Counter Current Immunoelectrophoresis – Principle, Procedure, Result.

What is Counter Current Immunoelectrophoresis?

• Counter Current Immunoelectrophoresis is a modified version of immunoelectrophoresis, a technique used to separate and test antigen mixtures. In this method, the antigen and antibody migrate in opposite directions and form a visible white precipitate in the area where they meet.
• The process begins by separating the antigen mixture into its component parts using electrophoresis. The antigen mixture is electrophoresed in an agarose gel, allowing the different antigens to separate along the gel slide. As the electrophoresis takes place, an antibody is introduced into the gel, resulting in lateral diffusion and the formation of a precipitate.
• Counter Current Immunoelectrophoresis is sometimes referred to as countercurrent or crossed-over immunoelectrophoresis. It shares similarities with the Ouchterlony method, a double diffusion technique, but with the key difference that the antigen movement is facilitated by electrophoresis. Therefore, it is also known as “voltage facilitated double immunodiffusion.”
• One of the advantages of Counter Current Immunoelectrophoresis is its speed. It is a rapid technique that can be performed within an hour. While primarily a qualitative test, it is possible to obtain a relative measure of the quantity of antigens present based on the thickness of the precipitin line.
• In this method, the antigen is placed in a well at the cathode end, while the antibody is placed at the anode side. As the electric field is applied during electrophoresis, the molecules acquire a charge based on their isoelectric point (pI). The antigen, if negatively charged, moves towards the anode, while the antibody, with a charge nearing zero at pH 7.6, moves towards the cathode.
• During electrophoresis, the agarose gel matrix absorbs OH- ions on its surface, resulting in a net increase in positive ions at a distance from the matrix. These positive ions migrate towards the negative pole along with a solvent shield, causing a net solvent flow known as endosmosis. Consequently, the antibody molecules, which have no charge, move towards the cathode along with the solvent shield due to this phenomenon.
• As a result, the antigen and antibody migrate towards each other. At a point where there is an optimal concentration of both, a line of precipitation, also known as a band, is formed.
• Counter Current Immunoelectrophoresis is a qualitative technique that is faster and more sensitive than the double diffusion technique. It was first used by Lang and Haan in 1957 to detect antibodies.

Objective of Counter Current Immunoelectrophoresis

The objective of Counter Current Immunoelectrophoresis (CIE) primarily revolves around two main goals:

1. Detection and Specificity of Antibodies: One of the key objectives of CIE is to rapidly check antisera for the presence and specificity of antibodies against a particular antigen. By introducing the antigen of interest into the gel and applying electrophoresis, CIE allows for the visualization of precipitin lines or bands that indicate the presence of antibodies. This technique enables the quick screening and determination of the antibody profile in antisera, providing valuable information about its reactivity and specificity towards specific antigens.
2. Diagnosis of Diseases: Another important objective of CIE is to detect antigens and/or antibodies in serum for the diagnosis of particular diseases. CIE can be utilized to identify specific antigens or antibodies associated with various diseases. By introducing the patient’s serum sample into the gel and allowing it to react with appropriate antibodies or antigens, CIE enables the formation of precipitin lines or bands indicative of the presence of specific disease markers. This facilitates the diagnosis and monitoring of diseases such as infectious diseases, autoimmune disorders, and certain types of cancers.

Principle of Counter Current Immunoelectrophoresis

The principle of Counter Current Immunoelectrophoresis (CIE) is based on the movement of antigens and antibodies in opposite directions through an agarose gel under the influence of an electric field.

The test is typically performed on a glass slide containing agarose gel with a high electro-endosmotic flow. Two wells are created on the gel, one filled with the antigen and the other with the antibody. When an electric current is applied to the gel, the migration of antigen and antibody is greatly facilitated by the electric field.

The antigen, if negatively charged, moves towards the anode (positive electrode), while the antibody, which typically has a charge nearing zero at pH 7.6, moves towards the cathode (negative electrode). As the antigen and antibody migrate through the gel, they approach each other due to their opposite movement.

At a point where the concentrations of antigen and antibody are optimal for reaction, a visible line of precipitation, known as precipitin arcs, is formed. This line serves as an indicator of a positive reaction between the antigen and antibody.

The entire process of Counter Current Immunoelectrophoresis typically takes about 30 to 60 minutes for the precipitin arcs to become visible, allowing for a relatively rapid detection of the antigen-antibody interaction.

In summary, the principle of Counter Current Immunoelectrophoresis relies on the application of an electric field to facilitate the migration of antigen and antibody towards opposite electrodes, leading to the formation of precipitin arcs at the point of optimal concentration. This principle enables the detection of specific antigen-antibody reactions in a relatively short period of time.

