High-quality reagents, good instruments and correct operation are necessary conditions to ensure the accuracy and reliability of ELISA test results. The operation of ELISA differs due to the formation of solid-phase carriers, and domestic medical tests generally use plate-type dots. This article will describe the main points of attention in each operation step of plate ELISA. Bead type, tube type and magnetic ball ELSIA, foreign reagents are used in conjunction with special instruments, and both have detailed instructions for use. Strictly follow the regulations to ensure accurate results. the result of.
There are a wide range of specimens that can be used for ELISA determination. Body fluids (such as serum), secretions (saliva), and excrement (such as urine, feces) can be used as specimens to determine certain antibodies or antigen components. Some specimens can be measured directly (such as serum, urine), and some require pretreatment (such as feces and certain secretions). Most ELISA tests use serum as the specimen. Except for fibrinogen and anticoagulant, the other components of plasma are the same as serum. The preparation of plasma specimens requires the help of anticoagulants, and serum specimens can be obtained after the serum has naturally coagulated and the blood clot has shrunk. Except for special circumstances, serum is used as the test specimen in medical tests. Plasma and serum can be used equally in ELISA. Serum samples can be collected according to conventional methods. Care should be taken to avoid hemolysis. When red blood cells are lysed, substances with peroxidase activity will be released. In ELISA assays marked with HRP, hemolyzed samples may increase non-specific coloration.
Serum samples should be tested when they are fresh. If there is bacterial contamination, the bacteria may contain endogenous HRP, and false positive reactions may also occur. If stored in the refrigerator for a long time, it may polymerize, which can deepen the background in the indirect ELISA.
Generally speaking, serum samples measured within 5 days can be stored at 4°C, and those measured over a week need to be stored on ice. After the frozen serum is thawed, the protein is locally concentrated and unevenly distributed. It should be mixed thoroughly and should be gentle to avoid bubbles. It can be mixed upside down and do not vibrate strongly on the mixer. Serum specimens with turbidity or precipitation should be centrifuged or filtered first, and then tested after clarification. Repeated freezing and thawing will cause the antibody titer to drop. Therefore, if the serum sample for antibody testing needs to be stored for multiple tests, a small amount of aliquots should be stored on ice. Attention should be paid to aseptic operation when preserving serum since collection, and appropriate preservatives can also be added.
Prepare the reagents needed in the experiment according to the requirements of the kit instructions. The distilled or deionized water used in ELISA, including those used for washing, should be fresh and high-quality. The self-prepared buffer should be measured and corrected with a pH meter. The test reagents taken out of the refrigerator should be used after the temperature is balanced with the room temperature. The parts of the kit that are not needed for this test should be returned to the refrigerator for storage in time.
In ELISA, there are generally 3 sample loading steps, that is, adding specimen, adding enzyme conjugate, and adding substrate. When adding samples, add the added material to the bottom of the hole of the LEISA plate, avoid adding it to the upper part of the hole wall, and pay attention to not splashing or generating bubbles.
The sample is usually added with a micropipette, and the specified amount is added to the well of the plate. The suction nozzle should be replaced each time the specimen is added to avoid cross-contamination. A disposable quantitative plastic tube can also be used for sample injection. For this determination (such as indirect method ELISA), diluted serum is needed, which can be diluted in a test tube according to the specified dilution before adding the sample. It is also possible to add the diluent to the wells of the plate, and then add the serum sample to it, and then shake on the micro shaker for 1 minute to ensure mixing. When adding enzyme conjugate application liquid and substrate application liquid, a quantitative multi-channel dosing device can be used, so that the liquid addition process can be completed quickly.
In ELISA, there are generally two antigen-antibody reactions, that is, after adding the specimen and adding the enzyme conjugate. The completion of the antigen-antibody reaction requires a certain temperature and time. This incubation process is called incubation. Some people call it incubation, which seems inappropriate in ELISA.
ELISA is a solid-phase immunoassay, and the combination of antigen and antibody only occurs on the solid-phase surface. Taking the antibody-coated sandwich method as an example, the antigen in the sample added to the well does not have an equal chance of binding with the solid phase antibody. Only the antigen in the solution closest to the well wall directly contacts the antibody. . This is a gradual equilibrium process, so diffusion is required to reach the end of the reaction. The same applies to the binding of the enzyme-labeled antibody added afterwards to the solid phase antigen. This is why the ELISA reaction always requires a certain period of incubation.
Temperatures commonly used for incubation are 43°C, 37°C, room temperature and 4°C (refrigerator temperature). 37°C is a commonly used temperature in the laboratory, and it is also a suitable temperature for most antigen-antibody binding. When establishing the ELISA method to study the reaction kinetics, experiments show that the two antigen-antibody reactions are generally at 37°C for 1-2 hours, and the product generation can reach the peak. In order to accelerate the reaction, the temperature of the reaction can be increased. Some experiments are carried out at 43°C, but a higher temperature is not suitable. The antigen-antibody reaction is more thorough at 4°C. In the radioimmunoassay, the reaction is often left in the refrigerator overnight to form the most precipitate. But because it takes too long, it is generally not used in ELISA.
In addition to the special electric heating block attached to some ELISA instruments, the heat preservation method generally uses a water bath. The ELISA plate can be placed in a water bath, and the bottom of the ELISA plate should be attached to the water surface to quickly balance the temperature. In order to avoid evaporation, the plate should be covered, and the hole of the plate can also be covered with plastic sticker or plastic wrap. At this time, the reaction plate can be floated on the water. If an incubator is used, the ELISA board should be placed in a wet box. The wet box should be made of materials with good heat transfer properties, such as metal. Pad wet gauze at the bottom of the box, and finally put the ELISA board on the wet gauze. The wet box should first be placed in the incubator to pre-warm to the specified temperature, especially when the temperature is low. Whether it is a water bath or a wet box incubation, the reaction plates should not be stacked to ensure that the temperature of each plate can be quickly balanced. For the reaction of room temperature incubation, the room temperature during operation should be strictly limited within the specified range. The standard room temperature refers to 20-25°C, but the specific operation can be controlled according to the requirements of the instructions. When incubating at room temperature, the ELISA plate only needs to be placed flat on the operating table. It should be noted that the temperature and time of incubation should be as accurate as required. To ensure this, it is not advisable to measure more than two plates at the same time when operated by one person.
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