Enzyme-linked immunosorbent assay (ELISA) 1. The principle and type of ELISA antibody or antigen can be adsorbed on a suitable carrier, the enzyme-labeled antibody or antigen corresponding antigen or antibody forms an enzyme-labeled antigen-antibody immune complex, on a certain substrate With the participation, the enzyme on the complex catalyzes the substrate to hydrolyze, oxidize or reduce it to another colored substance. Under certain conditions, the degradation substrate and the color of the enzyme are proportional, so you can use a spectrophotometer to determine the content of the antigen and antibody involved in the reaction. This is the principle of the ELISA technology established by Peter Perlmann and Eva Engvall. According to the different solid-phase carriers used, ELISA is divided into ordinary ELISA and Dot-ELISA. The different methods of adsorption of attracted antibodies or antigens on the solid-phase carrier are based on the use of different antibodies such as antibodies that can directly react with the antigen It is called primary antibody, an antibody labeled with an enzyme (commonly called an enzyme-labeled secondary antibody) that can react with the primary antibody in immune binding. ELISA differentiates a number of different measurement methods. ELISA is generally divided into the following types: Direct method: First, the antigen is adsorbed on the surface of the carrier, then the enzyme-labeled antibody is reacted with the antigen to form an enzyme-labeled immune complex, and finally the substrate is added to produce a colored substance, which is subjected to light Density measurement to calculate the amount of antigen present. Indirect method: first adsorb the antigen on the surface of the carrier, then add antiserum to bind the specific antibody (primary antibody) to the antigen, and then add enzyme-labeled antiglobulin (second antibody, ie, antibody against the primary antibody), It binds to the first antibody to form an enzyme-labeled immune complex. Finally, a substrate is added to produce a colored substance, the optical density is measured, and the amount of antibody present is calculated. Double antibody method: first adsorb the antibody on the surface of the carrier to form the surface of the antibody globulin sensitized carrier, then add the antigen solution to bind the antigen to the antibody, then add the enzyme-labeled antibody globulin, and the antigen adsorbed on the surface of the sensitized carrier Combined to form an enzyme-labeled immune complex, the substrate is finally added to produce a colored substance, the optical density is measured, and the amount of antigen present is calculated. The ELISA dual antibody method is also called the ELISA dual antibody sandwich method, which can reduce the interference of non-specific colors, and can be used as a blank control during the measurement. Enzyme-labeled antigen competition method: First, the antibody is adsorbed on the surface of the carrier to form the surface of the sensitized carrier of the antibody globulin, and then the antigen and enzyme-labeled antigen solution mixed in different proportions are added to react with the antibody adsorbed on the carrier. Formation of enzyme-labeled antigen and antibody immune complexes and non-labeled antigen and antibody immune complexes, and finally adding a substrate to determine the presence or absence of antigen and the amount of antigen in the unknown solution by the amount of substrate hydrolysis of the enzyme (determined by measuring optical density) How many. The enzyme-labeled antigen competition method uses mixed unlabeled antigen and enzyme-labeled antigen to compete with each other for the binding point on the antibody, thereby quantifying the antigen. The greater the amount of unlabeled antigen, the more inhibited the binding of enzyme-labeled antigen and antibody. However, the competition method is more complicated in the experiment. Sometimes after incubating the antigen mixture with the corresponding antibody, before measuring the activity of the free antigen and the antibody binding antigen, salting out, organic solvent precipitation, or second antibody precipitation should be used. Two different forms of antigen are separated. And after adding the substrate, it is easy to produce serum-colored substances. Therefore, a blank control is required for each determination. Because of these shortcomings, the enzyme-labeled antigen competition method is not widely used.
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