IMMUNODIFFUSION TECHNIQUES Principle Soluble Ab soluble Ag interacting

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

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 q Immunodiffusion o Radial Immunodiffusion (Mancini method). o Ouchterlony Double Diffusion q Electrophoresis o Rocket Immunoelectrophoresis o Immunoelectrophoresis • Precipitation is best demonstrated by Random movement of Ag or Ab to form Ag-Ab complexes in medium, such as gel.

MEDIUM q Agar q high molecular weight complex polysaccharid from seaweed q 0. 3 – 1. 5 % Agar concentration: diffusion of most reactants q Agaroseq purified agar Used to help stabilize the diffusion process and allow visualization of precipitin bands. q 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 q diffusion of reactants to form Ag - Ab reactions without electric current to speed up reaction. q Rate of Diffusion q Size of particles q Temperature q Gel viscosity q Amount of hydration q 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.

• 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

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.

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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