The complement fixation test (CFT) involves mixing antigen, antibody, and complement. If the antibody is specific to the antigen, it will bind and "fix" the complement, preventing it from lysing red blood cells. In a positive test, lysis does not occur, while a negative test results in lysis. CFT is used to detect antibodies against pathogens like syphilis, and can detect antibody levels below 1 microgram/ml. It has limitations like being time-consuming and labor-intensive. Variations involve using different complement sources or detection methods depending on the pathogen.

1. COMPLEMENT
FIXATION
TEST
SAKEENA ASMI
MAHATMA GANDHI
UNIVERSITY

2. INTRODUCTION
• The complement fixation test (CFT) was extensively used
in Syphilis serology after being introduced by Wasserman
in 1909.
• Complement is a protein present in normal serum.
• Made up of nine components : C1 to C9.
• They are heat labile and are destroyed by heating at 56OC
for 20 – 30 minutes.
• Complement takes part in many immunological reactions
and is absorbed during combination of antigen with their
antibodies.
• The ability of antigen antibody complexes to fix
complement is made use of in the complement fixation
test.

3. In the presence of appropriate antibodies, complement
can :
• Lyse erythrocytes.
• Lyse bacteria.
• Immobilise motile organisms.
• Promote phagocytosis.

4. August Paul von Wassermann

5. Components of CFT
Test System
• Antigen : It may be soluble or particulate.
• Antibody : Human serum (may or may not contain Antibody
towards specific Antigen) , heat inactivated.
• Complement : It is pooled serum from guinea pigs. Should be fresh
or specially preserved as complement is heat labile.
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.

6. The guinea pig serum should be titrated for complement
activity :
1 MHD (Minimum Hemolytic Dose) of complement is
defined as the highest dilution of the guinea pig serum
that lyses one unit volume of washed sheep erythrocytes
in the presence of excess hemolysin (amboceptor) within a
fixed time (usually 30 or 60 min) at a fixed temperature
(37OC).

7. The amboceptor should be titrated for hemolytic activity :
1 MHD (Minimum Hemolytic Dose) of amboceptor is
defined as the highest dilution of the inactivated
amboceptor that lyses one unit volume of washed sheep
erythrocytes in the presence of excess complement within
a fixed time (usually 30 or 60 min) at a fixed temperature
(37OC).

8. POSITIVE TEST
ANTIGEN + TEST SERUM
(contains antibody) + COMPLEMENT
COMPLEMENT FIXED
+ HEMOLYTIC SYSYTEM RESULT – NO HEMOLYSIS
POSITIVE CF TEST

9. NEGATIVE TEST
ANTIGEN + TEST SERUM
(contains no antibody) + COMPLEMENT
COMPLEMENT NOT FIXED
+ HEMOLYTIC SYSYTEM RESULT – HEMOLYSIS
NEGATIVE CF TEST

10. Amboceptor
Amboceptor
Amboceptor

11. RESULT
• No hemolysis is considered as a positive test.
• Hemolysis of erythrocytes indicative of a negative test.
1 2 3 4
A
B
Microtitre plates showing Hemolysis (Well A3, A4, B4)
and No Hemolysis (Well A1, A2, B1, B2, B3).

12. INDIRECT CFT
• Certain avian (eg: duck, turkey, parrot) and mammalian (eg:
horse, cat) sera do not fix guinea pig complement.
Ag-Ab
Complement
not fixed
Standard
antiserum
Complement
not fixed
POSITIVE TEST

13. No Ab Complement
not fixed
Standard
antiserum
Complement
fixed
NEGATIVE
TEST

14. CONGLUTINATION COMPLEMENT
ABSORPTION TEST
• Alternative method for systems that are unable to fix guinea pig
complement. Uses horses complement which being non
hemolytic.
• Indicator system :- Sensitised sheep erythrocytes mixed with
bovine serum.
• Bovine serum contains a beta globulin component called
Conglutinin, which acts as Ab to complement.
• Conglutinin causes agglutination of sensitised sheep erythrocytes
(Conglutination) if they have combined with complement.
• If the horse complement utilised by the Ag - Ab interactions
during first step = No agglutination of sensitised cells.

15. OTHER COMPLEMENT DEPENDENT
SEROLOGICAL TESTS
1. IMMOBILISATION TESTS
When live motile Treponema pallidum is mixed with patient’s
serum in presence of complement the organism is immobilised =
Treponema pallidum immobilisation.
2. CYTOLYTIC OR CITOCIDAL TEST
Live Vibrio cholera + its Antibody Bacterium lysed
+ Complement (Basis of measurement
of Vibrio cholera Ab)

16. 3. IMMUNE ADHERENCE
• The Ag – Ab complexes in some bacteria such as Vibrio cholera, T.
pallidum adhere to particulate material like RBCs, platelets.
• This bacterial adherence to cell Immune Adherence

17. LIMITATIONS
• CFT is used for the detection of Syphillis.
• Used to screen large numbers of viral or bacterial infections.
• Used to detect and quantify antibody that does not agglutinate or
precipitate with its antigen.
• CFT can detect antibody at level less than 1 microgram per
milliliter.
• Can also be used to detect antigen.
APPLICATIONS
• Time consuming
• Labor intense

18. 1. Textbook of Microbiology; Ananthanarayan and Paniker’s; 8th
Edition, University Press; page no : 109 – 111.
2. Textbook of Microbiology and Immunology; Panja.
3. Basic Serological Testing; Rowa Yousef Alhabbab; page no : 69 –
72.

19. THANK YOU