Complement Fixation Test- Principle, Procedure, Results, Types


Complement fixation is a technique that can be used to detect the presence of specific antibodies or antigens in a patient`s serum. It is based on the ability of complement proteins to bind to antigen-antibody complexes and mediate their destruction. Complement proteins are a group of serum proteins that are involved in the immune response and can cause lysis of cells, opsonization of pathogens, inflammation and immune clearance.

Complement fixation was widely used in the past to diagnose infections and autoimmune diseases, but it has been largely replaced by newer methods such as ELISA and PCR. However, it is still useful for some applications, such as detecting antibodies against certain pathogens that are difficult to culture or identify by other means.

The principle of complement fixation is that if a patient`s serum contains antibodies against a specific antigen, they will form complexes with the antigen and consume the complement proteins. This will leave no complement available for the indicator system, which consists of sheep red blood cells coated with antibodies. These cells will not be lysed by complement and will remain intact. On the other hand, if the patient`s serum does not contain antibodies against the antigen, the complement proteins will not be used up and will be able to lyse the indicator cells, causing hemolysis.

The procedure of complement fixation involves several steps:

  • The patient`s serum is heated to inactivate any endogenous complement proteins and then diluted.
  • The antigen of interest is added to the serum and incubated at 37°C for 30 minutes.
  • A source of exogenous complement proteins (usually from guinea pig serum) is added to the mixture and incubated again.
  • The indicator system (sheep red blood cells and anti-sheep antibodies) is added and observed for hemolysis.

The result of complement fixation is interpreted as follows:

  • If hemolysis occurs, it means that the patient`s serum does not contain antibodies against the antigen and the test is negative.
  • If no hemolysis occurs, it means that the patient`s serum contains antibodies against the antigen and the test is positive.

Complement fixation can also be used to detect antigens in a patient`s serum by adding a known antibody instead of an antigen in the first step. The rest of the procedure is similar.

Complement fixation can be made semi-quantitative by performing serial dilutions of the patient`s serum and determining the highest dilution that still gives a positive result. This dilution factor corresponds to the titer of antibodies or antigens in the serum.

Complement fixation has some advantages and limitations as a diagnostic method:

  • Advantages: It can detect antibodies or antigens that are not easily detected by other methods; it can measure low levels of antibodies or antigens; it can be used for various types of infections and diseases; it has good sensitivity.
  • Limitations: It is more complex and time-consuming than other methods; it requires specific reagents and equipment; it can be affected by factors such as temperature, pH, concentration and quality of reagents; it has lower specificity than other methods.