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

Complement Fixation Test

Introduction of Complement fixation test

Complement fixation test  brief form is CFT. Complement is  non specific protein found in our normal serum. This CFT was extensively used for the diagnosis of syphilis caused by Treponema pallidum, serological test called Wassermann test or Wassermann reaction named after the introducer surname bacteriologist August Paul von Wassermanne in 1909. 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 during the combination of antigens and antibody.This property of antigen–antibody complex to fix the complement is used in complement fixation test 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 which 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 complement fixation test.
Earlier it was used to diagnose  of syphilis but  now no longer in used.

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 standard amount of complement. Add erythrocytes coated with antibodies . Determine the amount 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

Where as 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:

No hemolysis is considered as a positive test.

Hemolysis of erythrocytes indicative of a negative test.

Advantage of Complement Fixation Test

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

Disadvantage of Complement Fixation Test

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

Other complement-dependent serological tests

Immobilization  test:- It is applicable for syphilis as  Treponema pallidum immobilization(TPI) test. Immune adherence test :some bacteria get 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 its specific antibody in presence of complement , bacteriolysis occurs.

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