What are the precautions for ELISA operation
(1) Sample addition: In addition to coating, 4 to 5 sample additions are generally required in ELISA. In qualitative determination, the accuracy of the amount of sample added is sometimes not emphasized, for example, 1 drop of sample is specified. At this time, a dropper with the same diameter should be used to maintain an accurate sample adding posture, so that the volume of each drop is basically the same. In quantitative determination, the amount of sample added should be accurate. Dilutions of specimens and conjugates should be prepared as specified. When adding the sample, the liquid should be added to the bottom of the well, avoid adding it to the upper part of the well wall, and be careful not to cause air bubbles.
(2) Incubation: There are generally two antigen-antibody reactions in ELISA, that is, after adding the sample and adding the conjugate, the temperature and time of the reaction should be in accordance with the specified requirements. balance. ELISA plates should not be stacked together. To avoid evaporation, the plates should be covered, or the plates should be placed in a wet box with damp gauze on the bottom. The wet box should be metal to transfer heat easily. If an incubator is used, an empty humidity box should be placed in it in advance to balance the temperature, this step is more important when the room temperature is low. After adding the substrate, the reaction time and temperature are usually not strictly required.
(3) Washing: Although washing is not a reaction step in the ELISA process, it is the key to success or failure. The purpose of washing is to remove substances that are not bound to the solid-phase antigen or antibody in the reaction and interfering substances that are non-specifically adsorbed to the solid-phase carrier during the reaction.
(4) Color development and termination reaction: Color development is an enzymatic reaction, which should be damaged at the specified temperature and time, and terminated with acid.
(5) Judgment of results: The results are determined with a colorimeter, and the accuracy depends on the smoothness and transparency of the bottom of the ELISA plate and the quality of the colorimeter.
Post time: Sep-20-2022