Materials Required

To perform Counter Current Immunoelectrophoresis, several materials are required. These include:

1. Agarose: Agarose is used to prepare the gel matrix in which the antigen and antibody will migrate during the test.
2. Antigen: The specific antigen of interest, which will be placed in one of the wells on the gel.
3. Test Antiserum: The serum sample containing antibodies that will react with the antigen. Multiple test antisera may be used for different samples or antigens.
4. Positive Control (Antiserum): A known positive antiserum that contains antibodies against the antigen of interest. It serves as a control to verify the test’s accuracy.
5. Assay Buffer: A buffer solution used to maintain the appropriate pH and ionic strength during the test, ensuring optimal conditions for antigen-antibody interaction.
6. Electrophoresis Apparatus: An apparatus designed to apply an electric current to the agarose gel, facilitating the migration of antigen and antibody.
7. Glass Slides: Glass slides are used to hold the agarose gel during the immunoelectrophoresis procedure.
8. Glasswares: Various glassware items such as conical flasks, measuring cylinders, beakers, and glass plates may be required for preparing reagents and handling samples.
9. Reagents: Sterile distilled water and alcohol may be needed for preparing buffers, diluting samples, or cleaning equipment.
10. Incubator: An incubator set at 37°C is required to promote the reaction between the antigen and antibody.
11. Microwave or Bunsen Burner: These heat sources are used to melt the agarose and prepare the gel.
12. Vortex Mixer: A vortex mixer is used to mix reagents and samples thoroughly.
13. Spatula: A spatula is used for measuring and transferring small amounts of solid reagents.
14. Micropipettes and Tips: Micropipettes are used for precise and accurate dispensing of small volumes of liquids. Disposable tips are used to prevent cross-contamination.
15. Moist Chamber: A container, such as a box with wet cotton, is used to create a humid environment for the gel to prevent drying out during the procedure.
16. Gel Puncher: A gel puncher is used to create wells on the agarose gel for loading samples and controls.

These materials are essential for conducting Counter Current Immunoelectrophoresis and are required to ensure the accuracy and reliability of the test results.

Procedure of Counter Current Immunoelectrophoresis

The procedure for performing Counter Current Immunoelectrophoresis (CIE) involves several steps. Here is a step-by-step outline of the procedure:

1. Prepare the Agarose Gel: Dissolve 0.1 g of agarose in 10 ml of 1X Assay Buffer by heating slowly until the agarose completely dissolves. This will result in a 1.0% agarose solution.
2. Mark the Glass Slide: Mark the ends of a glass slide as “+ve” and “-ve” to indicate the orientation. The “+ve” mark should face towards the anode (positive electrode), while the “-ve” mark should face towards the cathode (negative electrode).
3. Apply Agarose to the Glass Slide: Pipette 5 ml of the prepared agarose solution onto the glass slide. Spread it evenly across the slide and allow it to solidify for approximately 15 minutes.
4. Create Wells: Use a gel puncher and a template to cut wells in the solidified agarose gel. Ensure that the distance between the two wells is no more than 0.5 cm.
5. Set up the Electrophoresis Tank: Place the glass slide with the agarose gel into the electrophoresis tank. Fill the tank with 1X electrophoresis buffer until the buffer just covers the surface of the gel.
6. Add Antigen and Antisera: Pipette 10 µl of the antigen into each of the two wells located towards the cathode (negative electrode). Then, pipette 10 µl of the positive control antiserum and the test antisera into the wells located towards the anode (positive electrode).
7. Connect the Power Supply: Connect the power cord to the electrophoretic power supply, following the proper convention for the positive and negative electrodes.
8. Apply Voltage and Run Electrophoresis: Apply a voltage of 50 V and allow the electrophoresis to run for approximately 45 minutes.
9. Interpret the Results: After the electrophoresis is complete, observe the gel for the formation of precipitin arcs or lines. These indicate positive reactions between the antigens and the antibodies. The results can be interpreted based on the presence or absence of precipitin lines and their characteristics.

Following these steps will allow for the execution of Counter Current Immunoelectrophoresis and the subsequent interpretation of the results.

