Immunodiffusion techniques

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.

Precipitation Insoluble complexes Visible to the eyes

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.

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

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.

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.

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 (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 .

Method Interpretation Quantitative Ab in gel Ag in a well 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)

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)

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

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

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

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

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