The countercurrent 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. Countercurrent immunoelectrophoresis 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.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.
This qualitative technique is much faster and more sensitive than the double diffusion technique.This method is mostly used for the rapid check of any antisera for the presence and specificity of antibodies for a particular antigen.
Principle of countercurrent immunoelectrophoresis
In the countercurrent immunoelectrophoresis method, immunoprecipitation occurs when antigen at the level of 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 that is the positive pole because of endo osmotic flow.
So 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 form precipitates as a visible white line.
Counter Current immunoelectrophoresis kits content
|Agarose||1.8 g||Room Temperature|
|50X Electrophoresis Buffer||72 ml||Room Temperature|
|Positive Control (Antiserum)||0.1 m||2-8 °C|
|Test Antiserum 1||0.1 ml||2-8 °C|
|Test Antiserum 2||0.1 ml||2-8 °C|
|Test Antiserum 3||0.1 ml||2-8 °C|
|Glass plate||————||Room Temperature|
|Gel puncher||————||Room Temperature|
Materials required for Counter Current immunoelectrophoresis
- Conical flask
- Measuring cylinder
- Sterile distilled water
- Incubator (37 °C)
- Microwave or Bunsen burner
- Vortex mixer
- Moist chamber (box with wet cotton)
Note that before starting this experiment always read the entire procedure provided by the manufacturer carefully
- 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.
How to prepare 1X Electrophoresis Buffer
In order to prepare 300 ml of 1X Electrophoresis Buffer,
add 6 ml of 50X Electrophoresis Buffer to 294 ml of sterile distilled water.
How to preparation of 1.5% Agarose gel
In order to prepare 10 ml of agarose gel
- Add 0.15 g of agarose powder to 10 ml of 1X Electrophoresis Buffer
- boil to dissolve the agarose completely
Counter Current immunoelectrophoresis procedure
- Start by Preparing 10 ml of 1.5% agarose
- Mark the end of the slide that will be towards negative electrode during the electrophoresis.
- Cool the solution to 55-60 °C and pour 6 ml/plate on to grease free glass plate placed on a horizontal surface.
- Now 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
- Now pour 1X Electrophoresis buffer 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 37 °C
Observation and Result
Carefully observe for precipitin lines between the antigen and corresponding antiserum wells on the template
Interpretation of Test Result
The presence of 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.
Possible Limitation of Test Method
Source : Himedia