**Human EPOR ELISA Kit – For the Quantitative In Vitro Determination of Human Erythropoietin Receptor Concentrations in Serum, Plasma, Cerebrospinal Fluid, Tissue Homogenate, and Other Body Fluids**
**For Laboratory Research Use Only. NOT FOR USE IN DIAGNOSTIC PROCEDURES**
Before using this product, please read this entire package insert carefully. This ELISA kit is designed for research purposes only and should not be used for diagnostic or therapeutic applications.
**INTENDED USE AND TEST PRINCIPLE**
This Human EPOR ELISA Kit is intended for laboratory research use only. The test is based on a sandwich immunoassay format. The color change from blue to yellow upon addition of the Stop Solution indicates the reaction completion. Standards are run simultaneously with samples, allowing the operator to create a standard curve by plotting optical density (OD) values against known EPOR concentrations. The EPOR concentration in unknown samples is then determined by comparing their OD values to the standard curve.
**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 centrifugation 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 plasma 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 repeated freeze-thaw cycles.
- **Cell Culture Supernatants, Tissue Homogenates, and Other Biological Fluids**: Centrifuge to remove particulates and assay immediately, or aliquot and store at -20°C. Ensure samples are properly centrifuged, and avoid hemolysis or particulate contamination.
**MATERIALS REQUIRED BUT NOT SUPPLIED**
1. 37°C incubator
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 are stored at 2–8°C. Refer to the expiration date on the 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 |
**NOTES**
1. Standard concentrations: 100, 50, 25, 12.5, 6.25, 3.12 ng/ml
2. If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay.
3. Do not mix or match reagents from different kits.
4. Allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths for thawing.
5. Do not use any reagents beyond their expiration date.
6. Use only deionized or distilled water for dilutions.
7. Keep microtiter plates in sealed bags until needed. Unused strips should be stored with desiccant at 2–8°C.
8. Use fresh disposable pipette tips for each transfer to prevent cross-contamination.
9. Disposable items must be treated as potentially hazardous. Handle with care.
10. All samples and waste should be disposed of following local biosafety regulations.
**WASTE DISPOSAL**
- **Liquid Waste**: Add sodium hypochlorite to achieve a final concentration of 1.0%. Let stand for at least 30 minutes to inactivate viruses before disposal.
- **Substrate Solution**: Be cautious—this solution can be easily contaminated.
- **Chromogen B**: Contains 20% acetone; keep away from heat or flame.
**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 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 the blank. Cover with adhesive strip and incubate for 60 minutes at 37°C.
4. Wash the plate 4 times.
- **Manual Washing**: Aspirate contents, fill with 1X Wash Solution, aspirate again. Repeat four times. Blot dry after final wash.
- **Automated Washing**: Aspirate, wash four times with 1X Washer Buffer. Adjust brush and fill volume to 350 µL/well.
5. Add 50 µL of Chromogen A and 50 µL of Chromogen B to each well. Mix gently and incubate for 15 minutes at 37°C, protected from light.
6. Add 50 µL of Stop Solution to each well. Read OD at 450 nm.
**DATA ANALYSIS**
1. Plot average OD (450 nm) of standards against concentrations to generate a standard curve.
2. Calculate mean OD for each standard and sample. Subtract blank OD from all values.
3. Use graphing software to construct the standard curve.
4. Locate the OD value on the Y-axis and draw a horizontal line to intersect the curve. Draw a vertical line to the X-axis to determine the EPOR concentration.
5. Variations in technique, incubation time, or kit age may affect results. Each user should prepare their own standard curve.
6. Intra-assay CV <15%, Inter-assay CV <15%.
7. Assay range: 3.12 ng/mL – 100 ng/mL.
8. Sensitivity: <1.0 ng/mL.
9. Cross-reactivity: Recognizes recombinant and natural Human EPOR. No significant cross-reactivity observed.
10. Storage: 2–8°C (for frequent use); 6 months at -20°C.
**NOTE:** Always follow safety guidelines and proper disposal procedures when handling biological materials.
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