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ELECTROPHORESIS

Immuno-electrophoresis technique is based on the electrophoretic mobility and immuno


precipitin reaction. The term electrophoresis is defined as the migration of charged
particle under the influence of an electric field. In precipitin reaction, an antigen
combines with its specific antibody to form an antigen –antibody complex. Since most of
the Ag-Ab complexes are insoluble, they can be seen with the naked eye. In immuno-
electrophoresis the antigens are first separated based on their electrical charge and then
visualized by the precipitin reaction. There are different types of immuno-electrophoresis
techniques are used for various analytical purposes.
COUNTERCURRENT ELECTROPHORESIS
Counter current immunoelectrophoresis is a modification of immunoelectrophoresis in
which antigen and antibody migrate towards opposite directions and form a visible white
precipitate in the area between the wells. It is also known as voltage facilitated double
immunodiffusion because the migration of antigen and antibody through the agarose gel
is due to the applied voltage rather than simple double immunodiffusion. This qualitative
technique is much faster and more sensitive than the double diffusion technique.
In this method, the antigen and antibody are placed in parallel wells and under the
influence of an electric field move towards one another. A precipitin band appears where
they meet in the appropriate ratio.
In this method, the antigen is placed in a well at the cathode end and antibody is placed at
the anode side
In this method, immunoprecipitation occurs when antigen at the cathode (negative pole)
is caused to migrate in an electric field through a suitable medium of diffusion against a
stream of antibody migrating backward from the anode (positive pole) because of
endosmotic flow. When an electrical current is applied through the alkaline buffer, the
negatively charged antigen molecules migrate toward the positive electrode and thus
towards the wells filled with antibody and the positively charged antibodies are migrated
toward the negative electrode. At some point between the wells, a zone of equivalence
occurs and the antigen-antibody complex precipitates as a visible white line.
In this test, the antigen and abtibody are placed in wells punched out of an agar gel and
the antigen and antibody are electrophoresis into each other where they form a
precipitation line.
This test only work if condition can be found where the antigen and antibody have
opposite charges. It major advantage is its speed
The precipitin line indicates the presence of antibody specific to the antigen while the
absence of precipitin line indicates absence of corresponding antibody in the test
antiserum to the given antigen. The presence of more than one precipitin line indicates
the heterogeneity of the antibody for the antigen in the test sera.
. During electrophoresis molecules placed in an electric field acquire a charge depending
on their pI (Isoelectric point). Hence they move towards the appropriate electrode. The
antigen, if it is negatively charged moves towards the anode.
Antibody (Immunoglobulin) at pH 7.6 has a charge nearing zero. During electrophoresis, the agarose
matrix absorbs OH- ions on the surface resulting in a net increase in positive ions at a distant from the
matrix. These positive ions migrate towards the negative pole with a solvent shield, resulting in a net
solvent flow called endosmosis. Hence antibody molecules which have no charge move towards cathode
along with solvent shield due to this phenomenon. Thus the antigen and antibody travel towards each
other and at a point where there is optimum concentration of both a line of precipitation (band) is
formed

In this method, immunoprecipitation occurs when antigen at the cathode is caused to migrate in an
electric field through a suitable medium of diffusion against a stream of antibody migrating from the
anode because of endosmotic flow. This is one-dimensional double electroimmunodiffusion in which
antibody and antigen are moved towards each other by an applied electric field, because the gel is
buffered at a pH between the isoelectric points of the antigen and antibody. The antigen and antibody
are placed in separate wells in an Agarose gel plate. The gel is alkaline and antibody (Immunoglobulin) at
pH 7.6 has a charge-nearing zero. During electrophoresis, the agarose matrix absorbs OH- ions on the
surface resulting in a net increase in positive ions at a distant from the matrix. These positive ions
migrate towards the negative pole with a solvent shield, resulting in a net solvent flow called
endosmosis. Hence, antibody molecules, which have no charge, move towards cathode along with
solvent shield due to this phenomenon.

