ELISA Workshop

By Ricardo Chiesa

ELISA is a technique used in immunology for the detection of an antibody or

antigen in a sample. An antigen is a molecule or substance that enters the body and is

recognized as a foreign material. As a result, it promotes the production of an antibody that

will 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 test

and diagnose of HIV in patients. First, the antigens were added to the cells of a microplate

strip in order for them bind to the surface. In this case, chicken gamma-globulin (purified

from egg yolks) was used as an antigen representative. After the wells were washed with

detergent, the primary antibodies (polyclonal rabbit antibodies) were added and they were

supposed to bind to the antigens, if there were any. Then, the secondary antibodies are

added, and they were supposed to bind to the primary antibodies. These secondary

antibodies 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 antibodies

were produced. Finally, the detection of secondary antibodies involves an enzyme-

substrate reaction. These antibodies contained an HRP enzyme bound to them that in the

presence of nitrogen peroxide will catalyze the oxidation of a substrate known as TMB, and

will produce a blue color. In the experiment, the substrate was added to the wells and

oxidation occurred in some of the wells.