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 PROCEDURES.** Before using this kit, please read the entire package insert carefully. This ELISA (Enzyme-Linked Immunosorbent Assay) is specifically designed for research purposes and is not intended for diagnostic or therapeutic use. The kit allows for the accurate quantification of EPO levels by measuring optical density (OD) at 450 nm. The principle of the assay involves a competitive binding reaction where EPO in the sample competes with a pre-coated EPO on the microplate for binding sites. After incubation and washing steps, a chromogenic substrate is added, and the color change is stopped with a stop solution. The intensity of the color is proportional to the EPO concentration, which can be determined by comparing the OD values of the samples to a standard curve. --- **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 centrifuging 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:** Remove particulates by centrifugation. 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 (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 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 kits. Each component is matched for optimal performance. 2. Only use reagents provided by the manufacturer. 3. Allow all reagents and materials to reach room temperature (20–25°C) before use. 4. Do not use water baths to thaw samples or reagents. 5. Do not use reagents past their expiration date. 6. Use only deionized or distilled water for dilution. 7. Keep microtiter plates in their sealed bags until needed. Store unused strips at 2–8°C with desiccant. 8. Use fresh pipette tips for each transfer to prevent cross-contamination. 9. Do not mix disposable knives or any items that may come into contact with biological material. 10. All samples should be treated as potentially infectious. Follow biosafety protocols. 11. Dispose of all waste properly, including liquid waste. Add sodium hypochlorite to a final concentration of 1.0% and let stand for at least 30 minutes before disposal. 12. Substrate Solution A may turn blue if contaminated. If so, do not use. 13. Substrate 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 the assay. It is recommended to run standards and samples in duplicate. 2. Add 50 µL of standard or sample to the appropriate wells. Leave the blank well empty. 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 microtiter 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, fill with 350 µL per well, then invert and blot dry. 5. Add 50 µL of Chromogen Solution A and 50 µL of Chromogen Solution 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. Read OD at 450 nm within 15 minutes. --- **Calculation:** - Plot the average OD values (450 nm) of the six standards against their respective concentrations to create a standard curve. - Subtract the blank OD value from all measurements before interpreting results. - Use graph paper or software to construct the curve. - Locate the OD value of the sample on the Y-axis, draw a horizontal line to the curve, and then a vertical line to the X-axis to determine the EPO concentration. - Variability in results may occur due to differences in technique, incubation time, temperature, or kit age. Each user should generate their own standard curve. --- **Additional Information:** - **Intra-assay CV (%):** 0.75 mIU/mL – 24 mIU/mL - **Sensitivity:** Typically less than 1.0 ng/mL - **Cross-reactivity:** No significant cross-reactivity or interference observed - **Storage:** 2–8°C (for frequent use); 6 months at -20°C Please follow all instructions carefully to ensure accurate and reliable results. Always refer to the user manual for detailed guidance.

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