ROCKET ELECTROPHORESIS
Rocket Immunoelectrophoresis, also known as electro-immunodiffusion, is a simple,
quick and reproducible method for determining the concentration of antigen in an
unknown sample. This quantitative one dimensional immunoelectrophoresis method
involves a comparison of antigen sample of unknown concentration with a series of
dilutions of a known concentration of the antigen and requires a monospecific antibody
against the antigen under investigation. In this method, antigen migrates from the well
through agarose gel containing antiserum, forming rocket shaped precipitin peaks. The
height of this peak is proportional to the concentration of the antigen loaded in the
corresponding well
The pattern immunoprecipitation resembles a rocket and hence the name
Various concentrations of antigen are loaded side by side in small circular wells along the
edge of the agarose gel which contains specific antibody.
In Rocket Immunoelectrophoresis, negatively charged antigen samples are
electrophoresed in an agarose gel containing antibody which is specific to that antigen.
As the antigen moves out of the well and enters the agarose gel, it combines with the
antibody to form immune complex which is visible as white. Because the antigen is
migrated through the gel under the influence of an applied electric current, it moves in
one direction. During the initial phase there is considerable antigen excess over antibody
and no visible precipitation occurs. However, as the antigen sample migrates further
through the agarose gel, more antibody molecules are encountered that interact with the
antigen to form immune complex. When this immune complexes become large enough to
be retained within the gel, movement of the antigen stops. The area of precipitin has the
shape of a rocket and its height is proportional to the concentration of antigen in the
corresponding well.
On electrophoresis, the antigen begins to migrate towards the anode and interacts with
antibody molecule to form a soluble antigen0 antibody complex. However, as the
samples are electrophoresed further more antibody molecules are encountered that
interact with antigen and when equivalence is reached =, the antigen-antibody complex
precipitates. This precipitin line is seen in the form of rocket. Higher the amount of
antigen loaded in the gel, farther the antigen will travel through the gel. Hence with
increasing concentrations of antigen, a series of rockets of increasing heights are seen that
is proportional to the amount of antigen in the well. Therefore, a direct measurement of
the height of the rocket will reflect on the antibody concentration. The standard graph of
antigen concentration Vs peak height is then constructed and from the peak height of the
unknown sample, concentration of antigen is determined.
The height of the precipitin peak depends on the concentration of antigens loaded in the
corresponding wells. By plotting the graph of concentration of antigens versus length of
the precipitin peaks one can calculate the concentration of test antigen.
This method allows the determination of the concentration of a particular antigen in mixture.  The
antigen sample is placed in wells cut in agar containing specific antiserum to the antigen to be assayed.
 When a directelectric current in applied, most antigens migrate towards the anode and the IgG
antibodies migrates towards the cathode, initially soluble (Ag-Ab) antigen-antibody complexes are
formed in the presence of excess antigen. Principles of cross – over immune electrophoresis Magendira
Mani Vinayagam/ Academia.edu/ Asst. Prof., IC., VNB Page 5  When all antigens have migrated into
the gel, equivalence is reached and antigen antibody complexes precipitated.  The area under the
rocket shape is directly proportional to the antigen concentration. When the precipitation areas have
become stationary (1- 10 hours) plot of rocket height against concentration will be linear. Thus by the
use of standards and the preparation of a calibration curve, the concentration of antigen solutions may
be determined.
1.Why is a blue dye added to the protein solutions for electrophoresis?
2. Why do precipitates form arcs?
3. How does immunodiffusion assay differ from immunoelectrophoresis assay?
4. How many arcs were observed from the whole serum sample? What does each arc
represent?
5. What results can you expect if the gel were stained with a protein stain?
Rocket height is proportional to the amount of Ag in the sample
Rocket Immunoelectrophoresis: RID + electrophoresis to facilitate migration of antigen
into the antibody containing agar
Rocket Immunoelectrophoresis used used to quantitate immunoglobulins and proteins
whose concentrations are too low to be detected by nephelometry and too high for RID
such as alpha-fetoprotein in amniotic fluid, Igs in urine and CSF, and complement
components in body fluids
Countercurrent Immunoelectrophoresis: Double diffusion + electrophoresis; Ag and Ab
are placed in a well directly opposite from one another whith specific pH to control Ab
and Ag migration. Occurs at 90 degree angles

2. height of the rocket is linearly proportional to the [Ag] concentration in the sample
applied