By

Dr. Humera Kausar

(PhD Molecular Biology)

Antigen and Antibody Interaction basis of serological testing

Antigen Antibody Complex

Antigen and Antibody Interaction is the basis of serological testing

Principle

Soluble antigen combine with its soluble

antibody and form a lattice that develops

into a visible precipitate.

One of the easiest of serological tests

Occur best when antigen and antibody are present in optimal proportions (Equivelance).

Precipitation

Examples;

• Ring precipitation test• A solution of antigen is layered on the

surface of the antibody in a small tube or capillary tube. A narrow ring of precipitate occurs near the junction of two fluids.

• This type of test is used for grouping streptococci (according to C polysaccharide) and for determining unknown proteins in forensic medicine

Precipitation

Gel - diffusion precipitation

• Antigen and antibody meet in an agar medium and a thin line of precipitate is produced there (antigen - antibody complex).

• 1. Single diffusion

Antigen diffuses in the agar medium (antibody is homogenously spread in the agar). It is carried out either in the tubes - single gel - diffusion by Oudin or on the slide - single radial immunodiffusion by Mancini.

Gel - diffusion precipitationThe principle of the reaction: The antigen is placed in a well cut in an agar gel containing suitable diluted antibody. A ring of precipitate forms where the reactants meet in optimal proportions. The higher is the concentration of the examined antigen, the greater is the diameter of the ring. According to the diameter of the ring it is possible to count the concentration of the examined antigen.

This type of immunodiffusion is used for quantitative determination of immunoglobulins (IgM, IgG, IgA and IgD), complement components and other serum proteins.

Gel - diffusion precipitation

2. Double immunodiffusion

is used more often. Antigen and antibody are allowed to diffuse towards each other in an agar medium, e.g. from separate wells cut in an agar plate or in a Petri dish. When antigen and antibody meet in optimal proportions they produce a thin line of precipitate. Position of the precipitate line depends on concentrations of both antigen and antibody and on their diffusion coefficient.

•

Gel - diffusion precipitation

• This reaction is used for diagnosing various bacterial, viral, fungal and autoimmune diseases and for recognizing toxin production by Corynebacteriumdiphtheriae.

Gel - diffusion precipitation

Agglutination

6

Agglutination

Some of the major differences between Agglutination reaction and Precipitation reaction are:

The size of the antigen is different in these reactions.

Antigens are soluble molecules in precipitation reactions while they are large and insoluble molecules in case of agglutination.

Agglutination is a more sensitive reaction in comparison to precipitation.

15

Haemagglutination

RBC

16

Viral Hemagglutination

• the attachment of viral particles by their receptor sites to more than 1 cell.

• As more and more cells become attached in this manner agglutination becomes visible

18

Equivalence point: (suitable proportion between the virus particles and

RBCs)

Titer = 32 HA units/ml

Hemagglutination test: method

1:8

1:2 1:21:21:21:2

8 16 32 64 128 256

virus

serial dilution

mix with red

blood cells

side view

top view

One HA unit :minimum amount of virus that causes complete agglutination of RBCs

20

ELISA

The ELISA plate is coated with Antibody to

detect specific antigen

Enzyme labeled

Alkaline Phosphatase (AP) or

Horseradish Peroxidase (HRP)

They are readily availableThey are stable and remain usefull for long

Types of ELISA

Normal

Disease

Prepare a surface to which a known quantity of capture antibody is bound.

Block any non specific binding sites on the surface

Apply the antigen-containing sample to the plate.

Wash the plate, so that unbound antigen isremoved.

Apply enzyme linked primary antibodies asdetection antibodies which also bind specifically tothe antigen.

Wash the plate, so that the unbound antibody-enzyme conjugates are removed.

Sandwich ELISA Procedure

Apply a chemical which is converted by theenzyme into a coloured product.

Measure the absorbency of the plate wells todetermine the presence and quantity of antigen

Sandwich ELISA Procedure

Indirect ELISA Procedure

• The protein antigen to be tested for is added to each well of ELISA plate, where it is given time to adhere to the plastic through charge interactions

• A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen

Indirect ELISA-ContThen the serum is added, which contains a

mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well.

Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well. This secondary antibody often has an enzyme attached to it

Indirect ELISA-Cont A substrate for this enzyme is then added. Often,

this substrate changes colour upon reaction with the enzyme. The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen.

The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change. Often a spectrometer is used to give quantitative values for colour strength

Indirect ELISA

Western blot

The western blot (sometimes called the protein immunoblot) is awidely accepted analytical technique in molecular biology used to detect specific proteins in the given sample.

Used for the detection of HIV

Western blot

Western blot

Western blot

37

HEMAGGLUTINATION INHIBITION TEST (HI)

VIRUSE SERUM

38

In the absence of anti-virus antibodies

Erythrocytes

Virus

Virus agglutination of

erythrocytes

39

In the presence of anti-virus antibodies

Erythrocytes

Virus Anti-virus

antibodies

Viruses unable to bind to

the erythrocytes

40

Hemagglutination Inhibition

Immunoflouresence

This technique uses the specificity of antibodies to their antigen to target fluorescent dyes to specific biomolecule targets within a cell, and therefore allows visualization of the distribution of the target molecule through the sample.

Immunoflouresence

THANKS