Complement Fixation Test: Principle, Procedure, Results

The complement fixation test is one of the major traditional tests for the demonstration of the presence of specific antigens or antibodies. It requires a veritable zoo of reagents and numerous preparatory steps. There are almost as many versions as there have been users; the microtitre version developed at the Centers for Disease Control and Prevention (LBCF Test) includes rigorous controls and is commonly used.

The complement fixation test (CFT) does not depend on the hem-agglutinating activity of the virus, but the antibodies must fix the complement, and the sera must be free of anticomplementary activity. The test has been superseded, in many instances, by newer tests, such as enzyme immunoassay (EIA).

The terminal components of the complement cascade, C789 (the membrane attack complex), can damage cell membranes in the presence of specific antibody, which fixes complement to the cell surface. In the CFT, erythrocytes are used as the target cell, because complement-induced leakiness of the membrane can be visualized or measured calorimetrically as an increase in free hemoglobin. In the presence of specific antibodies to an infectious agent, any complement in the system is bound, leaving no residual complement for reaction with antibodies to the erythrocytes. Thus, the presence of a specific antibody is indicated by the absence of hemolysis.

Materials and Reagents

1. Sheep erythrocytes suspension (5% suspension of washed sheep RBCs)
2. Hemolysin (rabbit anti-sheep red-cell antibody)
3. Guinea pig complement, free of antibodies to the agent of interest (Note: Guinea pig is the commonest source of fresh complement)
4. Barbital-buffered diluents
5. Plastic microtitre plate
6. Centrifuge adapter for microtitre plates
7. Water bath for incubation of plates
8. Color standards for judging hemolysis (prepared by lysing various concentrations of red cells)

Procedure of Complement Fixation Test

Complement fixation test (CFT) consists of two-stage:

First step (complement fixation stage): a known antigen and inactivated patient’s serum are incubated with a standardized, limited amount of complement.

#Note: Patient’s serum is heated at 56°C for 30 minutes to inactivate endogenous complement which may disturb the test calibration.

1. If the serum contains specific complement activating antibodies, the complement will be activated or fixed by the antigen-antibody complex.
2. However, if there is no antibody in the patient’s serum, there will be no formation of antigen-antibody complexes, thus complement will not be fixed but will remain free.
   (In the indicator stage this complement will react with RBC coated with antibody to sheep RBC ).

Second step (Indicator Stage): The second step detects whether complements have been utilized in the first step or not. This is done by adding the indicator system.

1. If the complement is fixed in the first step owing to the presence of antibodies there will be no complement left to fix to the indicator system. There won’t be any lysis of RBCs.
2. However, if there is no specific antibody in the patient’s serum, there will be no antigen-antibody complex,  therefore, the complement will be present free or unfixed in the mixture. This unfixed complement will now react with the antibody-coated sheep RBCs to bring about their lysis.

Results and Interpretation

• No lysis of sheep red blood cells (positive CFT) indicates the presence of antibodies in the test serum,
• while lysis of sheep red blood cells (Negative CFT) indicates the absence of antibodies in the serum

Notes:  All reagents must be free of anticomplementary activity, the correct quantity of complement must be added, and the control specimens must react as expected.

Materials required for Quality Control

1. Known positive antibody or antigen
2. Known negative antibody or antigen
3. Serum control without antigen (to detect anticomplementary activity)
4. Antigen controls without serum (to detect anticomplementary activity)
5. Tissue control (the cells or tissue in which the antigen was prepared)
6. Buffer control without antigen or antibody
7. Back titration of complement to document the use of 5CH50 units

Controls should be used along with the test to ensure that

• Antigen and serum are not anti complimentary
• The appropriate amount of complement is used and
• The sheep red blood cells do not undergo autolysis

Advantages of Complement Fixation Test

1. Ability to screen against a large number of viral and bacterial infections at the same time.
2. Economical

Disadvantages of Complement Fixation Test

1. Not sensitive-cannot be used for immunity screening
2. Time-consuming and labor-intensive
3. Often non-specific e.g. cross-reactivity between herpes-simplex virus (HSV) and varicella-zoster virus (VZV).

References and further readings:

• Hsiung GD. Diagnostic Virology. Ed. 3. New Haven, CT: Yale University Press, 1982