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CFT.ppt

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  • 1. PRACTICAL 18: COMPLEMENT FIXATION TEST Sayeed Ismail Khatib Lecturer Microbiology & Immunology Ibn Sina National College for Medical Studies
  • 2. Introduction:
    • The complement fixation test (CFT) was extensively used in syphilis serology after being 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 56°C for 20 – 30 minutes.
    • Complement binds to Ag-Ab complex
    • When the Ag is an RBC it causes lysis of RBC’s.
  • 3. Principle
    • Complement takes part in many of the immunological reactions. It gets absorbed during the combination of antigens and antibody.
    • This property of antigen–antibody 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 .
  • 4.
    • The test requires five reagents and is carried out in two steps.
  • 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)
    • 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.
  • 6. Positive Test
    • Step 1:
    • At 37°C
    • Antigen + Antibody + Complement Complement gets fixed
    • (from serum) 1 Hour
    • Step 2:
    • At 37°C
    • Fixed Complement complex + Haemolytic system No Haemolysis
    • 1 Hour ( Test Positive)
  • 7. Negative Test
    • Step 1:
    • At 37°C
    • Antigen + Antibody absent + Complement Complement not fixed
    • 1 Hour
    • Step 2:
    • At 37°C
    • Free Complement + Haemolytic system Haemolysis
    • 1 Hour ( Test Negative)
  • 8.  
  • 9. Results and Interpretations:
    • No haemolysis is considered as a positive test .
    • haemolysis of erythrocytes indicative of a negative test .
    • 1 2 3 4
    • A
    • B
    • Microtiter plate showing Haemolysis (Well A3, A3 and B4) and No Haemolysis (Well