In complement fixation test, Complement is used which is a biologically labile serum factor that causes the immune cytolysis i.e. lysis of antibody-coated cells which is found in normal serum.

Our whole complement system is made up of a total of nine components from C1 to C9. And these proteins are heat-labile and may be destroyed by heating at 56 degrees for 20 -30 min, this process is called Heat inactivation.




The complement fixation test is a traditional test that is used to detect the presence of specific antibodies in the patient’s serum. It requires various types of reagent and different types of preparatory steps.

Principle of complement fixation test

The antigen-antibody complexes can fix the complement with no visible effect. This reaction is used to fixation of complement in which an indicator system consists of sheep erythrocytes coated with amboceptor (rabbit antibody to sheep erythrocytes) is used.

These erythrocytes which are coated with antibodies can be lysed by complement. If complement is fixed and utilized in the antigen-antibody reaction, there is no free complement to act on the indicator system and hence no lysis of erythrocytes, which indicates the positive complement fixation test.

The lysis of erythrocytes indicates that complement was not fixed in the first step and therefore, the serum is negative for antibodies which means a negative complement fixation test.

Components of the complement fixation test

In complement fixation test system two components are required:

1.) The first component is an indicator system consisting of a combination of sheep red blood cells, the complement-fixing antibody produced against the sheep red cells in another animal and exogenous source of complement, usually guinea pig serum.

• When these three components are combined in an optimum concentration, the anti-sheep cell antibody complexes and cause cell lysis.

2.) The second component consists of a known antigen and inactivated patient serum. Antigen-antibody reactions lead to the immune complex formation that produces complement fixation via the classical pathway.

• That is when complement takes part in antigen-antibody reactions; it is bound or fixed to the antigen-antibody complexes. When these complexes are on bacteria, red cells or other cells, the complement brings about the lysis of the cells involved.

Also Read: Western blotting – Introduction, Principle, Procedure & Uses

This may be exploited to determine the amount of antigen or antibody present in the patient sample.

• A complement fixation test can detect antibodies at a level of less than one microgram per milliliter.
• In the CFT, the patient’s inactivated serum is serially diluted and reacted with known antigen in the presence of complement. If the corresponding antibody is contained in the serum it will combine with the antigen and use up the complement.
• This will leave no complement to haemolyze the antibody-coated red cells that are added. The highest dilution of serum that prevents hemolysis is the antibody titer. If the patient’s serum, however, does not contain the corresponding antibody, the complement will not be used and will be available to fix to and haemolyze the antibody-coated red cells.

Materials and Reagents

1. Sheep erythrocytes suspension (5% suspension of washed sheep RBCs)
2. Hemolysin (rabbit anti-sheep red-cell antibody)
3. Guinea pig complement, free of antibodies to the agent of interest (Note: Guinea pig is the commonest source of fresh complement)
4. Barbital-buffered diluents
5. Plastic microtitre plate
6. Centrifuge adapter for microtitre plates
7. Water bath for incubation of plates
8. Color standards for judging hemolysis (prepared by lysing various concentrations of red cells)

Materials required for Quality Control

1. Known positive antibody or antigen
2. Known negative antibody or antigen
3. Serum control without antigen (to detect anticomplementary activity)
4. Antigen controls without serum (to detect anticomplementary activity)
5. Tissue control (the cells or tissue in which the antigen was prepared)
6. Buffer control without antigen or antibody
7. Back titration of complement to document the use of 5CH50 units

Controls should be used along with the test to ensure that

• Antigen and serum are not anti complimentary
• The appropriate amount of complement is used and
• The sheep red blood cells do not undergo autolysis

Also Read: Enzyme Linked Immunosorbent Assay (ELISA)

Procedure and test interpretation of CFT

The first step (Complement fixation stage): a known antigen and inactivated patient’s serum are incubated with a standardized, limited amount of complement. The patient’s serum should be heated at 56°C for 30 minutes to inactivate endogenous complement which may disturb the test calibration.

1. If the serum contains a specific complement activating antibody, the complement will be activated or fixed by the antigen-antibody complex.
2. However, if there is no antibody in the patient’s serum, there will be no formation of antigen-antibody complex, thus complement will not be fixed but will remain free (In the indicator stage this complement will react with RBC coated with antibody to sheep RBC).

The second step (Indicator Stage): The second step detects whether a complement has been utilized in the first step or not. This is done by adding the indicator system.

1. If the complement is fixed in the first step owing to the presence of antibody there will be no complement left to fix to the indicator system. There won’t be any lysis of RBCs.
2. However, if there is a nonspecific antibody in the patient’s serum, there will be no antigen-antibody complex,  therefore, the complement will be present free or unfixed in the mixture. This unfixed complement will now react with the antibody-coated sheep RBCs to bring about their lysis.

Interpretation

Positive test: The available complement is fixed by Ag-Ab complex and no hemolysis of sheep RBCs occurs. So the test is positive for the presence of antibodies.

Negative test: No Ag-Ab reaction occurs and the complement is free. This free complement binds to the complex of sheep RBC and it’s the antibody to cause hemolysis, causing the development of pink color.

Advantages of Complement Fixation Test

1. Ability to screen against a large number of viral and bacterial infections at the same time.
2. Economical

Disadvantages of Complement Fixation Test

1. Not sensitive – cannot be used for immunity screening
2. Time-consuming and labor-intensive
3. Often non-specific e.g. cross-reactivity between HSV and VZV