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MTEB2404
IMMUNOELECTROPHORESIS
Objective
1. To learn the technique of immunoelectrophoresis
2. To be able to observed the reaction given by electrophoresis
3.
Principle
The immunoelectrophoresis used to characterize antibodies. This
technique is based on the principle of electrophoresis of antigen and
immunodiffusion of the antigens with a polyspecific antiserum to form
precipitin bands.
ELECTROPHORESIS
During electrophoresis, molecules placed in an electric field acquire a
charge and move towards appropriate electrode. The mobility of
molecule is dependent on a numbers of factors:
1.Size/shape of molecule
2.pH
3.Heat generated by ionic strength buffer
4.Charge particles
IMMUNODIFFUSION
Antigen
Material provided
Antiserum A has antibody against whole serum and hence contains a
mixture of antibodies against serum proteins. It will thus form more
than one precipitin line indicating the heterogeneity of antisera
Antiserum B has antibody against IgG. It contain single antibody, so
only one precipitin line can be formed.
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Procedure
1.10ml
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Discussion
Immunoelectrophoresis
identifies
proteins based on their combined
electrophoretic and immunological
properties.
This method is useful to monitor
antigen and antigen-antibody purity
and to identify a single antigen in a
mixture of antigens. The three
major
immunoglobulins
were
detecting are immunoglobulin M
(IgM), immunoglobulin G(IgG), and
immunoglobulin A (IgA).
In this experiment, serum proteins
are separated by agarose gel electrophoresis and the point of
equivalence is observed by the maximum antigen-antibody complex
formation.
The properties of agarose gels
have a small number of fixed
charges,
which
cause
a
phenomenon
known
as
electroendosmosis
(EEO).
EEO
causes a slow net flow of water
through the gel away from the
positive electrode.
At a pH of 8.5, antibodies are nearly
uncharged, and their slow electrophoretic migration is nullified by the
EEO flow through the agarose. Most other proteins are highly charged
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Conclusion:
From this experiment, the antiserum B formed a dense precipitin arc
against IgG antiserum, thus this indicates the heterogenicity of
antiserum to antigen.
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