The complement fixation test (CFT) is a laboratory procedure used to identify specific antibodies or antigens in a patient’s blood serum. This immunological test operates on the principle of the complement system’s activation when it encounters an antigen-antibody complex. Developed in the early 20th century, the CFT was an early diagnostic method in immunology, particularly for detecting infectious agents and understanding immune responses.
Understanding the Complement System
The complement system is a complex group of proteins in the blood, part of the body’s innate immune system. These proteins circulate as inactive precursors and activate in response to triggers like bacterial presence or tissue injury. This activation initiates a cascade of reactions that enhance the ability of antibodies and phagocytic cells to clear microbes and damaged cells.
The system targets foreign invaders, promotes inflammation, and removes foreign substances. A key mechanism is “complement fixation,” where these proteins bind to antigen-antibody complexes. This binding can destroy pathogens by forming pores in their cell membranes or by “opsonization,” making them more susceptible to engulfment by phagocytic cells.
The Complement Fixation Test Process
The complement fixation test involves a multi-step process to determine the presence of specific antibodies or antigens. Initially, patient serum is heated to approximately 56°C for about 30 minutes to inactivate any pre-existing complement proteins. This heat treatment ensures that only the standardized complement added later will participate, as antibodies are more resistant to heat.
Following inactivation, standardized complement, often from guinea pig serum, is added to the patient’s treated serum. A known antigen of interest is then introduced. If specific antibodies against this antigen are present, they bind to the antigen, forming antigen-antibody complexes. The added complement then binds to, or “fixes,” these complexes, becoming consumed.
To determine if complement was consumed, an indicator system is introduced in a second stage. This system consists of sheep red blood cells (sRBCs) coated with anti-sheep red blood cell antibodies. If complement was consumed in the first stage due to antigen-antibody complexes, no free complement remains to react with the indicator system. Conversely, if no complexes formed, free complement binds to the sensitized sRBCs, causing them to lyse and release hemoglobin.
Interpreting Test Results
The interpretation of complement fixation test results relies on observing the lysis of indicator red blood cells. If no hemolysis occurs (the solution remains clear and red blood cells settle), this indicates a positive result. This outcome signifies that complement was consumed by antigen-antibody complexes formed in the initial stage, confirming the presence of specific antibodies (or antigen) in the patient’s serum.
Conversely, if hemolysis is observed (the solution turns clear red), it suggests a negative result. This means complement was not consumed in the first stage, as no specific antigen-antibody complexes were formed. Consequently, free complement remained available to bind to and lyse the sensitized red blood cells of the indicator system. This visual distinction provides a clear interpretation of whether the targeted antibodies or antigens were present.
Common Applications of the Test
Historically, the complement fixation test was widely used for diagnosing various infections. It was instrumental in the diagnosis of bacterial infections like syphilis, notably through the Wassermann test developed in 1909. The test also found applications in detecting viral infections such as influenza, mumps, and measles, as well as certain fungal infections.
Beyond infectious diseases, the CFT has been used to detect specific antibodies in autoimmune conditions and for research purposes. While it served as a foundational serological diagnostic tool for decades, newer, more sensitive methods like ELISA (Enzyme-Linked Immunosorbent Assay) and PCR (Polymerase Chain Reaction) have largely superseded it in many clinical diagnostic laboratories. Despite this, the complement fixation test retains historical importance and some niche diagnostic relevance where modern alternatives may not be readily available or feasible.