20.2 Detecting antigen-antibody complexes  (Page 7/12)

Typically, polyclonal antibodies are used for western blot assays. They are more sensitive than mAbs because of their ability to bind to various epitopes of the primary antigen, and the signal from polyclonal antibodies is typically stronger than that from mAbs. Monoclonal antibodies can also be used; however, they are much more expensive to produce and are less sensitive, since they are only able to recognize one specific epitope.

Several variations of the western blot are useful in research. In a southwestern blot , proteins are separated by SDS-PAGE, blotted onto a nitrocellulose membrane, allowed to renature, and then probed with a fluorescently or radioactively labeled DNA probe; the purpose of the southwestern is to identify specific DNA-protein interactions. Far-western blots are carried out to determine protein-protein interactions between immobilized proteins (separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and allowed to renature) and non-antibody protein probes. The bound non-antibody proteins that interact with the immobilized proteins in a far-western blot may be detected by radiolabeling, fluorescence, or the use of an antibody with an enzymatic molecular beacon.

Complement-mediated immunoassay

One of the key functions of antibodies is the activation (fixation) of complement. When antibody binds to bacteria, for example, certain complement proteins recognize the bound antibody and activate the complement cascade . In response, other complement proteins bind to the bacteria where some serve as opsonins to increase the efficiency of phagocytosis and others create holes in gram-negative bacterial cell membranes, causing lysis. This lytic activity can be used to detect the presence of antibodies against specific antigens in the serum.

Red blood cells are good indicator cells to use when evaluating complement-mediated cytolysis. Hemolysis of red blood cells releases hemoglobin, which is a brightly colored pigment, and hemolysis of even a small number of red cells will cause the solution to become noticeably pink ( [link] ). This characteristic plays a role in the complement fixation test , which allows the detection of antibodies against specific pathogens. The complement fixation test can be used to check for antibodies against pathogens that are difficult to culture in the lab such as fungi, viruses, or the bacteria Chlamydia .

To perform the complement fixation test, antigen from a pathogen is added to patient serum. If antibodies to the antigen are present, the antibody will bind the antigen and fix all the available complement. When red blood cells and antibodies against red blood cells are subsequently added to the mix, there will be no complement left to lyse the red cells. Thus, if the solution remains clear, the test is positive. If there are no antipathogen antibodies in the patient’s serum, the added antibodies will activate the complement to lyse the red cells, yielding a negative test ( [link] ).