USER MANUAL

Counter Current Immunoelectrophoresis

Teaching Kit

Catalog No. IMI-KIT-1010

Consumable for 5 experiments
Experiment duration: 2 h

Counter Current Immunoelectrophoresis Teaching Kit contains:

Standard reagents
and
Protocol

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Counter Current Immunoelectrophoresis Teaching Manual

User manual of Counter Current Immunoelectrophoresis

I. PRINCIPLE

Electrophoresis is the migration of charged colloidal particles or molecules through a solution under the
influence of an applied electric field usually provided by immersed electrodes. Immunoelectrophoresis
(IES) is the electrophoresis of a determined antigen mixture in an agarose gel that allows the separation
of different antigens along the gel slide, and then the lateral diffusion of an antibody in the gel. Counter
Current Immunoelectrophoresis is a modification of immunoelectrophoresis in which antigen and
antibody move in opposite directions and form precipitates in the area between the cells where they meet
in concentrations of optimal proportions.

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.

II. OVERVIEW

This method is more sensitive and faster than double immunodiffusion (DID) and is particularly useful for
antigens that diffuse slowly in the gel. This is the separation and identification of proteins based on
differences in electrical charge and reactivity with antibodies. It is used for the quantitation of group-
specific antigen and antibodies. It is simple, easy to perform and faster than Agarose gel precipitation. It
is a very rapid process; result can be obtained within 45 minutes. Counter immunoelectrophoresis (CIEP)
is currently used in many laboratories to detect hepatitis- associated antigen and in the past has been
employed to detect components of influenza virus

III. ADVANTAGES

• This kit helps the student to understand the principle and basic function of the technique. This is a
rapid and very sensitive method.
• Contains standard reagents and protocol for better results.

IV. KIT COMPONENTS AND STORAGE

All the reagents should be stored at 2-8°C when not in use.

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Counter Current Immunoelectrophoresis Teaching Manual

Sl No Components Quantity (5 expts)

1 Agarose 1g

2 Antigen 0.4 ml

3 Test anti-serum 0.2 ml

Anti-serum positive
4 0.2 ml
control

5 10X Assay buffer 200 ml

6 Glass slides 2

7 Manual 1

Materials Required:
Glassware: Conical flask, measuring cylinder.
Reagent: Distilled water.
Other Requirements: Tips, micropipette, forceps.

V. PREPARATION OF REAGENTS (for 1 experiment)

Note: The included buffers and reagents are optimized for use with this kit. Substitution with other
reagents may not give optimal results.

Assay Buffer: Prepare 1X of Assay buffer by diluting it with distilled water (Add 30 ml of 10X Assay
buffer to 270 ml of distilled water).The diluted assay buffer should be stored at 4°C before use. We
recommend preparing of fresh buffer as per experimental requirement.

Note: The antigen supplied has negative charge at pH 7.6. It will be loaded into wells near cathode and
antiserum will be loaded in to wells near anode. On electrophoresis, antigen will travel towards anode
and the antibody towards cathode. At the equivalence point, a precipitin line will be formed in those
antiserum samples, which have antibody towards given antigen.

VI. PROTOCOL (FOR 1 EXPERIMENT)

1. Prepare 10 ml of 1.0% Agarose (0.1 g/10 ml) in 1X Assay Buffer by heating slowly till agarose
dissolves completely. Take care not to froth the solution.
Note: Boiling Agarose can splatter and cause severe burns. When heating Agarose,
always wear safety hot gloves.
2. Mark the end of a glass slide as +ve and -ve, so that when placed the glass slide in
electrophoresis apparatus, the +ve mark faced towards anode and the negative mark faced
towards cathode.
3. Place the glass plate or slide on a horizontal surface. Pipette and spread 5 ml of 1.0% Agarose
onto slide, spreading while releasing the agarose. Allow to solidify for 15 minutes. Take care that the
slide is not disturbed and allow the gel to solidify.
4. Cut wells of according to the template indicated in Figure 1 using 200μl micropipette tip. The
distance between the two wells should not be more than 0.5 cm.

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Counter Current Immunoelectrophoresis Teaching Manual

Antigen Test Antiserum

Positive control anti-serum

- +
Cathode Anode
Figure 1: Template for Counter Current Immunoelectrophoresis

5. Remove Agarose gel plugs with a forceps or spatula.

6. Transfer the plate or slide containing the gel to the electrophoresis apparatus.
7. Take required amount of 1X assay buffer (about 200 ml) in electrophoresis chamber. Place the
filter paper wicks at both end of glass slide, so that it’s one end touch to the end of glass slide and
the other end submerged in the buffer. Press lightly on the wicks to ensure good contact between
the gel and the running buffer. If necessary, add more buffers, but do not cover the gel with
buffer.
8. Load 30 µl of antigen in the wells and 30 µl test antiserum as well as positive control antiserum as
indicated by the diagram. (Note: Always load the antigen to the –ve electrode & antiserum to
the +ve electrode.)
9. Carefully, snap on the lid of the electrophoresis apparatus, insert the leads into the power supply
with the black lead in the black (negative) input and the red lead into the red (positive) input.
10. Turn on and set the power supply for the required voltage (40 V). When current is flowing
properly, bubbles should form on the electrodes.
11. After electrophoresis is completed, turn off the power, unplug the power source, disconnect the
leads, and remove the cover.
12. Discard the filter paper wicks and remove the plate or slide from the apparatus.
13. Observe for precipitin line between the antigen and antisera wells.

Note: If precipitin line is not visible then put the gel or slide in a moist chamber and incubate it
for 6 h.

Observation: Precipitin line indicates the presence of antibody while its absence indicates the
absence of antibody.

VII. REFERENCES
1. Hoekstra J and Deinhardt F. Appl Microbiol. 22:1172–1173 (1971).
2. Tosswill JH et al. J Clin Pathol. 33:33-35 (1980)

VIII. TROUBLESHOOTING

Problem Probable Cause Suggestion

No precipitin line Less running time Increase the running time

Incubate the gel in solution B to

No precipitin line Visibility is not clear
see the precipitation.

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