use.
- Do not remove microtiter plates from the sealed bag until ready to use. Store unused strips in the pouch with desiccant.
This kit provides a reliable and reproducible method for determining human RF levels in laboratory settings. Always follow good lab practices and ensure proper handling of all materials.
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