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