1. Immunodiffusion techniques

2. 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.

3. Precipitation
Insoluble complexes
Visible to the eyes

4. Prozone and Postzone phenomena are negative reactions.
Precipitation Curve
Zone of Equivalence optimum precipitation
Prozone
excess antibody is present
Postzone
excess antigen is present
Prozone and Postzone phenomena are negative reactions.

5. Immunodiffusion Immunodiffusion Electrophoresis
Precipitation Reactions
Immunodiffusion
Radial Immunodiffusion (Mancini method).
Ouchterlony Double Diffusion
Electrophoresis
Rocket Immunoelectrophoresis
Immunoelectrophoresis
Precipitation is best demonstrated by Random movement of Ag or Ab to form Ag-Ab complexes in medium, such as gel.
Performed in an agarose gel supported by a glass slide or polyester film

6. MEDIUM Agar high molecular weight complex polysaccharid from seaweed
0.3 – 1.5 % Agar concentration: diffusion of most reactants
Agarose-
purified agar Used to help stabilize the diffusion process and allow visualization of precipitin bands.
Agarose- more preferred than agar
Agar has strong negative charge; Agarose has almost none (no charge) - interactions between gel and reactants are minimized.

7. Factors affecting Diffusion
diffusion of reactants to form Ag - Ab reactions without electric current to speed up reaction.
Rate of Diffusion
Size of particles
Temperature
Gel viscosity
Amount of hydration
Interactions between matrix and reactants.

8. 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.

9. Radial Immunodiffusion (RID)
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.
Area of ring: measure of Ag concentration compared to a standard curve using Ags of known concentration .

10. Method Interpretation Quantitative Ab in gel Ag in a well
Diameter of ring is proportional to the concentration
Quantitative
Ig levels

12. Ouchterlony Double Diffusion
Antigen and antibody diffuse independently through a semi solid medium (agar)

13. Reactants are added in the well
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)

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

15. IMMUNO ELECTROPHORESIS
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.
Why would we want to combine immunodiffusion with electrophoresis? SPEED, Specificity Two step process

16. Electrophoresis Techniques
Electrophoresis separates molecules according to differences in their electrical charge.
Rocket Immunoelectrophoresis
Countercurrent Immunoelectrophoresis

17. Method
Ags are separated by electrophoresis
Ab is placed in trough cut in the agar
Interpretation
Precipitin arc represent individual antigens

18. Thank you