Complement Fixation Test : Introduction, Principle, Procedure and Result

Complement Fixation Test : Introduction, Principle, Procedure and Result Interpretation

Introduction of Complement fixation test

Complement fixation test brief form is CFT. Complement is a non-specific protein found in our normal serum. This CFT was extensively used for the diagnosis of syphilis caused by Treponema pallidum, a serological test called Wassermann test or Wassermann reaction named after the introducer surname bacteriologist August Paul von Wassermann in 1909. The whole complement system is made up of nine components i.e. complement one ( C1)to C9 Complement proteins are heat-labile and are destroyed by heating at 56°C for 30 minutes. Complement binds to antigen and antibody ( Ag-Ab) complex. When the antigen is an RBC it causes lysis of red blood cells ( RBCs).

Principle of Complement fixation test

Complement takes part in many of the immunological reactions. It gets absorbed through the combination of antigens and antibodies. This property of antigen-antibody complex to fix the complement is used in complement fixation tests for the identification of specific antibodies. The hemolytic system containing sheep erythrocytes (RBCs) and its corresponding antibody (rabbit antibody to sheep erythrocyte; also called Amboceptor is used as an indicator that shows the utilization or availability of the complement. If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test. If the complement is available then there will be hemolysis which is a property of complement, denoting a negative test.
Wassermann reaction is a classical example of a complement fixation test.
Earlier it was used to diagnose syphilis but is now no longer in use.

Requirements for the Complement fixation test

Test SystemAntigen: It may be soluble or particulate.
Antibody: Human serum (May or may not contain Antibody towards specific Antigen) Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the complement activity is heat-labile (stored at -30 °C in small fractions). The complement activity should be initially standardized before using in the test.
Indicator System (Hemolytic system)Erythrocytes: Sheep RBC
Amboceptor (Hemolysins): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.

Procedure of the Complement fixation test

Mix with test serum to be assayed for antibody. Add the standard amount of complement. Add erythrocytes coated with antibodies. Determine the amount of erythrocytes lysis.

Observation of Complement fixation test

The reaction consists of two systems components of CFT.

In case of Positive Test

Step 1: Antigen + Antibody (from serum) + Complement at At 37°C for an hour→ Complement gets fixed

Step 2: Fixed Complement complex + Hemolytic system at At 37°C for an hour → No Hemolysis

Whereas in case of Negative Test

Step 1: Antigen + Antibody absent + Complement at At 37°C for an hour → Complement not fixed

Step 2: Free Complement + Hemolytic system At 37°C for an hour → Hemolysis

Results and Interpretations of CFT

No hemolysis is considered a positive test.

Hemolysis of erythrocytes is indicative of a negative test.

Advantage of Complement Fixation Test

It has the ability to screen against a large number of viral and bacterial infections at the same time.

The disadvantage of Complement Fixation Test

It is not sensitive and so can not be used for immunity screening.
It is time-consuming and labor-intensive.
It is often non-specific.

Other complement-dependent serological tests

Immobilization test:- It is applicable for syphilis as the Treponema pallidum immobilization(TPI) test. Immune adherence test: some bacteria get to adhere to particulate material such as erythrocytes, or platelets, or macrophages in presence of complement( e.g. Vibrio cholerae). Cytocidal reaction:- When live bacteria such as V. cholerae are mixed with their specific antibody in presence of complement , bacteriolysis occurs.

universe84a