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 48-well or 96-well configurations. The kit includes: - Standard Diluent: 1.5 ml × 1 bottle - Enzyme Standard Reagent: 3 ml × 1 bottle (for 48-well) / 6 ml × 1 bottle (for 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:** - Kit Storage: 2–8°C - Shelf Life: 6 months from the date of manufacture **Purpose:** The Human Hydrocortisone (HYD) ELISA Kit is designed for the quantitative determination of HYD levels in human serum, plasma, urine, cell culture supernatants, tissue homogenates, and other biological fluids. **Principle of Operation:** This kit utilizes a sandwich ELISA method. The microtiter plate is pre-coated with a specific antibody against HYD. After adding the sample and enzyme-labeled antibody, a complex is formed. Following washing steps, TMB substrate is added, leading to a color change that is proportional to the HYD concentration. The reaction is stopped, and absorbance is measured at 450 nm using a microplate reader. The HYD concentration is then determined by comparing the sample OD value to a standard curve. **Sample Preparation Guidelines:** - **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. - **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well, centrifuge, and collect the supernatant. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant. - **Cell Culture Supernatant:** Centrifuge at 2000–3000 rpm for 20 minutes. - **Tissue Specimens:** Homogenize in PBS, centrifuge, and collect the supernatant. - **General Note:** All samples should be processed as soon as possible. If not tested immediately, store at -20°C, avoiding repeated freeze-thaw cycles. **Operation Steps:** 1. Prepare standard dilutions and load them into designated wells. 2. Add sample diluent and test samples to the respective wells. 3. Seal the plate and incubate at 37°C for 30 minutes. 4. Wash the plate five times with diluted washing solution. 5. Add enzyme-labeled reagent to each well except blank controls. 6. Incubate again for 30 minutes. 7. Add TMB substrate A and B, incubate for 15 minutes at 37°C. 8. Stop the reaction with stop solution. 9. Measure absorbance at 450 nm within 15 minutes. 10. Calculate HYD concentration using the standard curve. **Notes:** - Equilibrate the kit at room temperature before use. - Avoid cross-contamination by using a new sealing film for each experiment. - Ensure accurate pipetting and avoid light exposure during the color development step. - Always prepare a standard curve and run duplicates for better accuracy. - Do not mix reagents from different batches. - Follow all safety guidelines and dispose of waste properly. **Performance Characteristics:** - Linear regression correlation coefficient (R²) ≥ 0.95 - Intra-assay CV < 9%, Inter-assay CV < 11% - Detection range: 0.2 IU/L – 6 IU/L **Service Commitment:** We offer free technical support during working hours and assistance with sample testing upon request. Please contact us for further details. **Important:** This kit is for research use only. Do not use if the reagents are expired or damaged.

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