Immunodiffusion techniques• APPLICATIONS • To determine relative concentrations of Antibodies /

Antigens. • To compare Antigens or • To determine the relative purity of an Antigen

preparation.• For disease diagnosis. • Serological surveys.The combination of antibody (Ab) with antigen(Ag) is the fundamental

reaction of immunology.

Primary binding tests Secondary binding tests Tertiary binding tests

Principle• Soluble Ab & soluble Ag interacting in aqueous solution

form lattice that develops into insoluble visible precipitate.

• Soluble Ag: Toxins, toxoids, proteins, carbohydrates, glycoproteins, Liproproteins

• Both qualitatively & quantitatively in solutions & gels.

• Formation of Ag-Ab lattice depends on the valency of both Ag & Ab:

Ab must be bivalent Fab fragments- Ag must be bivalent / polyvalent.

Precipitation

•Insoluble complexes

•Visible to the eyes

Precipitation Curve

• Zone of Equivalence optimum precipitation

• Prozone excess antibody is present

• Postzone excess antigen is present

Prozone and Postzone phenomena are negative

reactions.

Immunodiffusion

Precipitation Reactions Immunodiffusion

o Radial Immunodiffusion (Mancini method).o Ouchterlony Double Diffusion

Electrophoresiso Rocket Immunoelectrophoresiso Immunoelectrophoresis

• Precipitation is best demonstrated

• Random movement of Ag or Ab to form Ag-Ab complexes in medium, such as gel.

MEDIUM Agar - high molecular weight complex polysaccharide

- from seaweed Agarose- purified agar- Used to help stabilize the diffusion process

and allow visualization of precipitin bands.

0.3 – 1.5 % Agar concentration: diffusion of most reactants

Agarose- more preferred than agarAgar has strong negative charge; Agarose has almost none (no charge)

- interactions between gel and reactants are minimized.

Passive Immunodiffusion

• Passive diffusion method in which a concentration gradient is established for an antigen and/or antibody

- diffusion of reactants to form Ag - Ab reactions without electric current to speed up reaction.

Rate of Diffusion1. Size of particles2. Temperature3. Gel viscosity4. Amount of hydration5. Interactions between matrix and reactants.

Immunodiffusion Techniques

• Radial Immunodiffusion- A single diffusion technique where Ab is put into

gel and Ag is measured by the size of a precipitin ring formed when it diffused out in all directions from a well cut into the gel.

• Ouchterlony Double Diffusion- Both Ab and Ag diffuse from wells into a gel

medium.

Radial Immunodiffusion

• Ag is added to an antibody rich media.

• The two continue to react until the zone of equivalence is reached.

• The area of ring is a measure of the Ag concentration.

• Interpretation– Diameter of ring is

proportional to the concentration

• Quantitative– Ig levels

• Method– Ab in gel– Ag in a well

Ouchterlony Double Diffusion

• Antigen and antibody diffuse independently through a semi solid medium (agar)

Wells are cut in the gel

Reactants are added in the well

Incubate (12-48 hrs) in a moist chamber

Precipitin lines will form

(where the moving front of the antigen meets antibody)

The density of the line reflects the amount of immune complexes formed

• Both antigen and antibody can diffuse independently.

Ouchterlony

• Antibody that is multispecific is placed in the central well

• Different antigens are placed in surrounding wells

**Position of the precipitin bands between wells allows for the antigens to be compared with one another.

0.5% Amido-black

0.5% Coomassie Brilliant Blue

Diffusion patterns

Fusion of lines at their

junction to form an arc - Serologic identity /

presence of common epitope Crossed lines

- Demonstrates 2 separate reactions

- Compared antigens shared no common epitopes

Fusion of 2 lines with spur

- Partial identity

IMMUNOELECTROPHORESIS

• Double-diffusion technique that utilizes electric current to enhance results.

• SPEED, Specificity.

• Introduced by Grabar and Williams in 1953.

• Combine immunodiffusion with electrophoresis

• Can be used for semiquantitaion of wide range of antigens

• Qualitative

• Antigen source: serum.

Electrophoresis Techniques

• Electrophoresis separates molecules according to differences in their electrical charge.

– Rocket Immunoelectrophoresis

– Countercurrent Immunoelectrophoresis

Principle

Antigen from serum

Antigen from serum

electrophoresedelectrophoresed

Trough is cut in the gel parallel to the line of separation

Trough is cut in the gel parallel to the line of separation

Antiserum is placed in the

trough

Antiserum is placed in the

trough

Incubate: 18-24 hours

Incubate: 18-24 hours

•double diffusion occurs at right angles to the

electrophoresis separation

•Precipitin lines develop where Ag-

Ab combination takes place.

•double diffusion occurs at right angles to the

electrophoresis separation

•Precipitin lines develop where Ag-

Ab combination takes place.

Lines or arcs shape, intensity and location: compared with normal serum

control

Lines or arcs shape, intensity and location: compared with normal serum

control

2 step process

Immunoeletrophoresis

• Method

– Ags are separated by electrophoresis

– Ab is placed in trough cut in the agar

• Interpretation– Precipitin arc represent individual antigens

Uses

• Immunodeficiencies can be detected by this procedure, if no precipitin band is formed for a particular Ig.

• Overproduction of serum proteins.

• Deficiencies in complement can also be detected.

• Identification of monoclonal protein

– Free kappa and lambda light chains

• May be used to identify urine proteins.

• Testing normal & abnormal proteins in serum/urine.

• Purity of Ag.

Countercurrent electrophoresis

Method

– Ag and Ab migrate toward each other by electrophoresis

– Used only when Ag and Ab have opposite charges

• Qualitative- Rapid.• Detection of Antinuclear- Ribonuclear protein.

Ag Ab- +

Rocket Immunoelectrophoresis( LAURELL TECHNIQUE )• One dimension electroimmunodiffusion

- Adaptation of RID

• Electrophoresis is used to facilitate migration of antigen in agar- Ag diffuses out of the well: precipitation begins- Change in Ag concentration: dissolution and reformation of

precipitate.

End result: precipitin line = conical-shaped, resembles a racket• . The height of the racket is measured

- Height of the racket is directly proportional to the amount of antigen present

• Racket electrophoresis is more rapid than RID.

Rocket electrophoresis

Uses of Racket Immunoelectrophoresis

• Quantitate Igs- (using a buffer = pH 8.6)

• Assay of proteins- When concentration is too

low to be detected by nephelometry and too high for RID

- Ex: alpha-fetoprotein, Igs in urine and spinal fluid, Complement components in body fluid.

Applications

• Screening tool for differentiation of more than 30 serum proteins.

- Major classes of immunoglobulins

• Used for the detection of:

- Myelomas

- Malignant lypmhomas

- Other lymphoproliferative disorder.