Complement fixation test

The complement fixation test is an immunological medical test that can be used to detect the presence of either specific antibody or specific antigen in a patient's serum. It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by other serological methods such as ELISA and by DNA-based methods of pathogen detection, particularly PCR.

The CF test uses sheep red blood cells (sRBC), pre-bound by anti-sRBC antibody, and serum (usually from rabbit) as a source of complement, which is a system of serum proteins that react with antigen-antibody complexes. If this reaction occurs on a cell surface, it will result in the formation of trans-membrane pores and therefore destruction of the cell. Accordingly, if the antibody-sensitized sRBC are brought into contact with active complement, they will undergo disintegration (hemolysis).

Complement will also react with antigen-antibody complexes in solution. The complement is thereby expended and can no longer trigger hemolysis; inhibition of complement hemolysis therefore indicates the presence of antigen-antibody complexes. A patient's serum containing a certain antibody (specific for, say, rubella virus) will yield antigen-antibody complexes after addition of the corresponding antigen (inactivated rubella virus in our example). Complement added to the mixture will be consumed, and sensitized sRBC added subsequently will not undergo hemolysis. Therefore, absence of hemolysis constitutes a positive CF test.

While detection of antibodies is the more common test format, it is equally possible to test for the presence of antigen. In this case, the patient's serum is supplemented with specific antibody to induce formation of complexes; successive addition of complement and indicator sRBC is performed as before.

The test can be made quantitative by setting up a series of dilutions of patient serum and determining the highest dilution factor that will still yield a positive CF test. This dilution factor corresponds to the titer.