Abnormal prothrombin (APT) elisa sample determination

This abnormal prothrombin (APT) ELISA kit is intended for research purposes only and is designed to quantitatively measure the level of abnormal prothrombin in human serum, plasma, urine, cell culture supernatants, and other biological fluids. The assay is based on the sandwich ELISA method, which uses a pair of specific antibodies to capture and detect the target antigen. The process begins with the immobilization of a purified anti-APT antibody onto the microplate wells. After incubation with the sample, APT binds to the immobilized antibody. Subsequently, an HRP-conjugated secondary antibody is added, forming a complex of antibody-antigen-enzyme-labeled antibody. Following a thorough wash step, TMB substrate is introduced, which reacts with the HRP enzyme to produce a blue color that turns yellow upon acid termination. The intensity of the color is directly proportional to the concentration of APT in the sample. A microplate reader is used to measure the optical density (OD) at 450 nm, and the results are calculated by comparing the sample OD values to a standard curve generated from known concentrations of APT. **Kit Components:** - Microtiter plate (48 or 96 wells) - Standard (36 ng/ml, 0.5 ml/bottle) - Standard diluent (1.5 ml/bottle) - Enzyme-labeled reagent (3 ml/bottle) - Sample diluent (3 ml/bottle) - TMB substrate A and B (3 ml/bottle each) - Wash buffer (20 ml × 20 or 30 times) - Sealing film - Instruction manual **Storage Conditions:** All components should be stored at 2–8°C. The enzyme-labeled plate must be kept sealed to prevent contamination. **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 and re-centrifuge if precipitate forms. - **Plasma:** Use EDTA or sodium citrate as anticoagulant. Mix and centrifuge similarly. - **Urine:** Centrifuge at 2000–3000 rpm for 20 minutes. Collect supernatant carefully. - **Cell Culture Supernatant:** Centrifuge after collection. For intracellular proteins, lyse cells via freeze-thaw cycles before centrifuging. - **Tissue Samples:** Homogenize in PBS, centrifuge, and collect the supernatant. Store samples at 2–8°C after thawing. **Important Notes:** - All samples should be processed as soon as possible after collection. If not tested immediately, store at -20°C and avoid repeated freezing and thawing. - Avoid using samples containing NaN3, as it can inhibit HRP activity. - Ensure proper dilution of samples if their OD exceeds the highest standard well. Perform duplicate measurements for accuracy. - Always use a new sealing film for each experiment to prevent cross-contamination. - Handle all reagents and waste as biohazardous materials. - Do not mix reagents from different batches. - Follow the manual instructions strictly. Results must be interpreted based on the microplate reader readings. **Procedure Summary:** 1. Prepare standards by serial dilution. 2. Add sample and standard solutions to the microplate. 3. Incubate at 37°C for 30 minutes. 4. Wash the plate thoroughly. 5. Add enzyme-labeled reagent and incubate again. 6. Add TMB substrate and develop color for 15 minutes. 7. Stop the reaction with stop solution. 8. Read absorbance at 450 nm within 15 minutes. This detailed protocol ensures accurate and reproducible detection of abnormal prothrombin levels, making it a reliable tool for research applications.

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