**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 Diagnostic or Therapeutic Procedures.* Before using this product, please read this entire package insert carefully. --- ### **INTENDED USE AND TEST PRINCIPLE** This Human Erythropoietin Receptor (EPOR) ELISA Kit is designed for laboratory research purposes only and should not be used in diagnostic or therapeutic procedures. The kit utilizes a sandwich ELISA method to quantitatively measure EPOR levels in various biological samples. The reaction involves binding of EPOR to specific antibodies immobilized on microtiter plates. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change. The intensity of the color is directly proportional to the concentration of EPOR in the sample. A standard curve is generated using known concentrations of EPOR, and the unknown sample concentrations are determined by comparing their optical density (OD) values to this curve. --- ### **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 at 2000×g for 20 minutes. Remove the 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 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 and assay immediately, or aliquot and store at -20°C. Ensure no hemolysis or granulation occurs. --- ### **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** | Reagent | 96 Determinations | 48 Determinations | |--------|-------------------|-------------------| | MicroELISA Strip Plate | 12 x 8 strips | 12 x 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: 100, 50, 25, 12.5, 6.25, 3.12 ng/ml. If sample readings exceed the highest standard, dilute with Sample Diluent and repeat the test. --- ### **PRECAUTIONS AND GUIDELINES** 1. Always allow all reagents and materials to reach room temperature (20–25°C) before use. Do not use water baths for thawing. 2. Do not use any reagents beyond their expiration date. 3. Use only deionized or distilled water for dilutions. 4. Keep microtiter plates in sealed bags until needed. Unused strips should be stored at 2–8°C with desiccant. 5. Use fresh, sterile pipette tips for each transfer to prevent contamination. 6. Treat all disposable materials as potentially hazardous. Follow biosafety protocols when handling blood-derived products. 7. Dispose of all waste according to local regulations. Liquid waste should be treated with sodium hypochlorite (final concentration: 1.0%) for at least 30 minutes before disposal. 8. Substrate solutions may be easily contaminated. Avoid exposure to light and keep Chromogen B away from heat or flame (contains 20% acetone). 9. Maintain consistent incubation times and temperatures for accurate results. Each user should generate their own standard curve. --- ### **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. Add standards and samples in duplicate to the microtiter plate. 2. Add 50 µL of standard or sample to each well (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 at 37°C for 60 minutes. 4. Wash the plate four times. For manual washing, aspirate and refill with 1X Wash Solution. For automated washing, use 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, protecting from light. 6. Add 50 µL of Stop Solution to each well. Read OD at 450 nm using a microplate reader. --- ### **DATA ANALYSIS** - Plot average OD values (450 nm) of standards against their concentrations to create a standard curve. - Subtract blank OD values from all measurements before interpretation. - Determine sample concentrations by locating the OD value on the Y-axis and drawing a line to the standard curve. - Inter-assay and intra-assay CV% are less than 15%. - Assay range: 3.12 – 100 ng/mL. - Sensitivity: <1.0 ng/mL. - Cross-reactivity: No significant cross-reactivity with other species or proteins. --- ### **STORAGE AND STABILITY** - Store unused kits at 2–8°C for frequent use, or at -20°C for long-term storage (up to 6 months). - Protect from moisture and light. --- **Important Note:** This kit is intended for research use only and must not be used in clinical diagnostics. Always follow proper safety and disposal guidelines.

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