COMPLEMENT FIXATION TEST - CFT

ABOUT COMPLEMENT FIXATION TEST

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 56 O C 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 w ashed sheep erythrocytes in the presence of excess hemolysin ( amboceptor ) within a fixed time (usually 30 or 60 min) at a fixed temperature (37 O C).

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 w ashed sheep erythrocytes in the presence of excess complement within a fixed time (usually 30 or 60 min) at a fixed temperature (37 O C).

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

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