Observation and Result

During Counter Current Immunoelectrophoresis (CIE), the observation and interpretation of results are crucial in determining the presence of a specific antigen-antibody reaction. Here are some key observations and corresponding results:

1. Precipitin Line Formation: The presence of a visible precipitin line between the wells containing the antigen and antisera indicates a positive reaction or a specific antigen-antibody interaction. The formation of this line is a result of the antibodies present in the antisera binding with the corresponding antigen.
2. Absence of Precipitin Line: If no precipitin line is observed, it indicates either no reaction or the absence of the corresponding antibody for the antigen being tested. This suggests that the antisera does not contain antibodies that recognize the specific antigen under investigation.
3. Multiple Precipitin Lines: The presence of more than one precipitin line suggests heterogeneity of the antibodies present in the antisera. This indicates that the antisera contains antibodies that recognize different epitopes or variants of the antigen, resulting in the formation of multiple lines. This observation highlights the diversity of the antibody response to the antigen.

By carefully observing the gel after the completion of electrophoresis, one can determine the presence or absence of precipitin lines and interpret the results accordingly. Positive reactions are indicated by the formation of a precipitin line, while negative reactions are indicated by the absence of such a line. The presence of multiple lines signifies the heterogeneity of the antibody-antigen interaction.

It is important to note that the interpretation of results in CIE should consider appropriate positive and negative controls to ensure the accuracy and reliability of the test. Control samples with known positive and negative reactions can be included to validate the results obtained from the test samples.

Overall, the observation and interpretation of precipitin lines provide valuable information about the presence, specificity, and heterogeneity of antigen-antibody reactions, contributing to the understanding and diagnosis of various diseases and immune responses.

Applications of Counter Current Immunoelectrophoresis

Counter Current Immunoelectrophoresis (CIE) has several applications due to its rapidity and high specificity in detecting antigens and antibodies. Here are some key applications of CIE:

• Diagnosis of Infectious Diseases: CIE is widely used in the diagnosis of various infectious diseases caused by bacteria, viruses, fungi, and parasites. It can detect antigens and antibodies in serum, cerebrospinal fluid, and other body fluids. This makes it a valuable tool for diagnosing infections such as bacterial meningitis, viral hepatitis, fungal infections, and parasitic diseases.
• Detection of Specific Antigens: CIE has been historically used to detect specific antigens in various body fluids. For example, it was popular in the past for detecting alpha-fetoprotein in serum and capsular antigens of Cryptococcus and Meningococcus in cerebrospinal fluid. Even today, it remains commonly used for detecting antigens such as Hepatitis B surface antigen (HBsAg), fetoprotein, hydatid and amoebic antigens in the serum, and cryptococcal antigen in cerebrospinal fluid.
• Rapid Detection of Pneumococcal Capsular Antigens: CIE is a sensitive method for detecting pneumococcal capsular antigens in sputum. This allows for the rapid diagnosis of pneumococcal infections, particularly pneumonia, helping with appropriate treatment decisions.

The advantages of CIE, including its speed and specificity, make it a valuable technique in the diagnosis of infectious diseases. Its ability to detect both antigens and antibodies in various body fluids contributes to its wide range of applications. CIE continues to be used in clinical laboratories to aid in the identification and management of infectious diseases.

Advantages of Counter Current Immunoelectrophoresis

Counter Current Immunoelectrophoresis (CIE) offers several advantages compared to other methods of antigen-antibody detection. Here are some key advantages of CIE:

• Rapid Detection: CIE is a fast method for antigen-antibody detection. The entire process, including electrophoresis and result interpretation, can be completed within 30 minutes. This makes it a time-efficient technique, allowing for quick diagnosis or screening of samples.
• Increased Sensitivity: CIE is more sensitive than electro-immunodiffusion (EID) due to the simultaneous electrophoresis of the antigen and the antibody in opposite directions. This setup facilitates the formation of distinct bands at the optimal concentration of antigen and antibody. The visual identification of these bands enhances the sensitivity of the test, enabling the detection of low concentrations of antigens or antibodies.
• Faster and More Sensitive Than Double Diffusion: Compared to the traditional double diffusion technique, CIE is much faster and more sensitive. In double diffusion, the antigen and antibody diffuse passively through the gel, whereas in CIE, electrophoresis facilitates the movement of antigen and antibody towards each other. This voltage-facilitated movement speeds up the reaction and improves sensitivity, making CIE a preferred choice in many applications.

By combining speed, sensitivity, and ease of interpretation, CIE offers distinct advantages for antigen-antibody detection. Its ability to provide rapid results, increased sensitivity, and improved efficiency compared to other techniques makes it a valuable tool in various diagnostic and research settings.

