Collection, preservation and precautions of serum samples in ELISA experiments - Database & Sql Blog Articles

Collection, preservation and precautions of serum samples in ELISA experiments

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The test was performed on serum. Serum samples can be collected according to conventional methods. Care should be taken to avoid hemolysis. When red blood cells are dissolved, substances with peroxidase activity are released. In ELISA assays marked with HRP, hemolyzed specimens may increase non-specific color development. Serum samples should be tested fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, which may also produce false positive reactions. In general, serum samples measured within 5 days can be placed at 4 ° C, and the specimens are stored in the refrigerator for too long, resulting in the polymerization of serum IgG, which makes the reagent background of the indirect method deeper. It should be stored at -20 °C for more than one week. After freezing the serum, the protein is partially concentrated and unevenly distributed. It should be thoroughly mixed and avoid bubbles. Gradient or precipitated serum samples should be centrifuged or filtered before clarification. Repeated freezing and thawing will cause the antibody titer to fall, so if the serum sample of the test antibody needs to be stored for multiple tests, it should be stored in small amounts. Preservation of serum should be performed aseptically, and appropriate preservatives may be added.

Specimens with incomplete anticoagulation are false positive due to fibrinogen interference. It is recommended that anticoagulation, especially heparin anticoagulant, be used as much as possible.

Specific considerations:

1

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Care should be taken to avoid severe hemolysis. Hemoglobin contains a heme group, which has a peroxide-like activity. Therefore, in an ELISA assay using HRP as a labeling enzyme, if the hemoglobin concentration in a serum sample is high, it is easily adsorbed during the incubation. In the solid phase, it reacts with the HRP substrate added later to develop color.

2

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In the collection of samples and serum separation, attention should be paid to avoid bacterial contamination. In the case of bacterial growth, some enzymes secreted by the bacteria may decompose proteins such as antigens and antibodies; secondly, endogenous enzymes of some bacteria such as Escherichia coli Î’-galactosidase itself produces non-specific interference with assays labeled with the corresponding enzymes.

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If the serum sample is isolated by aseptic operation, it can be stored at 2-8 °C for one week. If it is operated by bacteria, it is recommended to freeze it. Long-term storage of the sample should be below -70 °C.

4

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Frozen serum samples should be taken care of to avoid repeated freezing and thawing caused by power outages. The mechanical shear force generated by repeated freezing and thawing of the specimen will destroy the molecules such as proteins in the specimen, causing false negative results. In addition, the mixing of freeze-thaw specimens should also be noted, do not violently shake, and repeatedly invert and mix.

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If the specimen is turbid or floc due to bacterial contamination during storage, the supernatant should be taken after centrifugation and sedimentation.

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