Explain indirect ELISA.
enzyme-linked immunosorbet assay
used for find antibody and determine whether they are producing antibodies against a particular antigen
In a small well, coat the bottom with antigen. Take blood sample from the animal and incubate it into the well with buffer
If they are producing antibodies for that antigen, it will bind and be attached.
Washing the solution to get rid of unbound proteins is the next step. Although it is bound, we cannot see it. As a result, we add a second antibody with marker molecule (or enzyme) attached
The two most common enzymes are peroxidse adn alkaline phosphatase. This enzyme does not alter the function of the antibody.
This second antibody with enzyme attached will bind to the first antibody
At this point, wash it again. Inject a substrate that will react with the enzyme, causing a product to form and a change in color of the solution. Yes, they are attached (This is called colorometric assay)