Counter Current Immunoelectrophoresis Principle, Procedure, Result.

Counter Current Immunoelectrophoresis

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.
Counter Current Immunoelectrophoresis is a rapid version of Ouchterlony double diffusion (ODD) technique which can be performed within an hour. It is primarily a qualitative test, although from the thickness of the precipitin line relative measure of quantity can be obtained.

In this method, the antigen is placed in a well at the cathode end and the antibody is placed at the anode side. 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 distance 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 that have no charge move towards the cathode along with the solvent shield due to this phenomenon.
Thus the antigen and antibody travel towards each other and at a point where there is an optimum concentration of both a line of precipitation (band) is formed.
This qualitative technique is much faster and more sensitive than the double diffusion technique.

The technique was used by Lang and Haan for the detection of antibodies in 1957.

Objective

To check antisera for the presence of antibodies towards a specific antigen by Countercurrent immunoelectrophoresis (CCIEP).

Principle of Counter Current Immunoelectrophoresis

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 antibodies 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.

Materials Required

Glass wares: Conical flask, Measuring cylinder, Beaker
Reagents: Sterile distilled water, alcohol

Other requirements: Incubator (37o C), Microwave or Bunsen burner, Vortex mixer, spatula, Micropipettes, Tips, Moist chamber (box with wet cotton).
Agarose
50XTAE

Positive Control (Antiserum
Test Antiserum 1
Test Antiserum 2

Test Antiserum 3
Antigen
Glass plate

Template
Gel puncher

Procedure of Counter Current Immunoelectrophoresis

Prepare 10 ml of 1.5% agarose (as given in important instructions).

Mark the end of the slide that will be towards negative electrode during the electrophoresis.
Cool the solution to 55-60oC and pour 6 ml/plate on to grease free glass plate placed on a horizontal surface. Allow the gel to set for 30 minutes.
Place the glass plate on the template provided.

Punch wells with the help of gel puncher corresponding to the markings on the template. Use gentle suction to avoid forming rugged wells.
Add 10 μl of antigen sample to the wells that will be placed towards the negative electrode and 10 μl of antiserum samples to the wells towards the positive electrode as shown in the figure 2.
Pour 1X TAE into the electrophoresis tank such that it just covers the gel.

Electrophorese at 80-120 volts and 55-60 mA, until precipitin lines are observed.
Place the glass plate in a moist chamber and incubate overnight at 37oC.

Observation and Result

Observe for precipitin lines between the antigen and corresponding antiserum wells.

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.

Applications of Counter Current Immunoelectrophoresis

It is a fast and sensitive technique used for the detection of pneumococcal capsular antigens in sputum.

It is used for the detection of both antigen and antibodies within the serum, cerebrospinal fluid, and other body fluids in the diagnosis of many infectious diseases including bacterial, viral, fungal, and parasitic.
Used for the detection of various antigens such as alpha-fetoprotein in serum and capsular antigens of Cryptococcus and Meningococcus in cerebrospinal fluid.
It is used for Hepatitis B surface antigen (HBsAg), fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in the CSF.

Advantages of Counter Current Immunoelectrophoresis

It is the quickest method for antigen-antibody detection.
It is a faster and more sensitive method as compared to the double diffusion technique.
More sensitive than electro-immunodiffusion (EID) because it involves simultaneous electrophoresis of the antigen and the antibody in gel in opposite directions resulting in band formation.

Limitations of Counter Current Immunoelectrophoresis

It is a costly process as compared to agglutination based tests.
It is considered to have decreased sensitivity, speed, and simplicity, then latex agglutination tests.

Precautions

Before starting the experiment the entire procedure has to be read carefully.

Always wear gloves while performing the experiment.
Preparation of 1X Electrophoresis Buffer: To prepare 300 ml of 1X TAE, add 6 ml of 50X TAE to 294 ml of sterile distilled water.
Preparation of 1.5% Agarose gel: To prepare 10 ml of agarose gel, add 0.15 g of agarose powder to 10 ml of 1X TAE, boil to dissolve the agarose completely.

Wipe the glass plates with cotton; make it grease free using alcohol for even spreading of agarose.
Cut the well and troughs neatly without rugged margins.
Ensure that the moist chamber has enough wet cotton to keep the atmosphere humid.

Interpretation

Samples should be loaded directly into the wells without spilling to the sides otherwise it will cause Inadequate filling of the wells, as a result of it No precipitin lines will be observed.
Ensure that the moist chamber has enough moist cotton to avoid drying of the gel otherwise, it will cause drying of the agarose gel during incubation.
Ensure that the antigen wells are towards the cathode and antibody wells towards the anode during electrophoresis otherwise, the reversal of the antigen and antibody wells may leading to flow of current in wrong direction.

The buffer added in the tank should be just enough to have complete contact with the gel and not to immerse the slide completely, otherwise the excess buffer added in the electrophoresis tank leads to loss of the samples.
Samples should be loaded directly into the wells without spilling to the sides.
Place the glass plate on a flat surface while pouring the gel. Do not disturb the plate once the gel is poured.

References

http://www.ispybio.com/search/protocols/CounterCurrentImmunoelectrophoresis.pdf
https://himedialabs.com/TD/HTI007.pdf
http://www.ispybio.com/search/protocols/ie%20protocol2.pdf

https://microbenotes.com/counter-current-immunoelectrophoresis/
Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India: Elsevier India.
Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical Publishers.