Scribd
Upload
8 views

Complement Fixation Test Practical 7

Micro

Uploaded by

mmuneebvirk
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
8 views

Complement Fixation Test Practical 7

Micro

Uploaded by

mmuneebvirk
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 9

Complement Fixation Test

The complement fixation test (CFT) is used to detect the presence of specific antibodies in
the patient’s serum. This test is based on the use of complement, a Biologically heat labile (
heat unstable) serum factor that is responsible for the lysis of antibody coated cells.
Principle of Complement Fixation Test
 It is the nature of the complement to be activated when there is formation of
antigen-antibody complex.
 The test serum is inactivated by heating at 56°C for 30 minutes to destroy the
complement activity of test serum and to remove anti-complementary effect of
some non-specific inhibitors in the serum.
 A measured amount of complement and antigen are then added to the serum.

 If no antibody is present then the complement remains free. To determine


whether the complement has been fixed, sheep RBCs and antibodies against
sheep RBCs are added.
 If there is presence of antibody in the serum, the complement is fixed due to the
formation of Ag-Ab complex.
Principle of Complement Fixation Test
 Antigen-antibody complex fixes the complement. But the fixation of complement
with Ag-Ab complex do not have any visible effect like agglutination and
precipitation. So it is necessary to use indicators system.

 The indicator system consists of sheep RBC coated


with anti-sheep RBC antibody (Amboreceptor).
 Lysed and unlysed RBCs can be differentiated with naked eyes.

 In the positive test : The available complement is fixed by Ag-Ab complex and
no hemolysis of sheep RBCs occurs. So the test is positive for presence of antibodies.

 In the negative test : No Ag-Ab reaction occurs and the complement is free.
This free complement binds to the complex of sheep RBC and it’s antibody
to cause hemolysis, causing the development of pink color.
Requirements for Complement Fixation Test
 Antigen: Viral Ag/ washed Sheep RBCs

 inactivated patient’s serum

 Complement ( Guineapig serum)

 Indicator system: Sheep RBCs

 Water bath for incubation

 Microtiter plate
Procedure of Complement Fixation Test
 The following figure shows the steps involved in the procedure of complement
fixation test.
 A known antigen is mixed with inactivated patient’s serum

 Add a measured amount of complement ( Guineapig serum) in the test system

 The test system is incubated at 37°C for about 1 hour.

 After 1 hour an indicator system (sensitized RBC) is added to the test system and
again incubated at 37°C for 30 minutes

 If Ag and Ab matches, they form Ag-Ab complex and utilizes complement.

 Observe the result


Observation of Complement Fixation Test
 If hemolysis is observed: it indicates the absence of specific antibody
in patient serum, so that complement has not been used which lysed
the sensitized RBC giving hemolysis.

 If no hemolysis is observed: it indicates that the patient serum contains antibody


which reacts with Ag to form Ag-Ab complex and then fix complement
so that no complement is available to hemolyse sensitized RBC.
Results Interpretation of Complement Fixation Test
 Positive test : The available complement is fixed by Ag-Ab complex and no hemolysis
of sheep RBCs occurs. So the test is positive for presence of antibodies.
 Negative test : No Ag-Ab reaction occurs and the complement is free. This free
complement binds to the complex of sheep RBC and it’s antibody to cause hemolysis,
causing the development of pink color.
Application of Complement Fixation Test
 It is used to detect and quantify antibody that does not precipitate or agglutinate
with its antigen.
 This test can be used to detect antibody at level less than 1 microgram per milliliter.

You might also like