Complement Fixation Test
It 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. As coupling of complement has no visible effects or changes, it is necessary to use an indicator system consisting of sheep RBC and coated with anti-sheep RBC antibody. Complement lyses antibody coated RBC.
The complement fixation test consists of two components.
The first component is an indicator system that 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.
The second component is Test System (A known antigen and patient serum added to a suspension of sheep red blood cells in addition to complement). These two components of the complement fixation method are tested in sequence. Patient serum is first added to the known antigen, and complement is added to the solution. If the serum contains antibody to the antigen, the resulting antigen-antibody complexes will bind all of the complement. Sheep red blood cells and the anti-sheep antibody are then added. If complement has not been bound by an antigen-antibody complex formed from the patient serum and known antigens, it is available to bind to the 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.
Steps of Complement Fixation Test
Step 1: A known antigen and inactivated patient’s serum are incubated with a standardized, limited amount of complement. If the serum contains specific, complement activating antibody the complement will be activated or fixed by the antigen-antibody complex. However, if there is no antibody in the 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: The second step detects whether complement has been utilized in the first step or not. This is done by adding the indicator system. If the complement is fixed in the first step owing to the presence of antibody there will be no complement left to fix to the indicator system. However, if there is antibody in the patient’s serum, there will be no antigen-antibody complex, and therefore, complement will be present free or unfixed in the mixture. This unfixed complement will now react with the antibody- coated sheep red blood cells to bring about their lysis.
Thus, no lysis of sheep red blood cells (positive CFT) indicates the presence of antibody in the test serum, while lysis of sheep red blood cells (Negative CFT) indicates the absence of antibody in the serum.
Advantages of Complement Fixation Test
- Ability to screen against a large number of viral and bacterial infections at the same time.
Disadvantages of Complement Fixation Test
- Not sensitive – cannot be used for immunity screening.
- Often non-specific e.g. cross-reactivity between Herpes Simplex Virus and Voricella Zoster Virus.