BCH361 [Practical] Biochemistry department
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Experiment (7): Single Radial Immunodiffusion and Immuno-
electrophoresis
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Aims:
• To get familiar with radial immunodiffusion and how it is used to determine the
concentration of a specific antigen.
• To learn the technique of immune-electrophoresis.
Introduction:
Antibody and antigen reactions is widely used in laboratory diagnostics including:
precipitation reactions, agglutination reactions, immunofluorescence, radioimmunoassay
(RIA), ELISA and Western blotting.
Precipitation reactions are based on the interaction of antibodies and antigens. They are
based on two soluble reactants (Ab and Ag) that come together to make one insoluble product,
the precipitate. These reactions depend on the formation of lattices (cross-links) when antigen
and antibody exist in optimal proportions (it is known as zone of equivalence and appears to
as precipitation). Excess of either component reduces lattice formation and subsequent
precipitation. (1)
In precipitation reactions the formation of an Ab-Ag lattice depends on the valency of both
the antibody and antigen. The antibody must be bivalent; a precipitate will not form with
monovalent Fab fragments. In other hand, the antigen must be either bivalent or polyvalent;
that is, it must have at least two copies of the same epitope or have different epitopes that react
with different antibodies present in polyclonal antisera. (2)
PAUSE AND THINK !!What is the differences between monoclonal and polyclonal antibodies?
Precipitation reactions differ from agglutination reactions in the size and solubility of the
antigen and sensitivity. For precipitation, antigens are soluble molecules, and for agglutination,
antigens are large, easily sedimented particles. In addition, agglutination is more sensitive than
