Modifications of Complement Fixation Test

Modifications of complement fixation test

Complement fixation test is a classic method for demonstrating the presence of antibody in patient serum. It is an immunological test for detection of presence or absence of specific antibody or antigen in a serum based on fixation of complement.


Complement fixation test is based on the principle that when antigen and antibodies of the IgM or the IgG classes are mixed, complement is fixed to the antigen–antibody complex. If this occurs on surface of RBCs, the complement cascade will be activated and hemolysis will occur.


There are various modifications and types of complement fixation test. Some of them includes

Indirect complement fixation test: It is used when serums which can’t fix guinea pig complement is to be tested. These includes avian sera (Parrot, Duck) and mammalian sera (Cat, Horse). The test is set up in duplicate.

  • Standard antiserum to antigen which is known to fix complement is added to one set. If antibodies were not present in the test serum then antigen would react with standard antiserum fixing complement.
  • On other hand if antibodies are present in test serum antigen would be utilized in first step. So, no reaction would occur between standard antiserum and antigen and therefore no fixation of complement would cause lysis of sheep red blood cells.

Hemolysis indicates a positive result while absence of hemolysis is negative result.


Indirect complement fixation test



Congluatinating complement absorption test: It is a test for detection of complement fixation by observing the phenomenon of conglutination i.e. Agglutination of RBCs by conglutininin.

In this test, horse complement which is non-haemolytic is used. The indicator system used is sensitized sheep red blood cells mixed with bovine serum. Bovine serum contains a beta globulin called conglutinin would also combine with this complement causing agglutination (conglutination) of sheep red blood cells. If horse complement is utilized by Antigen-Antibody reaction in first step, agglutination of sensitized cells doesn’t occur.


  • Agglutination of sensitized RBcs (Hemolysis) indicates a negative result.
  • No agglutination (absence of hemolysis) indicates positive result (fixation of complement).


Immune adherence: It is immunological reaction between normal erythrocytes and a wide variety of microorganisms sensitized with their individually specific antibody and complement. Attachment of cells such as RBCs, plateletsto complement coated immune complex or microorganisms is immune adherence.

When some bacteria (such as Vibrio cholera or Treponema pallidum) combine with their specific antibody in presence of complement and some particles such as erythrocytes or platelets, they adhere to erythrocytes or platelets. This is called immune adherence. It facilitates phagocytosis of bacteria.


 Immune adherence



Immobilisation test: It is a serological procedure in which organisms are immobilized by the use of antibodies in presence of complements.  It is a procedure for identifying antibodies to motile microorganisms by measuring the ability of antibodies to restrict motility of microorganisms.

In this test, antigen is incubated with patient’s serum in presence of complement. If specific antibody is present it would immobilize the antigen. Eg. Treponema palladium immobilization test, considered gold standard for sero-diagnosis of syphilis. A positive test shows serum to contain treponemal antibodies.


Cytolytic tests: Cytolysis refers to pathological disintegration or dissolution of cells. The incubation of a live bacterium with its specific antibody in presence of complement leads to lysis of bacterial cells. E.g. Vibriocidal antibody test used to measure anti-cholera antibodies.


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