Human EPO ELISA Kit

**Human EPO ELISA Kit – For the quantitative in vitro determination of Human Erythropoietin concentrations in serum, plasma, cerebrospinal fluid, tissue homogenate, and other body fluids. FOR LABORATORY RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES.** Before using this kit, please read the entire package insert carefully. --- ### **INTENDED USE AND TEST PRINCIPLE** This Human EPO ELISA Kit is specifically designed for laboratory research use only and is not intended for diagnostic or therapeutic purposes. The assay is based on a sandwich immunoassay principle, where EPO in the sample binds to a specific antibody coated on the microplate. A horseradish peroxidase (HRP)-conjugated secondary antibody is then added, followed by a chromogenic substrate that produces a color change. The reaction is stopped with a stop solution, and the optical density (OD) is measured at 450 nm. By comparing the OD values of the samples to a standard curve, the concentration of EPO in the samples can be accurately determined. --- ### **SAMPLE COLLECTION AND STORAGE** - **Serum**: Use a serum separator tube. Allow the blood 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 repeated freezing and thawing. - **Cell culture supernatants, tissue homogenates, and other biological fluids**: Centrifuge to remove particulates. Assay immediately or store at -20°C. Ensure proper centrifugation and avoid hemolysis or granulation. --- ### **MATERIALS REQUIRED BUT NOT SUPPLIED** 1. Incubator set at 37°C 2. Microplate reader capable of measuring absorbance at 450 nm 3. Pipettes, disposable tips, and absorbent paper 4. Distilled or deionized water --- ### **REAGENTS PROVIDED** All reagents are stored at 2–8°C. Check the expiration date on the label before use. | Reagent | 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:** - Standard concentrations: 24, 12, 6, 3, 1.5, 0.75 mIU/mL - If sample values exceed the highest standard, dilute with Sample Diluent and repeat the assay. --- ### **PRECAUTIONS** 1. Do not substitute reagents from different kit lots. All components are calibrated for optimal performance. 2. Allow all reagents and materials to reach room temperature (20–25°C) before use. 3. Do not use water baths to thaw samples or reagents. 4. Do not use any kit components past their expiration date. 5. Use only deionized or distilled water for reagent dilutions. 6. Keep microtiter plates in their sealed bags until ready for use. Store unused strips in 2–8°C with desiccant. 7. Use fresh pipette tips for each transfer to prevent cross-contamination. 8. Avoid mixing disposable knives with the assay procedure. 9. Treat all biological samples as potentially infectious. Follow biosafety guidelines. 10. Dispose of all samples and waste in accordance with local regulations. 11. Liquid waste should be treated with 1.0% sodium hypochlorite for 30 minutes before disposal. 12. Substrate solutions may be easily contaminated. If blue in color, discard and do not use. 13. Chromogen B contains 20% acetone; keep away from heat or open 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 the assay. It is recommended to 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 an 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. Invert and blot dry. - **Automated washing:** Aspirate, wash four times with 350 µL/well. Invert and blot dry. 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. The color will change from blue to yellow. 7. Measure OD at 450 nm within 15 minutes. --- ### **CALCULATION AND INTERPRETATION** 1. Plot the average OD (450 nm) of each standard against its concentration on a graph. 2. Subtract the blank OD value from all readings before interpretation. 3. Use graph paper or software to construct the standard curve. 4. Locate the OD value of the sample on the Y-axis and draw a horizontal line to intersect the curve. Draw a vertical line to the X-axis to determine the EPO concentration. 5. Each user should generate their own standard curve due to potential variations in technique, timing, and kit age. 6. Intra-assay CV (%) and Inter-assay range: 0.75–24 mIU/mL. 7. Sensitivity: <1.0 ng/mL. 8. No significant cross-reactivity or interference observed. 9. Storage: 2–8°C for frequent use; 6 months at -20°C. --- ### **STORAGE AND STABILITY** - Store all reagents at 2–8°C. - Unopened kits: 6 months at -20°C. - Once opened, use within the specified time frame. --- **Please read this instruction carefully before performing the assay.** **For research use only. Not for diagnostic or clinical applications.**

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