ELISA

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ELISA

  1. 1. ELISA Workshop By Ricardo Chiesa ELISA is a technique used in immunology for the detection of an antibody orantigen in a sample. An antigen is a molecule or substance that enters the body and isrecognized as a foreign material. As a result, it promotes the production of an antibody thatwill bind to the antigen and neutralize it, avoiding any type of infection for the organism.This process is known as immunization. In the lab, ELISA was done to simulate the testand diagnose of HIV in patients. First, the antigens were added to the cells of a microplatestrip in order for them bind to the surface. In this case, chicken gamma-globulin (purifiedfrom egg yolks) was used as an antigen representative. After the wells were washed withdetergent, the primary antibodies (polyclonal rabbit antibodies) were added and they weresupposed to bind to the antigens, if there were any. Then, the secondary antibodies areadded, and they were supposed to bind to the primary antibodies. These secondaryantibodies were obtained from goats by injecting rabbit’s antibodies into them. As a result,rabbit’s antibodies were recognized by the goat’s immune system as antigens, so antibodieswere produced. Finally, the detection of secondary antibodies involves an enzyme-substrate reaction. These antibodies contained an HRP enzyme bound to them that in thepresence of nitrogen peroxide will catalyze the oxidation of a substrate known as TMB, andwill produce a blue color. In the experiment, the substrate was added to the wells andoxidation occurred in some of the wells.

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