Rat heme binding protein (HPX) ELISA kit instruction manual

**Rat Heme Binding Protein (HPX) Enzyme-Linked Immunosorbent Assay (ELISA) Kit Instruction Manual** This reagent is intended for research purposes only. The kit is designed to quantitatively determine the levels of Heme Binding Protein (HPX) in rat serum, plasma, cell culture supernatants, and other related biological fluids. **Principle of the Assay:** The Rat HPX ELISA Kit utilizes a double-antibody sandwich immunoassay. A microtiter plate is pre-coated with a specific antibody against rat HPX. After incubation with the sample, the HPX present in the sample binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming an immune complex. Following washing steps, the substrate TMB is added, and a color change occurs under the catalytic action of HRP. The reaction is stopped by adding an acidic solution, which turns the blue color into yellow. The intensity of the yellow color is directly proportional to the concentration of HPX in the sample. The optical density (OD) at 450 nm is measured using a microplate reader, and the HPX concentration is calculated based on a standard curve. **Kit Components:** - 48-well or 96-well configuration - Microtiter plate coated with HPX antibody - Standard (270 pg/mL) - Standard Diluent - Enzyme-labeled Reagent - Sample Diluent - TMB Substrate A & B - Stop Solution - Wash Buffer (concentrated, 20x or 30x) - Sealing Film - Storage Bag **Storage Instructions:** All components should be stored at 2–8°C. The kit has a shelf life of 6 months from the date of manufacture. **Sample Preparation Guidelines:** 1. **Serum:** Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant carefully. 2. **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix well and centrifuge similarly. 3. **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. 4. **Cell Culture Supernatant:** Centrifuge after collection. For intracellular components, lyse cells via freeze-thaw cycles before centrifuging. 5. **Tissue Samples:** Homogenize in PBS, centrifuge, and collect the supernatant. 6. **General:** Avoid repeated freezing and thawing. Store samples at -20°C if not tested immediately. Do not use samples containing NaN3, as it inhibits HRP activity. **Procedure Summary:** 1. Prepare standards by serial dilution. 2. Add samples and blanks to the microplate. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate five times with diluted wash buffer. 5. Add enzyme-labeled reagent and incubate again. 6. Add TMB substrate and incubate for 15 minutes. 7. Stop the reaction with stop solution. 8. Measure OD at 450 nm within 15 minutes. 9. Plot a standard curve and calculate HPX concentrations. **Notes:** - Allow the kit to reach room temperature before use. - Ensure accurate pipetting and avoid cross-contamination. - Always run a standard curve in duplicate. - Keep all reagents away from light. - Follow the manual strictly for reliable results. - Dispose of all waste as biohazardous material. **Performance Characteristics:** - Intra-assay and inter-assay variation <9% and <11%, respectively. - Detection range: 5 pg/mL to 230 pg/mL. - Correlation coefficient (R²) ≥ 0.990. **Important:** This manual is for reference only. In case of discrepancies, the English version takes precedence.

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