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. It 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 kit employs a double-antibody sandwich ELISA method to detect HPX. The microtiter plate is pre-coated with a specific antibody against rat HPX. After adding the sample, HPX binds to the immobilized antibody. A horseradish peroxidase (HRP)-labeled secondary antibody is then added, forming a complex of antibody–antigen–enzyme-labeled antibody. Following washing steps, the TMB substrate is introduced, which turns blue under HRP catalysis and changes to yellow upon acid termination. The color intensity is directly proportional to the HPX concentration in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, and the HPX concentration is determined by comparing the sample OD value to a standard curve. **Kit Components** - 48-well configuration: 1×48 coated plate, 2 sealing films, 1 standard, 1 standard dilution, 1 enzyme label reagent, 1 sample dilution, 1 developer A, 1 developer B, 1 stop solution, 1 concentrated wash solution (20×), 1 sample diluent - 96-well configuration: 1×96 coated plate, 2 sealing films, 1 standard, 1 standard dilution, 1 enzyme label reagent, 1 sample dilution, 1 developer A, 1 developer B, 1 stop solution, 1 concentrated wash solution (30×), 1 sample diluent All components should be stored at 2–8°C. **Sample Preparation** - **Serum**: Allow blood to clot at room temperature for 10–20 minutes, then centrifuge at 2000–3000 rpm for 20 minutes. Collect the supernatant; if precipitate forms, re-centrifuge. - **Plasma**: Use EDTA or sodium citrate as anticoagulant. Mix for 10–20 minutes, then centrifuge. Collect supernatant. - **Urine**: Centrifuge at 2000–3000 rpm for 20 minutes. - **Cell Culture Supernatant**: Centrifuge at 2000–3000 rpm for 20 minutes. For intracellular components, lyse cells via freeze-thaw cycles and centrifuge again. - **Tissue Homogenate**: Weigh the tissue, add PBS, homogenize, and centrifuge. Collect the supernatant. **Storage and Handling** - Store all samples at 2–8°C if not tested immediately. Avoid repeated freezing and thawing. - Do not use samples containing NaN3, as it may inhibit HRP activity. **Procedure Summary** 1. Prepare standard dilutions and load into designated wells. 2. Add sample diluent and test samples to respective wells. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate 5 times with diluted washing solution. 5. Add HRP-conjugated antibody and incubate again. 6. Add TMB substrate and develop color for 15 minutes. 7. Stop the reaction with stop solution. 8. Measure OD at 450 nm within 15 minutes. **Notes** - Allow the kit to reach room temperature before use. - Ensure accurate pipetting and avoid cross-contamination. - Always run a standard curve and consider sample dilution if OD values exceed the standard range. - Keep substrates away from light. - Follow the manual strictly for reliable results. - Dispose of all waste as biohazardous material. **Calculation** Plot the standard curve using HPX concentrations vs. OD values. Calculate the sample concentration based on the regression equation and multiply by the dilution factor. **Performance** - Intra-assay and inter-assay CVs < 9% and < 11%, respectively. - Linear range: 5 pg/mL – 230 pg/mL. **Storage and Shelf Life** - Store the kit at 2–8°C. - Validity: 6 months from the date of manufacture. This manual provides detailed instructions to ensure accurate and reproducible results when measuring HPX levels in rat samples.

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