[PRACTICAL 4] MTEB2404

IMMUNOELECTROPHORESIS

Objective

1. To learn the technique of immunoelectrophoresis

2. To be able to observed the reaction given by electrophoresis

3. Able to establish the homogeneity/heterogeneity of the antisera

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 thus resolved by electrophoresis is subjected to
immunodiffusion wit antiserum added in a cut of the agarose gel.
•Immunodiffusion is due to the diffusion, density, gradient of antigen
and antibody which formed at the zone of equivalence, antigen
antibody complex precipitate to form an opaque arc shaped line in
gel.
•This precipitate line indicates the presence of antibody specific to
that 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 of 1.5% agarose (0.15g/10ml) was prepared in 1X
electrophoresis buffer by heating slowly till agarose dissolves
completely.
2.The end of the glass was marked. It will be towards
negative electrode during electrophoresis
3.The glass plate was placed on a horizontal surface. 10 ml of
agarose solution was pipette and spread onto the plate.
4.The glass plate was placed on the template holder provided in
ETS-2 and fixes the template. Punch a 3mm well with the gel
puncher, towards the negative end
5.Cut two roughs with the gel cutter provided, but do not
remove the gel from the trough.
6.12-15ul of antigen was added to the gel.
7.The glass plate was placed in the electrophoresis tank such that
the antigen well is at the cathode / negative electrode. 1X
electrophoresis buffer was poured such that it covers the gel.
8.The voltage was set to 50-100V and electrophoreses until the blue
dye travels 3-4cm from the wells. If electrophoresis beyond 3 hours,
heat can be generated and this is not good for the result of this
experiment.
9.The gel was removed from both the troughs and the plate was
kept at room temperature for 15 min. 250ul of antiserum-A was
added in one of the troughs and antiserum B in the other
10.The plate was placed in a moist chamber and diffusion was
allowed to occur at room temperature for overnight.

Result and observation

The results on this experiment were showed a two straight seen.

Forming of precipitin was indicated as presence of antibody specific to
an antigen. The 2 lines formed were indicated the heterogenecity of
antiserum to antigen. While, the single lines formation are the
homogenecity.
The formation line at corner left shown the IgG, which was formed
due to charges. The others horizontal lines were the IgM and IgA which
the lines much shorter causing the lines formed curved.

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The result picture:

Precipitation (IgG)
Antiserum A

Mixed antigen

Antiserum B

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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at pH 8.5, and will migrate through an agarose gel.

If antiserum is included in the gel matrix, immuno precipitates will
form, in a pattern dependent upon the antigen concentration. This
technique, immunoelectrophoresis, is much faster than
immunodiffusion, and has been applied in a variety of geometries, to
analyze simple or complex samples.

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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