•Crossed immunoelectrophoresis, also known as
Two-dimensional (2-D) immunoelectrophoresis is a
particularly useful technique for the quantitation of
mixtures of proteins and the analysis of the composition
of protein mixtures.
•The method consists of two sequential electrophoretic
•Agarose gel electrophoresis is used for the antigen
separation in step 1 and an Antibody-containing
agarose gel is used for the immune- precipitation in
Crossed immunoelectrophoresis is performed in two steps:
Step 1: The antigens are separated by electrophoresis in an
Step 2: The separated antigens are electrophoresed at right angles
a freshly applied layer of agarose containing a
amount of antibody.
STEP 1: Agarose Gel Electrophoresis
Antigen mixture is electrophoresed in an agarose gel that allows the
separation of its different components based on their charge along
the gel slide.
1) Casting of gel
• Prepare sufficient electrophoresis buffer (usually 1xTAE ) to fill the electrophoresis
tank and to cast the gel.
• The gel is prepared by dissolving the agarose powder in an appropriate buffer, such
asTAE orTBE and ethidium bromide is added to it.
• A 1% suspension of agarose is dispersed in the buffer before heating it to near-
boiling point, but avoid boiling.
• The melted agarose is allowed to cool sufficiently before pouring the solution into
a cast as the cast may warp or crack if the agarose solution is too hot.
• A comb is placed in the cast to create wells for loading sample, and the gel should
be completely set before use.
2) Loading of samples
4) Fixation, drying, staining
STEP 2: Immune- Precipitation
•While the electrophoresis is being run an agarose
gel plate containing the antiserum is prepared for the
•After the agarose has set the glass plate covering
the gel is removed and two parallel cuts ( 2 to 5 mm
apart and near, and parallel to, the future cathodal
edge of the plate) are made in the gel. [Fig F]
•The gel between the two cuts is removed with
consequent formation of a ditch.
• After the proteins of the sample have been
sufficiently separated a strip of the gel containing
the electrophoretic protein fractions is cut out and
transferred to the ditch in the gel containing
• The gel plate to be cut is placed on a graph
paper, which is used as a simple coordinate
system during the cutting.
• Supporting blocks may be used as a support for a
ruler and the ruler serves as a support for the
• When the strips have been cut out all the
surrounding gel is cautiously removed, and all
droplets of fluid from the glass plate are wiped off
with a filter paper (Fig. B).
• The next step is to transfer the gel strip from
the steel blade to the edge of the microscopic
• The moist cut surface of the gel strip will then
adhere to the edge of the slide (Fig. 1, D and
• The strip is then easily placed in proper
position in the prepared ditch of the gel
containing antiserum (Fig. 1 G).
• When the strip is in correct position and in
good electric contact with the antiserum-
containing gel the final electrophoresis is run
with the electric field perpendicular to the
• As in electro-immuno assay, this
electrophoresis is continued until all the
antigen has been precipitated.
• The plate is then washed, dried and stained.
•Agarose Gel Electrophoresis.
•Electrophoresis occurred in the same
gel containing rabbit antibody against
human serum with the anode at the top.
•The antibody is isoelectric at pH 8.6
and therefore stationary during
PRINCIPLE AREAS OF APPLICATION
• Antigen quantitation
• Quantitation of subpopulations of extensively heterogeneous antigenic
• Studies of association and dissociation phenomena
• Studies of hereditary polymorphism, micro-heterogenecity and
• Studies of immunochemical relationships between antigens and