Complement Fixation Test

Complement Fixation

Complement is a protein (globulin) present in normal serum. It refers to a set of serum proteins that cooperates with both innate and the adaptive immune systems to eliminate blood and tissue pathogens.

Complement system is a part of the immune system that enhances (complements) ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promotes inflammation, and attacks pathogen’s plasma membrane.

Complement fixation test is a classic method for demonstrating the presence of antibody in patient serum. It is based on the principle that antigen-antibody complex fixes the complement.

It is an immunological test for detection of presence or absence of specific antibody or antigen in a serum based on fixation of complement.

As coupling of complement has no visible effects or changes, it is necessary to use an indicator system consisting of sheep RBC and RBC coated with anti-sheep antibody. Complement lyses antibody coated RBC.


It is nature of complements to get activated when there is formation of antigen-antibody complex. Thus presence of specific antibody or antigen towards known antigen or antibody will form a complex and activate complement system.

Complement binds to Ag-Ab complex and 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 complement fixation test consists of two component, Indicator system and Test system.

Indicator system uses combination of sheep red blood cells, complement-fixing antibody such as immunoglobulin G produced against the sheep red blood cells and an exogenous source of complement usually guinea pig serum. When these elements are mixed in optimum conditions, the anti-sheep antibody binds on the surface of red blood cells. Complement subsequently binds to this antigen -antibody complex formed and will cause the red blood cells to lyse.

Test System consists of a known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement.

Complement Fixation Test


These two components of the complement fixation are tested in sequence.

  • Patient serum is first added to known antigen, and complement is added to solution. If serum contains antibody towards antigen, resulting antigen-antibody complexes will bind all of the complement.
  • Sheep red blood cells and anti-sheep antibody are then added. If complement has not been bound by an antigen-antibody complex formed from patient serum and known antigens, it is available to bind to indicator system of sheep cells and anti-sheep antibody.

Lysis of the indicator sheep red blood cells signifies both a lack of antibody in patient serum and a negative complement fixation test.

If the patient’s serum does contain a complement-fixing antibody, a positive result will be indicated by the lack of red blood cell lysis.



  1. Easy to read/Interpret.
  2. Economical
  3. Ability to screen against a large number of viral and bacterial infections at same time.
  4. Large variety of test antigens can be used.


  1. Time consuming Method.
  2. Not sensitive – cannot be used for immunity screening.
  3. Often non-specific e.g. cross-reactivity between Herpes Simplex Virus and Voricella Zoster Virus.
  4. Some components needs to be fresh (RBC, Complements).


Download Link: Complement Fixation Test.pdf

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