Collection, Preservation, and Handling of Serum Samples in ELISA Experiments
ELISA tests are typically performed on serum samples. These samples can be collected using standard procedures, but care must be taken to avoid hemolysis. When red blood cells rupture, they release substances with peroxidase-like activity, which may lead to non-specific color development in HRP-labeled ELISA assays. Therefore, it's crucial to handle the samples gently during collection and centrifugation to prevent this issue. Fresh serum samples are ideal for testing. However, if storage is necessary, samples should be kept at 4°C for up to 5 days. Prolonged storage at this temperature may cause IgG polymerization, leading to increased background in indirect ELISA methods. For longer-term storage (more than a week), serum should be stored at -20°C. After freezing, proteins may become concentrated or unevenly distributed, so it's important to mix the sample thoroughly before use and avoid creating air bubbles. If the serum appears cloudy or has precipitates, it should be centrifuged or filtered before proceeding. Repeated freeze-thaw cycles can degrade antibodies and reduce their titer, so it's best to aliquot the samples into small volumes if multiple tests are planned. Additionally, maintaining aseptic conditions during handling is essential to prevent bacterial contamination, which can introduce endogenous enzymes like HRP or β-galactosidase that may interfere with results. Bacterial contamination not only introduces unwanted enzymes but also risks degrading antigens and antibodies. To minimize these issues, anticoagulants such as heparin are recommended for blood collection, especially when complete anticoagulation is needed. Incomplete anticoagulation can lead to false positives due to fibrinogen interference. Specific considerations include: 1. Avoid severe hemolysis, as hemoglobin contains heme groups that can mimic peroxidase activity. High levels of hemoglobin may adsorb onto solid-phase surfaces and react with HRP substrates, causing false signals. 2. During sample collection and serum separation, ensure no bacterial contamination occurs. Bacteria may secrete enzymes that degrade antigens or antibodies, or they may produce endogenous enzymes that interfere with enzyme-labeled assays. 3. If collected under aseptic conditions, serum can be stored at 2–8°C for up to one week. If not, it’s better to freeze the sample immediately. For long-term storage, keep the samples below -70°C. 4. Avoid repeated freezing and thawing, as mechanical stress can damage proteins and lead to false-negative results. Also, do not shake or invert the sample forcefully; gentle mixing is preferred. 5. If the serum becomes turbid or forms clumps due to bacterial growth, centrifuge the sample to collect the supernatant for further use. By following these guidelines, you can ensure the quality and reliability of your ELISA results while minimizing potential errors caused by improper sample handling.Usb Cable,Usb Charging Cables,Android Iphone Data Cable,Flat Micro Usb Cable
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