Antibodies in Research 

Antibodies are proteins made by B cells, part of the body’s immune sys­tem. The normal function of antibodies is to latch onto foreign substances (antigens) and flag them for destruction, thus helping to fight infection. This ability to bind to specific molecules makes them ideal probes in cell research, where they are used to latch onto, and thus help isolate and identify, mol­ecules of interest in and on cells. Antibodies have become one of the most important tools for studying protein function in cells.

To see how antibodies are used, consider the challenge of determining where actin is located in a nerve cell. Actin is a protein that forms part of the cytoskeleton, giving internal structure to the cell much like the human skeleton does. First, purified actin is used to trigger an immune reaction in a rabbit. The B cells that make the anti-actin antibodies are then isolated and fused with tumor cells. Unlike B cells, tumor cells will grow forever in the lab, and thus can supply large amounts of anti-actin antibodies indefi­nitely. These can be harvested from the cells in large quantities. The re­sulting antibodies are called “monoclonal” antibodies, because they derive from identical (cloned) cells.

An enzyme-linked immunosorbent assay (ELISA) plate, an immunologic test that can be used to indicate pregnancy or HIV infection by detecting very small quantities of antigens and antibodies (HTLV-III viral antibodies, in this case).

Next, in order to make the antibodies visible once inside the cell, a flu­orescent molecule is attached to them. They are then injected into the cell using a very fine glass needle. Once an antibody encounters actin, it attaches to it. The cell can then be examined under the light microscope, where the fluorescent molecules will light up, revealing the location of the actin. Of course it is not just actin that can be found this way; any protein to which we can make an antibody can be located in the cell.

Several modifications and extensions of this basic procedure are possi­ble. To mark more than one protein at a time, a set of different antibodies is used, each marked with a differently colored fluorescent tag. In this way, for instance, the spatial relations between actin and other cytoskeleton pro­teins can be visualized. Instead of fluorescent tags, antibodies can be at­tached to gold particles, which will show up under the electron microscope. Outside of cells, antibodies attached to glass beads can grab proteins out of a homogenized cell puree, allowing the protein to be isolated for further study. In one widely used technique called western blotting, fluorescently tagged antibodies are used to locate proteins of interest that have been sep­arated in electrophoresis gels.

Antibodies are also used in a test, or assay, called the enzyme-linked immunosorbent assay (ELISA). This is the assay used in the home preg­nancy test, which detects the presence of human chorionic gonadotropin (HCG), produced by human embryos. The test kit contains an antibody to HCG, which traps HCG if it is present in a woman’s urine. Next, a second antibody to HCG is added, which will bind to the HCG if it is trapped. This antibody is linked to an enzyme called peroxidase. Chemicals are then added which the peroxidase will cause to react, making a color change. The color change will only occur if the enzyme is present, and the enzyme will only be present if the HCG is present. Therefore, a color change indicates pregnancy. An ELISA test is also used to screen for HIV (human immun­odeficiency virus) infection. In this case, the test kit contains HIV proteins, which bind to anti-HIV antibodies in the patient’s blood.

It is the specificity of the antibody-antigen reaction, combined with the ability to link one or the other to fluorescent tags, enzymes, or other mark­ers, that makes antibodies such versatile tools in both basic and clinical re­search.

References

ELISA at the Biology Project. http://www.biology.arizona.edu/immunology/activities/ elisa/main.html.

Roitt I., D. Male, and J. Brostof. Immunology, 5th ed. New York: Mosby, 1998.