Complement fixation test - PowerPoint Presentation

Slide1

Complement fixation testSlide2

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 availableSlide3

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

.Slide4

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 ).Slide5

principleSlide6

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.

principleSlide7

Interpretation

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

.

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 autolysisSlide8

Positive TestStep 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)Slide9

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)Slide10

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)Slide11

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.Slide12

Advantages and disadvantages of CFT

Advantages

Ability

to screen against a large number of viral and bacterial infections at the same time.

Economical

.

Disadvantages

Not

sensitive - cannot be used for immunity screening

Time

consuming and labor intensive

Often

non-specific e.g. cross-reactivity between HSV and VZVSlide13

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.Slide14

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.Slide15

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.Slide16

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 syphilisSlide17

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.Slide18

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. Slide19

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.