Limitations of Counter Current Immunoelectrophoresis

Counter Current Immunoelectrophoresis (CIE) has certain limitations that should be taken into consideration. Here are some key limitations of CIE:

• Cost: CIE is generally more expensive compared to agglutination-based tests. The equipment, reagents, and consumables required for CIE can be costlier, making it less accessible in certain settings or resource-limited laboratories.
• Decreased Sensitivity: While CIE is a sensitive technique, it may have decreased sensitivity compared to more advanced methods such as latex agglutination tests. These newer techniques often utilize highly specific and sensitive detection systems, allowing for enhanced sensitivity in detecting antigens or antibodies.
• Speed and Simplicity: In terms of speed and simplicity, CIE may not be as advantageous as latex agglutination tests. Latex agglutination tests offer rapid results within minutes and are generally easier to perform, requiring less technical expertise. On the other hand, CIE involves multiple steps, including electrophoresis and result interpretation, which can take around 30 minutes or more.

It’s important to note that while CIE may have these limitations, it still remains a valuable technique in certain applications. Factors such as cost-effectiveness, availability of resources, and specific diagnostic requirements should be considered when choosing the appropriate method for antigen-antibody detection. Newer advancements in diagnostic technologies continue to offer alternatives that address some of the limitations associated with CIE.

Precautions

When conducting Counter Current Immunoelectrophoresis (CIE), it is important to follow certain precautions to ensure accurate and reliable results. Here are some key precautions to consider:

1. Familiarize Yourself with the Procedure: Before starting the experiment, carefully read and understand the entire procedure. Ensure that you have a clear understanding of the steps involved and the necessary precautions to be taken.
2. Wear Gloves: Always wear gloves while performing the CIE experiment to maintain sterility and prevent contamination of samples or reagents.
3. Prepare Electrophoresis Buffer: Follow the instructions provided to prepare the 1X electrophoresis buffer. Take caution in accurately measuring and mixing the appropriate amounts of 50X TAE and sterile distilled water.
4. Prepare Agarose Gel: Prepare the 1.5% agarose gel by dissolving the agarose powder in the 1X TAE buffer. Ensure that the agarose is completely dissolved by boiling, and take care to prevent any spills or splashes.
5. Clean Glass Plates: Prior to pouring the agarose gel, wipe the glass plates with cotton to remove any dust or debris. Use alcohol to make the glass plates grease-free, as this helps in the even spreading of the agarose.
6. Neatly Cut Wells and Troughs: When cutting the wells and troughs in the agarose gel, ensure that they are neat and have smooth margins. Rugged or uneven edges can affect the migration of antigen and antibody, potentially compromising the results.
7. Maintain Moist Chamber: It is essential to maintain a moist chamber throughout the experiment. Ensure that the moist chamber contains enough wet cotton to keep the atmosphere humid. This helps to prevent drying of the gel and maintains optimal conditions for the antigen-antibody reaction.

By adhering to these precautions, you can minimize the risk of errors and ensure accurate and reliable results in your Counter Current Immunoelectrophoresis experiments.

Key Points

Here are the key points to remember when performing Counter Current Immunoelectrophoresis (CIE):

1. Careful Sample Loading: When loading samples into the wells, ensure that they are directly placed into the wells without spilling onto the sides. Inadequate filling of the wells may result in the absence of precipitin lines, affecting the observation of positive reactions.
2. Maintain Moist Chamber: It is crucial to have enough moist cotton in the moist chamber to prevent drying of the agarose gel during incubation. Drying of the gel can lead to inaccurate results or incomplete reactions.
3. Correct Orientation of Antigen and Antibody Wells: Ensure that the antigen wells are placed towards the cathode and the antibody wells towards the anode during electrophoresis. Incorrect orientation can result in the flow of current in the wrong direction, compromising the migration of antigen and antibody.
4. Appropriate Buffer Level: The buffer added to the electrophoresis tank should be sufficient to make complete contact with the gel but should not fully immerse the slide. Excessive buffer can cause the loss of samples and affect the outcome of the experiment.
5. Neat Sample Loading: It is important to load samples directly into the wells without spilling onto the surrounding areas. This ensures accurate placement and prevents contamination or mixing of samples.
6. Maintain Stability of the Gel: Once the agarose gel is poured onto the glass plate, avoid disturbing it. Place the glass plate on a flat surface and handle it carefully to maintain the stability and integrity of the gel.

By paying attention to these key points, you can improve the accuracy and reliability of your Counter Current Immunoelectrophoresis experiments and obtain meaningful results.

FAQ

References

1. http://www.ispybio.com/search/protocols/CounterCurrentImmunoelectrophoresis.pdf
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4. https://microbenotes.com/counter-current-immunoelectrophoresis/
5. Parija S.C. (2012). Textbook of Microbiology & Immunology.(2 ed.). India: Elsevier India.
6. Sastry A.S. & Bhat S.K. (2016). Essentials of Medical Microbiology. New Delhi : Jaypee Brothers Medical Publishers.