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

 Electrophoresis
o Rocket Immunoelectrophoresis
o 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 agar

Agar 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 Diffusion
1. Size of particles
2. Temperature
3. Gel viscosity
4. Amount of hydration
5. 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.
 • Method
– Ab in gel
– Ag in a well

• Interpretation
– Diameter of ring is
proportional to the
concentration

• Quantitative
– Ig levels
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
Ouchterlony

• Both antigen and

antibody can diffuse
independently.
• Antibody that is multispecific
is placed in the central well

• Different antigens are placed

in surrounding wells

0.5% Amido-black
0.5% Coomassie Brilliant
Blue

**Position of the precipitin bands between wells allows for the

antigens to be compared with one another.
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
2 step process

Trough is cut in
the gel parallel
Antigen from electrophoresed to the line of
serum separation

•double diffusion Antiserum is

occurs at right Incubate: 18-
24 hours placed in the
angles to the
trough
electrophoresis
separation
•Precipitin lines
develop where Ag- Lines or arcs shape, intensity and
Ab combination location: compared with normal serum
takes place. control
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

- +
Ag Ab

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