PRACTICAL 18:  COMPLEMENT FIXATION TEST Sayeed Ismail Khatib Lecturer  Microbiology & Immunology Ibn Sina National College...
Introduction: <ul><li>The complement fixation test (CFT) was extensively used in syphilis serology after being introduced ...
Principle <ul><li>Complement takes part in many of the immunological reactions. It gets absorbed during the combination of...
<ul><li>The test requires five reagents and is carried out in two steps. </li></ul>
Components of CFT <ul><li>Test System </li></ul><ul><li>Antigen:  It may be soluble or particulate. </li></ul><ul><li>Anti...
Positive Test <ul><li>Step 1: </li></ul><ul><li>    At 37°C </li></ul><ul><li>Antigen + Antibody + Complement    Complemen...
Negative Test <ul><li>Step 1: </li></ul><ul><li>    At 37°C </li></ul><ul><li>Antigen + Antibody absent + Complement    Co...
 
Results and Interpretations: <ul><li>No haemolysis is considered as a  positive test .  </li></ul><ul><li>haemolysis of er...
Upcoming SlideShare
Loading in...5
×

CFT.ppt

1,763

Published on

0 Comments
2 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total Views
1,763
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
68
Comments
0
Likes
2
Embeds 0
No embeds

No notes for slide

CFT.ppt

  1. 1. PRACTICAL 18: COMPLEMENT FIXATION TEST Sayeed Ismail Khatib Lecturer Microbiology & Immunology Ibn Sina National College for Medical Studies
  2. 2. Introduction: <ul><li>The complement fixation test (CFT) was extensively used in syphilis serology after being introduced by Wasserman in 1909. </li></ul><ul><li>Complement is a protein (globulin) present in normal serum. </li></ul><ul><li>Whole complement system is made up of nine components: C1 to C9 </li></ul><ul><li>Complement proteins are heat labile and are destroyed by heating at 56°C for 20 – 30 minutes. </li></ul><ul><li>Complement binds to Ag-Ab complex </li></ul><ul><li>When the Ag is an RBC it causes lysis of RBC’s. </li></ul>
  3. 3. Principle <ul><li>Complement takes part in many of the immunological reactions. It gets absorbed during the combination of antigens and antibody. </li></ul><ul><li>This property of antigen–antibody complex to fix the complement is used in complement fixation test for the identification of specific antibodies. </li></ul><ul><li>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. </li></ul><ul><li>If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test . </li></ul><ul><li>If the complement is available then there will be haemolysis which is a property of complement, denoting a negative test . </li></ul>
  4. 4. <ul><li>The test requires five reagents and is carried out in two steps. </li></ul>
  5. 5. Components of CFT <ul><li>Test System </li></ul><ul><li>Antigen: It may be soluble or particulate. </li></ul><ul><li>Antibody: Human serum (May or may not contain Antibody towards specific Antigen) </li></ul><ul><li>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. </li></ul><ul><li>Indicator System (Haemolytic system) </li></ul><ul><li>Erythrocytes: Sheep RBC </li></ul><ul><li>Amboceptor (Hemolysin): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol. </li></ul>
  6. 6. Positive Test <ul><li>Step 1: </li></ul><ul><li> At 37°C </li></ul><ul><li>Antigen + Antibody + Complement Complement gets fixed </li></ul><ul><li> (from serum) 1 Hour </li></ul><ul><li>Step 2: </li></ul><ul><li> At 37°C </li></ul><ul><li>Fixed Complement complex + Haemolytic system No Haemolysis </li></ul><ul><li> 1 Hour ( Test Positive) </li></ul>
  7. 7. Negative Test <ul><li>Step 1: </li></ul><ul><li> At 37°C </li></ul><ul><li>Antigen + Antibody absent + Complement Complement not fixed </li></ul><ul><li> 1 Hour </li></ul><ul><li>Step 2: </li></ul><ul><li> At 37°C </li></ul><ul><li>Free Complement + Haemolytic system Haemolysis </li></ul><ul><li> 1 Hour ( Test Negative) </li></ul>
  8. 9. Results and Interpretations: <ul><li>No haemolysis is considered as a positive test . </li></ul><ul><li>haemolysis of erythrocytes indicative of a negative test . </li></ul><ul><li> 1 2 3 4 </li></ul><ul><li>A </li></ul><ul><li>B </li></ul><ul><li>Microtiter plate showing Haemolysis (Well A3, A3 and B4) and No Haemolysis (Well </li></ul>
  1. A particular slide catching your eye?

    Clipping is a handy way to collect important slides you want to go back to later.

×