Crossed immunoelectrophoresis
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Transcript of Crossed immunoelectrophoresis
Crossed Immunoelectrophoresis
ByM.Vharshini
B.Sc. Biomedical sciencesSri Ramachandra University
INTRODUCTION
•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
steps•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 step 2.
PRINCIPLE
Crossed immunoelectrophoresis is performed in two steps:Step 1: The antigens are separated by electrophoresis in an agarose gel.Step 2: The separated antigens are electrophoresed at
right angles into a freshly applied layer of agarose containing a
predetermined 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 1x TAE ) to fill the
electrophoresis tank and to cast the gel.• The gel is prepared by dissolving the agarose powder in an appropriate
buffer, such as TAE or TBE 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.
AGE Cont…
2) Loading of samples3) Electrophoresis4) Fixation, drying, staining
The sequential steps in crossed
immunoelectrophoresis
STEP 2: Immune- Precipitation
•While the electrophoresis is being run an agarose gel plate containing the antiserum is prepared for the electro-immunoassay.•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 antiserum.• 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 knife.• 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 slide. • The moist cut surface of the gel strip will then
adhere to the edge of the slide (Fig. 1, D and E).• 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 strip.• As in electro-immuno assay, this
electrophoresis is continued until all the antigen has been precipitated.• The plate is then washed, dried and stained.
Crossed Immunoelectropherogram Of
Human Serum
First dimension: •Agarose Gel Electrophoresis. Second dimension: •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 electrophoresis.
PRINCIPLE AREAS OF APPLICATION
• Antigen quantitation• Quantitation of subpopulations of extensively heterogeneous
antigenic populations• Studies of association and dissociation phenomena• Studies of hereditary polymorphism, micro-heterogenecity
and fragmentation, and • Studies of immunochemical relationships between antigens
and between antisera.
REFERENCE
• Crossed ImmunoelectrophoresisP. 0. GANROTScand. J. din. Lab. Invest. 29, suppl. 124, 3 9 4 1 . 1972.
• ImmunoelectrophoresisL.-A. NILSSONScand. J. Immunol. Vol. 17, Suppl. 10, 71-76, 1983
• Crossed ImmunoelectrophoresisA. 0. GRUBBScand. J. Irnrnunol. Vol. 17, Suppl. 10, 13-124,1 983