ENZYME LINKED IMMUNOSORBANT ASSAY (ELISA)
An important application of
antibodies is their use in the diagnosis and monitoring of diseases.
The reaction between antibodies and antigens is very specific and it
can be measured easily by a variety of techniques. This makes
possible a large number of very sensitive and accurate clinical
laboratory tests collectively called
serological tests.
Serology literally
means the study of serum and these tests use serum as a source of
antibodies.
Serological tests are very powerful and versatile. They can be used to identify microorganisms even in mixed culture. These tests can also measure the immune status of an individual patient. For example one could determine the ability of a person to mount an immune response or one could determine exactly which bacterial or viral diseases a person has had in their lifetime or is suffering from presently. Serology is also used for cancer detection, the diagnosis of allergies and in tissue and blood typing for transplantation and transfusion.
We will cover, in a demonstration, a technique which is based on antibody-antigen reactions but which also involves the use of an enzyme substrate reaction to indicate the presence of an antigen-antibody reaction. This type of assay, called an ELISA assay is described in your Tortora, et. al. textbook.
The Immunodiagnosis of HIV Status by the ELISA Technique.
In this simulation, the serum from three patients is being tested on the microtiter ELISA plates supplied for your laboratory. A negative control serum and a positive control serum are also being tested on this plate.
All 96 wells of this plate were coated with a preparation of HIV-1 virus which was inactivated and dried down into each of the wells. This is the antigen and the point of the assay will be to determine whether the individual patients have antibody to this virus.
The first step is to make dilutions of each of the test sera and the control sera and to apply these dilutions to the wells of the microtiter plate. Figure 1 describes how the serum samples were applied.
After a one hour incubation the whole plate is washed three times with a buffer solution to completely remove any antibodies which did not attach to the antigen on the plate.
A dilution of Protein A - Horseradish Peroxidase conjugate is applied to all wells and allowed to incubated for one hour. Protein A is a protein isolated fromStaphylococcus aureus and this protein has the ability to bind very tightly to the Fc region of Human IgG. In order to be useful in this assay the Protein A has been covalently bonded to a peroxidase isolated from horseradish. Horseradish peroxidase in the presence of peroxide can oxidase a number of substrates to form a brown colored product.
This is where you come in.:
After a one hour incubation the whole plate is washed three times with a buffer solution to completely remove any Protein A which did not attach to the antibody attached to the antigen on the plate.
A solution of 5-amino salicylic acid and H2O2 is applied to all wells. Two small drops with a pasteur pipette will be sufficient. The plate is then incubated at room temperature for one hour and observed for color reaction. A brown color is a positive test. Since dilutions of serum were tested you should also be able to determine the titer of each serum.