COMPLEMENT FIXATION TEST

Introduction:

The complement fixation test (CFT) was introduced by Wasserman in 1909. Complement is a protein (globulin) present in normal serum. Whole complement system is made up of nine components: C1 to C9 Complement proteins are heat labile and are destroyed by heating at 56C for 20 30 minutes. Complement binds to Ag-Ab complex When the Ag is an RBC it causes lysis of RBCs.

Principle

Complement takes part in many of the immunological reactions. It gets adsorbed during the combination of antigens and antibody. This property of antigenantibody complex to fix the complement is used in complement fixation test for the identification of specific antibodies.

The haemolytic system containing sheep erythrocytes (RBC) and its corresponding antibody (amboceptor) is used as an indicator which shows the utilization or availability of the complement.

If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test.

If the complement is available then there will be haemolysis which is a property of complement, denoting a negative test.

The complement fixation test is an immunological medical test looking for evidence of infection. It tests for the presence of either specific antibody or specific antigen in a patient's serum. It uses sheep red blood cells (sRBC), anti-sRBC antibody and complement, plus specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in serum). If either the antibody or antigen is present in the patient's serum, then the complement is completely utilized, so the sRBCs are not lysed. But if the antibody (or antigen) is not present, then the complement is not used up, so it binds anti-sRBC antibody, and the sRBCs are lysed. The Wassermann test is one form of complement fixation test.

The test requires five reagents and is carried out in two steps.

Components of CFT
Test System Antigen: It may be soluble or particulate.

Antibody: serum (May or may not contain Antibody towards specific Antigen)

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.

Indicator System (Haemolytic system) Erythrocytes: Sheep RBC

Amboceptor (Hemolysin): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.

Positive Test

Step 1:
At 37C Complement gets fixed

Antigen + Antibody + Complement (from serum) 1 Hour

Step 2:
At 37C No Haemolysis (Test Positive)

Fixed Complement complex + Haemolytic system 1 Hour

Negative Test

Step 1:
At 37C Complement not fixed 1 Hour

Antigen + Antibody absent + Complement

Step 2:
At 37C Haemolysis (Test Negative)

Free Complement + Haemolytic system 1 Hour

Complement Fixation:

Antigen/antibody complexes can also be measured by their ability to fix complement because an antigen/antibody complex will "consume" complement if it is present, whereas free antigens or antibodies do not. Tests for antigen/antibody complexes that rely on the consumption of complement are termed complement fixation tests and are used to quantitate antigen/antibody reactions. This test will only work with complement fixing antibodies (IgG and IgM are best). Antigen is mixed with the test serum to be assayed for antibody and antigen/antibody complexes are allowed to form. A control tube in which no antigen is added is also prepared. If no antigen/antibody complexes are present in the tube, none of the complement will be fixed. However, if antigen/antibody complexes are present, they will fix complement and thereby reduce the amount of complement in the tube.

After allowing complement fixation by any antigen/antibody complexes, a standard amount of red blood cells, which have been pre-coated with antierythrocyte antibodies is added. The amount of antibody-coated red blood cells is predetermined to be just enough to completely use up all the complement initially added, if it were still there. If all the complement is still present (i.e. no antigen/antibody complexes formed between the antigen and antibody in question), all the red cells will be lysed. If antigen/antibody complexes are formed between the antigen and antibody in question, some of the complement will be consumed and, thus, when the antibody-coated red cells are added not all of them will lyse. By simply measuring the amount of red cell lysis by measuring the release of hemoglobin into the medium, one can indirectly quantitate antigen/antibody complexes in the tube. Complement fixation tests are most commonly used to assay for antibody in a test sample but they can be modified to measure antigen.

Results and Interpretations:

No haemolysis is considered as a positive test. Haemolysis of erythrocytes indicative of a negative test. 1 2 3 4 A

Microtiter plate showing Haemolysis (Well A3, A4 and B4) .

Summary: Complement fixation tests detect lysins- Ab that fix complement and can lyse target cells. Involves mixing test Ag and Ab with complement and then with sensitized sheep RBCs. If complement is fixed by the Ag-Ab, the RBCs remain intact and the test is positive. If RBCs are hemolyzed, specific Ab are lacking and the test is negative.