Counter immunoelectrophoresis (CIEP) is a laboratory technique used to detect antigen-antibody binding by facilitating the rapid migration of antigens and antibodies towards each other using an electric current in an agar or polyacrylamide gel. CIEP is similar to Ouchterlony immunodiffusion but uses electrophoresis to speed up the process, forming a precipitin line within 30-60 minutes if binding occurs. CIEP is commonly used to detect antigens and antibodies in serum to diagnose infectious diseases like hepatitis B and detect antigens in body fluids like cerebrospinal fluid. The procedure involves placing antigen in one well and antiserum in another on an agarose gel slide, applying an electric current,

1. Practical Immunology
And Serology
Counter
immunoelectrophoresis
By
Ahmed Riyadh Abdul Rahman
Al-Noor University College
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Counter immunoelectrophoresis
 *Counterimmunoelectrophoresis (CIEP) is a laboratory technique used to detect the
binding of an antibody to its antigen.
 *CIEP is a modification of Ouchterlony immunodiffusion,method that speeds up
migration of an antigen and antibody in the diffusion medium, usually an agar or
polyacrylamide gel by applying an electrical current. The effect is rapid migration of
the antibody and antigen out of their wells towards one another to form a line of
precipitation, or a precipitin line, indicate the formation of Ag-Ab complex in the
zone of equivalence.
 *The technique is similar to the Ouchterlony method, the only difference being that
the antigen/ antibody movement is facilitated by electrophoresis. It is thus also called
‘voltage facilitated double immunodiffusion’.

3. • CIEP depends on the movement of strongly negatively charged antigen
towards the anode and of antibody towards the cathode through the agar
under the electric field.
• The test is performed on a glass slide/plate in agarose gel, a pair of wells is
punched out where one well is filled with antigen and the other with the
antibody. Electric current is then passed through the gel, the migration of
antigen and antibody is greatly facilitated under the electric field, and the
line of precipitation as precipitin arcs (or lines) is made visible in 30–60
minutes, which indicates a positive reaction.
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PRINCIPLE

4. Counter immunoelectrophoresis principle
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5. Counter immunoelectrophoresis is mostly carried out with one
or more of the following objectives:
1. To rapidly check any antisera for the presence and specificity
of antibodies for a particular antigen.
2. To detect antigens and/or antibodies in serum for diagnosis
of a particular disease.
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Objective and Application

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1. It is a rapid and a highly specific method for detection of both antigen and
antibodies in the serum, cerebrospinal fluid, and other body fluids in the diagnosis
of many infectious diseases including bacterial, viral, fungal, and parasitic.
2. The test was very popular in the past for detecting various antigens such as alpha-
fetoprotein in serum and capsular antigens of Cryptococcus and Meningococcus in
cerebrospinal fluid.
3. Still today, it is commonly used for Hepatitis B surface antigen (HBsAg),
fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in
the CSF.
4. It is a rapid sensitive method for detecting pneumococcal capsular antigens in
sputum.
CIEP has many uses

7. PROCEDURE
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1. Prepare 10 ml of 1.0% Agarose (0.1 g/10 ml) in 1X Assay Buffer is heating slowly until agarose dissolves
completely.
2. Mark the ends of a glass slide as +ve and -ve so that when placed in the electrophoresis apparatus, the +ve mark is
faced towards the anode and the negative mark faced towards the cathode.
3. Place the glass plate or slide on a horizontal surface, pipette and spread (5) ml of agarose onto the glass slide, It is
allowed to solidify for 15 minutes.
4. Cut Wells in the gel according to the template using gel puncher. The distance between the two wells should not be
more than 0.5 cm.
5. Place the slide in the electrophoresis tank and fill the tank with 1X electrophoresis buffer till the buffer just covers
the gel surface, Do not add excess of buffer.
6. Add 10µl of antigen in each of the two wells towards the cathode (Negative electrode) and 10µl of positive control
antiserum and test antisera in wells towards the anode (Positive Electrode).
7. Connect the power cord to the electrophoretic power supply according to the convention.
8. 50 V is applied and the electrophoresis is allowed to continue for about 45 minutes.
9. Interpret the results after the completion.

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9. INTERPRETATION OF RESULTS
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I. Precipitin line between the antigen and antisera wells indicate positive
reaction or specific antigen-antibody reaction due to the presence of
antibody specific to the antigen in the test sera (Indicates specificity).
II. The absence of the precipitin line indicates no reaction or the absence of
any antibody for the antigen in the test sera (Indicates non-specificity).
III. The presence of more than one precipitin line indicates the heterogeneity of
the antibody for the antigen.

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Advantage
• The method is Much faster and more sensitive than double
immunodiffusion. (takes 30 minutes).
Limitations
 It is more expensive than agglutination based tests.
 It is believed to have decreased sensitivity, speed, and simplicity, than latex
agglutination tests.
 Need of large quantity of Ag and Ab.
Advantages and Limitations

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Any
question?