One of the classic methods of demonstrating the presence of antibody in a patient’s serum is the complement fixation (CF) test. This test consists of two separate systems. The first (the test system) consists of the antigen suspected of causing the patient’s disease and the patient’s serum. The second (the indicator system) consists of a combination of sheep red blood cells, complement-fixing antibody (IgG) raised against the sheep red blood cells in another animal, and an exogenous source of complement (usually guinea pig serum). When these three components are mixed together in optimum concentrations, the anti-sheep erythrocyte antibody binds to the surface of the red blood cells, and the complement then binds to the antigen-antibody complex, ultimately causing lysis (bursting) of the red blood cells. For this reason the anti sheep red blood cell antibody is also called hemolysin. For the CF test, these two systems are tested in sequence (Figure 1). The patient’s serum is first added to the putative antigen; then the limiting amount of complement is added to the solution. If the patient’s serum contains antibody to the antigen, the resulting antigen antibody complexes bind all the complement added. In the next step, the sheep red blood cells and the hemolysin (indicator system) are added. The patient’s complement is available to bind to the sheep cell–hemolysin complexes and cause lysis if the complement has not been bound by a complex formed with antibody from the patient’s serum. A positive result, meaning the patient has complement-fixing antibodies, is revealed by failure of the red blood cells to lyse in the final test system. Lysis of the indicator cells indicates lack of antibody and a negative CF test result.

Fig1.  Complement fixation test. 

Although this test requires many manipulations, takes at least 48 hours to complete both stages, and often yields nonspecific results, it has been used for many years to detect many types of antibodies, particularly antiviral and antifungal antibodies. Many new systems have gradually been introduced to replace the CF test, because they demonstrate improved recovery of pathogens or their products and provide more sensitive and less demanding procedures for detecting antibodies, such as particle agglutination, indirect fluorescent antibody tests, and enzyme-linked immunosorbent assay (ELISA). CF tests are performed chiefly for diagnosis of unusual infections and are done primarily in laboratories.

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