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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 the surface of RBCs, the complement cascade will be activated and hemolysis will occur.
Indirect complement fixation test
It is used when serums which can’t fix guinea pig complement. These includes avian sera (Parrot, Duck) and mammalian sera (Cat, Horse). Here, the test is set up in duplicate. After step 1, 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 the antigen would react with the standard antiserum fixing the complement. On the other hand if antibodies are present in the test serum the antigen would be utilized in the first step. So, no reaction would occur between the standard antiserum and the antigen and therefore no fixation of complement would cause lysis of sheep red blood cells. Thus in this case haemolysis indicates a positive result.
Congulatinating complement absorption test
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 the sheep red blood cells, indicating a negative result. If horse complement is utilized by Ag-Ab reaction in first step, agglutination of sensitized cells doesn’t occur.
When some bacteria (such as Vibrio cholera or Treponema pallidum) combine with their specific antibody in the presence of complement and some particles such as erythrocytes or platelets, they adhere to the erythrocytes or platelets. This is called immune adherence. Immuno adherence facilitates phagocytosis of bacteria.
Here 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 the sero diagnosis of syphilis. A positive test shows serum to contain treponemal antibodies.
The incubation of a live bacterium with its specific antibody in the presence of complement leads to the lysis of the bacteria cells. This is the basis of Vibriocidal antibody test used to measure anti-cholera antibodies.