SINGLE RADIAL IMMUNODIFFUSION AND

IMMUNOELECTROPHORESIS

BCH462- Practical

Widely used in laboratory diagnostics including:

➢ Precipitation reactions,

➢ Agglutination reactions,

➢ Immunofluorescence,

➢ Radioimmunoassay (RIA),

➢ ELISA

➢ Western blotting.

Based on ?

Soluble reactants make insoluble product.

Lattice formation ?

The antibody must be bivalent; a precipitate will not form with monovalent Fab fragments.

The antigen must be either bivalent or polyvalent; that is, it must have at least two copies of the same

epitope or have different epitopes that react with different antibodies present in polyclonal antisera.

Monoclonal Antibody Polyclonal Antibody

Consists of one antibody class/subclass which is selective for

a single epitope on the antigen

Contains a mixture of antibodies (mainly IgGs), often

recognizing multiple epitopes on the antigen

Because of their specificity, they are less likely to cross-react

with other proteins, giving lower background than polyclonal

antibodies

May contain non-specific antibodies resulting in background

staining

Specificity makes them ideal as the primary antibody in an

assay, for detecting antigens in tissue, or for affinity

purification of antigens

Useful as secondary antibodies or for immunoprecipitation,

as they target multiple epitopes providing a more robust

detection

Size.

Solubility of the antigen.

Sensitivity.

Single radial

immunodiffusion

(RID) -Mancini test-

Double

immunodiffusion

-Ouchterlonytest-

immuno-

electrophoresis (IEP)

immunofixationrocket electro-

immunodiffusion (EID)

counter

immune-

electrophoresis (CIEP)

Immunodiffusion Electro-immunodiffusion

Simple immunodiffusion (ID) technique is any technique that involving diffusion of

antigen or antibody through a semi-solid medium, usually agar or agarose gel,

resulting in a precipitin reaction.

Electro-immunodiffusion method reaction differs in the uses of an electric current to enhance the mobility of the reactants toward each other.

Single radial immunodiffusion (RID) -Mancini-

Is a quantitative immunodiffusion technique.

Used in immunology to determine the quantity (concentration) of an antigen.

Precipitin ring formation on a thin agarose layer.

Based on the diffusion of an antigen in a radial pattern from a cylindrical well through an agar or agarose gel

containing an appropriate mono-specific antibody.

As the antigen diffuses radially in all directions into the medium (which containing the fixed antibody), its

concentration continuously decreases until the equivalence point (zone of equivalence.) is reached.

At this point an insoluble immune precipitation complexes occurs .

The result is the formation of a precipitin ring around the antigen well.

The diameter of the ring increases with time as the antigen diffuses into the medium, reacts with the antibody,

and forms insoluble precipitin complexes.

At this stage, a linear relationship exists between the square of the ring diameter and the antigen concentration.

Since the diameter of the ring is directly proportional to the antigen concentration, a calibration curve of

diameter versus antigen concentration can be constructed using a series of reference standards of

known antigen concentration.

1. Incorporate the antibodies that are specific for the antigen of interest into molten agarose. Then pour the

mixture in Petri dish.

Petri dish containing the uniformly

distributed antiserum in the agarose gel.

Agarose + antiserum [antibody]

2. Cut the wells using a gel puncher. Then apply your standard antigen and sample antigen to the wells.

Antigen diffusion through the well radially.

3. After 24 to 48 hours, the diameter of circular precipitates formed around the wells then they are measured.

Figure: The precipitin ring is formed around the wells of the samples.

4. A plot of precipitation ring diameters versus concentrations is made for the samples with known antigen

concentrations.

➢ Wells (1-4), contain standard concentrations

of protein under study ”antigen”.

➢ The graph shows the resulting calibration

curve from which, an unknown concentration of the antigen can be determent.

Figure: precipitation curve.

Immuno-electrophoresis

Based on the principles of electrophoresis of antigens and immunodiffusion of the electrophoresed antigens with

a specific antiserum to form precipitin bands.

Application?

To check the presence, homogeneity, and specificity of antibodies.

This technique is usually used for the detection of three immunoglobulin levels in blood: IgM, IgG, and IgA

It is the rapid version of bi-directional gel diffusion; it involves the cooperation of electrophoresis and immunodiffusion.

In this system, a thin layer of agar is poured on a slide. After digging small holes in the agar, antigens are added into theseholes and the slide is subjected to an electric current.

The antigen molecules migrate at different rates through the electric current because of differing electric charges at a specific pH value (electrophoresis).

During the electrophoresis molecules placed in an electric field acquire a charge and move towards appropriate electrode. Mobility of the molecule is dependent on a number of factors: size of molecules to be separated, concentration of agarose gel, voltage applied and the buffer used for electrophoresis.

After electrophoresis, some bands are cut away from the agar parallel to the migration track, and the cut places are filled with antiserum.

When a specific antigen encounters the antibody, a white precipitin band is formed on a black background (immunodiffusion).

This precipitin band represents the presence of an antibody for a specific antigen. If the antibody is homogenous, there is only one precipitin band observed. The presence of more than one precipitin band represents antibody heterogeneity.

On the other hand, if there is no precipitin band seen, that means there is no antibody specific to the target antigen

1. A gel is prepared with a well to add the antigen in it.

2. The antigen mixture is first electrophoresed to separate its components by charge.

3. Troughs are then cut into the agar gel parallel to the direction of the electric field. Antiserum is added to the

troughs.

4. Lines of precipitation [arcs], will be produced where they meet in appropriate proportions [at the zone of

equivalence].