Complement Fixation Test (Process)

Components of Complement Fixation test System:  The complement fixation test consists of two component, Indicator system and Test system.

 Test System: It consists of antigen, antibody and complements. A known antigen and patient serum added to a suspension of sheep red blood cells.

  • Antigen: It can be soluble or particulate.
  • Antibody: Human serum (May or may not contain Antibody towards specific Antigen).

Paired sera are used to detect recent infection (one at acute stage of disease, other taken two weeks later (convalescent stage)

  • 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.


Complement Fixation Procedure


Indicator System (Hemolytic system): It consists of sheep RBC and Hemolysin. 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.

  • Erythrocytes: Sheep RBC which act as antigen.
  • Hemolysin: Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.


Complement Fixation Test consists of following two steps:

Step 1: A known antigen and inactivated patient’s serum are incubated with a standardized, limited amount of complement. If serum contains specific complement activating antibody, complement will be activated or fixed by antigen-antibody complex. However, if there is no antibody in patient’s serum, there will be no formation of antigen-antibody complex, and therefore complement will not be fixed. But will remain free.


Step 2: It detects whether complement has been utilized in first step or not. This is done by adding indicator system. If complement is fixed in first step owing to presence of antibody there will be no complement left to fix to indicator system. However, if there is absence of antibody in patient’s serum, there will be no antigen-antibody complex, and therefore, complement will be present free or unfixed in mixture. This unfixed complement will now react with antibody- coated sheep red blood cells to bring about their lysis.


Complement Fixation Test Process



  • If the complement is fixed on test system with Ag-Ab complex, then there will be no lysis of sheep erythrocytes, thus denoting a positive test.


  • If the complement is available not bound to test system, it will be free to combine with indicator system resulting in hemolysis denoting a negative test.

Thus, absence of lysis of sheep red blood cells (Positive CFT) indicates presence of antibody in test serum, while lysis of sheep red blood cells (Negative CFT) indicates absence of antibody in serum.


Download Link: Complement Fixation Test Components and Process.pdf

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