Complement-Mediated Immunoassay

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Diagram of the complement fixation test. 1. Patient A’s serum contains antibodies to the suspected antigen. Patient B’s serum does not. Both patients have complement, but different amounts. 2. Heating the serum destroys all of the complement in the patient’s serum. Antibodies remain in Patient A’s serum. 3. An equal amount of complement is then added to the serum for both patients. Antigens are also added. In patient A’s serum, antibodies bind to antigens and complement fixation occurs. Patient B’s serum lacks antibodies, so complement fixation does not occur. 4. Sheep RBCs and antibodies to sheep RBCs are added to both samples. 5. In patient A, complement is already fixed and cannot lyse RBCs. The antibodies bind to RBCs and settle to the bottom. In patient B, antibodies bind to RBCs and complement lyses the RBCs. Serum turns pink.
The complement fixation test is used to determine whether a patient’s serum contains antibodies to a specific antigen. If it does, complement fixation will occur, and there will be no complement available to lyse the antibody-bound sheep red blood cells that are added to the solution in the next step. If the sample does not contain antibodies to the antigen, hemolysis of the sheep blood cells will be observed.

Source: OpenStax Microbiology

OpenStax Microbiology

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. 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.

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology

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