Human Survival Protein (Surv) ELISA Kit Instructions for Use

**Human Survivin (Surv) ELISA Kit Instruction Manual** This reagent is intended for research use only and is designed to quantitatively measure the level of Human Survivin (Surv) in human serum, plasma, and other biological fluids. The assay is based on the double-antibody sandwich ELISA method. A microtiter plate pre-coated with a specific anti-Human Surv antibody is used as the solid phase. After adding the sample, Surv is captured by the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following washing steps, TMB substrate is introduced, which turns blue under HRP catalysis and changes to yellow when acid is added. The intensity of the color is directly proportional to the concentration of Surv in the sample. Absorbance is measured at 450 nm using a microplate reader, and the Surv concentration is determined by comparing the OD values to a standard curve. **Kit Components:** - 48-well or 96-well configuration - Coated microtiter plate (1 × 48 or 1 × 96) - Standard: 0.5 mL × 1 bottle (180 U/mL) - Standard Diluent: 1.5 mL × 1 bottle - Enzyme Conjugate: 3 mL × 1 bottle - Sample Diluent: 3 mL × 1 bottle - TMB Substrate A & B: 3 mL × 1 bottle each - Stop Solution: 3 mL × 1 bottle - Wash Buffer (20×): 20 mL × 20 times or 20 mL × 30 times - Sealing Films: 2 pieces (for 48-well), 2 pieces (for 96-well) - Storage: All components should be stored at 2–8°C **Sample Preparation Guidelines:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant; if precipitate forms, re-centrifuge. - **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix and centrifuge similarly. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. - **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles and centrifuge again. - **Tissue Samples:** Homogenize in PBS, centrifuge, and collect supernatant. Store at 2–8°C or freeze at -20°C if not used immediately. **Procedure Summary:** 1. Prepare standards by serial dilution. 2. Add samples and blanks to the microtiter plate. 3. Incubate at 37°C for 30 minutes. 4. Wash 5 times with diluted wash buffer. 5. Add enzyme conjugate and incubate again. 6. Add TMB substrate and incubate for 15 minutes. 7. Stop the reaction with stop solution. 8. Measure absorbance at 450 nm. **Notes:** - Equilibrate the kit at room temperature before use. - Avoid repeated freezing and thawing of samples. - Do not use samples containing NaN3. - Always prepare a standard curve and use duplicate wells for accuracy. - Keep all reagents away from light. - Follow instructions carefully and dispose of waste as biohazardous material. **Performance Characteristics:** - Linear range: 5–150 U/mL - Intra- and inter-assay variation <9% and <11%, respectively - Correlation coefficient (R²) ≥ 0.95 **Storage and Shelf Life:** - Store the kit at 2–8°C. - Shelf life: 6 months from the date of manufacture. This manual provides detailed guidance for accurate and reliable detection of Human Survivin levels in various biological samples. Ensure proper handling and storage to maintain kit integrity and test performance.

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