Enzyme-linked immunosorbent assay (ELISA) operation points - Database & Sql Blog Articles

First, the adoption and preservation of specimens

Specimens that can be used as elisa assays are widely available, and body fluids (such as serum), secretions (saliva), and excreta (such as urine, feces) can be used as specimens to determine an antibody or antigen component. Some specimens can be directly measured (such as serum, urine), while others require pretreatment (such as feces and certain secretions). Most ELISA tests use serum as a specimen. In addition to fibrinogen and anticoagulant, the plasma is equivalent to serum. Preparation of plasma specimens requires the use of anticoagulants, and serum specimens can be obtained as long as the serum naturally solidifies and the blood clot shrinks. In addition to special cases, serum was used as a test specimen in medical tests. Plasma and serum can be used equally in the ELISA. Serum samples can be collected according to conventional methods, care should be taken to avoid hemolysis, red blood cells

When dissolved, a substance with peroxidase activity is released. In an ELISA assay labeled 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. If stored in the refrigerator for a long time, the polymerization can occur, and the background can be deepened in the indirect ELISA. In general, serum samples measured within 5 days can be placed at 4 ° C, and cryopreservation is required for more than one week. After the frozen serum is melted, the protein is partially concentrated and unevenly distributed. It should be mixed well and gently, avoiding air bubbles, and mixing upside down, do not oscillate strongly on the mixer. 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. 4
Second, the reagent preparation According to the requirements of the kit instructions to prepare the reagents used in the experiment. Distilled or deionized water used in ELISA, including for washing, should be fresh and of high quality. The self-contained buffer is measured using a pH meter. The test reagent taken out of the refrigerator should be used after the temperature and room temperature are balanced. The parts of the kit that are not needed for this test should be returned to the refrigerator in time for storage.
Third, the sample is usually added in the ELISA three times, such as adding the specimen, adding the enzyme combination, adding the substrate. When adding the sample, add the added material to the bottom of the LEISA plate hole, avoid adding it to the upper part of the hole wall, and be careful not to splash it, and do not generate bubbles. The spiked sample is generally added to the well of the plate in a prescribed amount using a micropipette. The nozzle should be replaced every time the specimen is added to avoid cross-contamination. It can also be loaded with a disposable quantitative plastic tube. In this assay (eg, indirect ELISA), diluted serum is used, which can be diluted in a test tube at the specified dilution before loading. Dilutions can also be added to the wells, serum samples are added to them, and then shaken on a micro-vibrator for 1 minute to ensure mixing. When adding the enzyme conjugate application liquid and the substrate application liquid, a quantitative multi-channel dosing device can be used to complete the dosing process quickly.
4. Insulation In the ELISA, there are generally two antigen-antibody reactions, that is, after adding the specimen and adding the enzyme combination. The completion of the antigen-antibody reaction requires a certain temperature and time. This incubation process is called incubation, which is called incubation and may not be appropriate in ELISA. ELISA is a solid phase immunoassay in which antigen and antibody binding occurs only on the surface of a solid phase. Taking the sandwich method of antibody coating as an example, the specimen added to the well of the plate does not have an equal opportunity to bind to the solid phase, and only the antigen in the solution closest to the pore wall is directly in contact with the antibody. . This is a process of gradual equilibrium, so it needs to be diffused to reach the end of the reaction. The same applies to the binding of the enzyme-labeled antibody added thereafter to the solid phase antigen. This is why ELISA reactions always require a certain amount of incubation.

The temperatures commonly used for incubation are 43 ° C, 37 ° C, room temperature and 4 ° C (refrigerator temperature). 37 ° C is the usual incubation temperature in the laboratory, and is also the appropriate temperature for most antigen-antibody binding. When the ELISA method was established as the reaction kinetics study, the experiment showed that the two antigen-antibody reactions generally reached the peak at 37 ° C for 1-2 hours. In order to accelerate the reaction, the temperature of the reaction can be raised. Some tests are carried out at 43 ° C, but higher temperatures are not suitable. The antigen-antibody reaction was more thorough at 4 ° C, and the reaction was mostly allowed to stand overnight in the refrigerator in a radioimmunoassay to form the most precipitate. However, because the time required is too long, it is generally not used in ELISA.