Human Hydrocortisone (HYD) elisa Kit Instruction Manual

**Human Hydrocortisone (HYD) ELISA Kit Instruction Manual** **Kit Specifications:** This Human Hydrocortisone (HYD) ELISA Kit is available in a 48-well or 96-well configuration. The kit includes the following components: - Standard Diluent: 1.5 ml × 1 bottle - Enzyme Standard Reagent: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well) - Sealing Film: 2 pieces (48-well) / 2 pieces (96-well) - Standard: 0.5 ml × 1 bottle, concentration of 2700 ng/L - Sample Diluent: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well) - Developer A: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well) - Chromogen B: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well) - Stop Solution: 3 ml × 1 bottle (48-well) / 6 ml × 1 bottle (96-well) - Concentrated Washing Solution: 20 ml × 20 times (48-well) / 20 ml × 30 times (96-well) **Storage Conditions and Expiration Date:** - Storage: 2–8°C - Shelf Life: 6 months from the date of receipt **Purpose:** The Human Hydrocortisone (HYD) ELISA Kit is designed for the quantitative determination of HYD in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **Principle of Operation:** This ELISA kit uses a double-antibody sandwich method. The microtiter plate is pre-coated with a specific antibody against HYD. After adding the sample, HYD binds to the immobilized antibody. A second enzyme-labeled antibody is then added, forming an immune complex. After washing, TMB substrate is added, and the color develops under the catalytic action of HRP. The reaction is stopped, and the absorbance is measured at 450 nm. The HYD concentration is calculated based on a standard curve. **Sample Preparation and Handling:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. - **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge as above. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. - **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. - **Tissue Homogenate:** Weigh the tissue, add PBS, homogenize, and centrifuge. - **Storage:** Samples should be processed immediately after collection. If not tested right away, store at -20°C, avoiding repeated freeze-thaw cycles. **Operation Steps:** 1. **Standard Dilution:** Prepare a serial dilution of the standard solution in the 96-well plate. 2. **Sample Loading:** Add 40 μl of sample diluent and 10 μl of sample to each well. 3. **Incubation:** Seal the plate and incubate at 37°C for 30 minutes. 4. **Washing:** Wash the plate 5 times with diluted washing solution. 5. **Enzyme Addition:** Add 50 μl of enzyme-labeled reagent to each well. 6. **Second Incubation:** Incubate again at 37°C for 30 minutes. 7. **Color Development:** Add 50 μl of TMB A and B, incubate for 15 minutes at 37°C. 8. **Stop Reaction:** Add 50 μl of stop solution to each well. 9. **Measurement:** Read the OD values at 450 nm within 15 minutes. **Notes:** - Equilibrate the kit at room temperature for 15–30 minutes before use. - Avoid cross-contamination by using a new sealing film for each test. - Always prepare a standard curve and run duplicates for accuracy. - Do not mix reagents from different batches. - Handle all samples and waste as biohazardous materials. **Performance:** - Intra-assay and inter-assay CVs < 9% and < 11%, respectively. - Linear range: 0.2 IU/L – 6 IU/L. - Correlation coefficient (R²) > 0.95. **Service Commitment:** We provide free technical support during working hours. Sample testing services are also available upon request to ensure optimal results. **Important:** This product is intended for research use only. Do not use if the reagents show signs of degradation or contamination.

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