Download lecture11-complement-fixation-immobilization

Complement fixation test
complement fixation test
• The complement fixation test (CFT) was extensively used in
syphilis serology after being introduced by Wasserman in
1909.
• It took a number of decades before the CFT was adapted for
routine use in virology.
• CFT meet the following criteria
▫ It is convenient and rapid to perform
▫ The demand on equipment and reagents is small
▫ A large variety of test antigens are readily available
Principle of CFT
• The complete 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 labile serum factor that causes the
immune cytolysis i.e. lysis of antibody coated cells.
principle
First step (Complement fixation stage):
• A known antigen and inactivated patient’s serum are
incubated with a standardized, limited amount of
complement.
• Note: patient’s serum is heated at 56°C for 30 minutes to
inactivate endogenous complement which may disturb the
test calibration.
• If the serum contains specific complement activating
antibody, the complement will be activated or fixed by the
antigen-antibody complex.
• However, if there is no antibody in the patient’s serum, there
will be no formation of antigen-antibody complex,
thus complement will not be fixed but will remain free (In
the indicator stage this complement will react with RBC
coated with antibody to sheep RBC ).
principle
principle
Second step (Indicator Stage):
• The second step detects whether complement has been
utilized in the first step or not. This is done by adding the
indicator system.
• If the complement is fixed in the first step owing to
the presence of antibody there will be no complement left
to fix to the indicator system. There won’t be any lysis of
RBCs.
• However, if there is no specific antibody in the patient’s
serum, there will be no antigen-antibody
complex, therefore, complement will be present free or
unfixed in the mixture. This unfixed complement will
now react with the antibody- coated sheep RBCs to bring
about their lysis.
Interpretation
• In the positive test : The available complement is fixed by AgAb 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.
• Controls should be used along with the test to ensure
that
▫ Antigen and serum are not anti complimentary
▫ The appropriate amount of complement is used and
▫ The sheep red blood cells do not undergo autolysis
Positive Test
• Step 1:
At 37°C
Antigen + Antibody + Complement
(from serum)
1 Hour
Complement gets fixed
• Step 2:
Fixed Complement complex + Haemolytic system
At 37°C
1 Hour
No haemolysis
(Test Positive)
Negative Test
• Step 1:
At 37°C
Antigen + Antibody absent + Complement
Complement not fixed
1 Hour
• Step 2:
Free Complement + Haemolytic system
At 37°C
1 Hour
Haemolysis
(Test Negative)
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, A4 and B4) and 
No Haemolysis (Well A1,A2,B1 &B2)
Materials and Reagents
• The test requires five reagents and is carried out in two steps.
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.
Advantages and disadvantages of CFT
Advantages
1. Ability to screen against a large number of viral and
bacterial infections at the same time.
2. Economical.
Disadvantages
1. Not sensitive - cannot be used for immunity
screening
2. Time consuming and labor intensive
3. Often non-specific e.g. cross-reactivity between HSV
and VZV
Modifications of complement fixation test
Indirect complement fixation test:
• This modification is used when serums which don’t fix guinea pig
complement is to be tested.
• Seras of duck, turkey, parrot, horse, cat unable to fix guinea
pig complement.
• After step 1, standard antiserum to antigen which is known to fix
complement is added to one set.
• If antibodies were not present in the test serum then the antigen
would react with the standard antiserum fixing the complement.
• On the other hand if antibodies are present in the test serum the
antigen would be utilized in the first step. So, no reaction would
occur between the standard antiserum and the antigen and
therefore no fixation of complement would cause lysis of sheep
red blood cells. Thus in this case haemolysis indicates a positive
result.
Congulatinating complement absorption test
• Here horse complement which is non-haemolytic is
used.
• The indicator system used is sensitized sheep red blood
cells mixed with bovine serum.
• Bovine serum contains a beta globulin called conglutinin
would also combine with this complement causing
agglutination (conglutination) of the sheep red blood
cells, indicating a negative result.
Immune adherence
• When some bacteria (such as vibrio cholera or
treponemapallidum) combine with their specific
antibody in the presence of complement and
some particles such as erythrocytes or platelets,
they adhere to the erythrocytes or platelets. This
is called immune adherence.
Immobilisation test
• Here antigen is incubated with patient’s serum in
presence of complement.
• If specific antibody is present it would immobilize the
antigen. Eg.Treponema palladium immobilization test,
considered gold standard for the serodiagnosis of
syphilis
Cytolytic tests
• The incubation of a live bacterium with its
specific antibody in the presence of complement
leads to the lysis of the bacteria cells.
• This is the basis of vibriocidal antibody test used
to measure anticholera antibodies.
Neutralization
• A procedure in which the chemical or biological activity of a
reagent or a living organism is inhibited, usually by a specific
neutralizing antibody.
• As an example, the lethal or the dermonecrotic actions of
diphtheria toxin on animals may be completely neutralized
by an equivalent amount of diphtheria antitoxin—an
antibody produced in animals or in humans after contact
with diphtheria toxin or toxoid.
Neutralization
• Lesser amounts of antitoxin provide intermediate
degrees of inhibition.
• These facts provide the basis for the Schick test for
susceptibility to diphtheria.
• Tetanus and botulinus toxins may be similarly inhibited
by their specific antitoxins.
• In contrast, the typical toxins of dysentery and other
gram-negative bacteria are only slightly neutralized, even
by large excesses of antibody.