Immuno-electrophoretic Assays
Immunoelectrophoretic techniques separate and identify proteins based on their antigenic properties and electrophoretic mobility. It combines two different techniques, electrophoresis and immunodiffusion, to provide increased sensitivity, improved specificity, and the ability to detect multiple antigens in a single analysis.
Some of the commonly used immunoelectrophoretic assays are discussed below:
A. Immunoelectrophoresis (IEP)
Immunoelectrophoresis (IEP) is a serological technique that involves two steps to analyze the presence of multiple antigens in a biological sample. The antigens present in the sample are first separated based on charge and size using electrophoresis. The separated antigens are then subjected to immunodiffusion to identify the specific antigens.
The step-by-step procedure for performing immunoelectrophoresis (IEP) is as follows:
Prepare the agarose gel:
Usually, a 1-2% agarose gel is used for immunoelectrophoresis. However, the percentage of agarose gel may vary depending on the type and size of the antigens being analyzed. To prepare a 1% agarose gel, mix 1 gram of agarose with 100 ml of TAE buffer and heat the mixture until the agarose is completely dissolved. Finally, pour the mixture on a casting tray and allow the gel to solidify.
Cut the gel:
Cut a well in the center of the agarose gel for addition of the sample. Additionally, cut a trough parallel to the direction of electrophoresis for the addition of the antibody and place it in an electrophoresis tank filled with buffer solution.
Load the sample:
Add 5ul of sample in the central well connect the electrodes to the tank and run the gel at a constant voltage for a predetermined amount of time (usually 20-30 minutes at 100-150V). As the sample migrates through the gel under the influence of an electric field, the antigens present in the sample as separated based on their charge and size.
Add antibody:
After electrophoresis, remove the gel from the electrophoresis tank and load the antibody specific to the antigen of interest into the trough. Incubate the gel overnight to ensure adequate time for the antigens and antibodies to diffuse and form precipitin lines.
Result Interpretation:
The formation of a visible precipitation arc indicates the presence of the specific antigen in the sample. The position, shape, and number of the arcs can provide information about the types and quantities of antigens present. The interpretation of results may require comparison to standards or controls to confirm the identity of the antigen.
The exact procedures for IEP may vary depending on the specific needs of the experiment or investigation. For example, the concentration of agarose gel, running buffer, and the voltage used in the electrophoresis step may need to be adjusted for different samples. The amount and type of antibodies used may also need to be optimized depending on the antigens present in the sample.
B. Crossed Immunoelectrophoresis (CIE)
Crossed immunoelectrophoresis (CIE), also known as two-dimensional IEP, is a modification of IEP. The term ‘two-dimensional’ refers to the fact that the technique involves two rounds of electrophoresis in perpendicular directions to separate and identify proteins based on their antigen-antibody interactions.
The step-by-step procedure for performing Crossed Immunoelectrophoresis (CIE) is as follows:
First Dimension Electrophoresis:
For the first round of electrophoresis, prepare a 1-2% agarose gel and load the sample into the well that is cut into the center of the gel. Run the gel at a constant voltage for a predetermined amount of time, typically 20-30 minutes at 100-150V. During this process, the antigens present in the sample are separated based on their size and charge, as they migrate towards the anode or cathode depending on their net charge. The speed at which the antigens migrate depends on their size, with smaller antigens migrating more quickly through the gel than larger antigens.
Cut the Gel Strip:
Carefully excise the gel strip containing separated antigens from the gel using a scalpel.
Second Dimension Electrophoresis:
Prepare a 1-2% agarose gel in a suitable buffer containing the antibody solution. Cut a trough parallel to the future cathode end of the prepared agarose gel and place the gel strip containing separated antigens in the agarose gel containing uniformly distributed antibodies. Place the gel in an electrophoresis tank filled with buffer solution and run at a constant voltage for a predetermined amount of time. During this process, the antigens migrate from the gel strip into the agarose gel matrix
Detection:
After the second dimension electrophoresis is complete, the gel is stained with a suitable dye to visualize the proteins. The protein spots will appear in the gel as an array of crossed arcs representing each protein's position in the two dimensions.
Result Interpretation:
The formation of a visible precipitation arc indicates the presence of the specific antigen in the sample. The position, shape, and number of the arcs can provide information about the types and quantities of antigens present. The position of the antigens on the gel can be determined by comparing the result to a standard chart.
C. Crossed-over Immunoelectrophoresis (COE)
Crossed-over Immunoelectrophoresis (COE), also known as counter-immunoelectrophoresis, is a technique designed to detect and analyze antigens based on their interaction with specific antibodies. This method involves the creation of two arrays of opposing wells in an agarose gel, facilitating the migration of antigens and antibodies towards each other.
The step-by-step procedure for performing Crossed-over Immunoelectrophoresis (COE) is as follows:
Prepare the Agarose Gel:
Prepare a 1-2% agarose gel and create opposing wells by punching holes in the agarose gel. Place the gel in an electrophoresis tank filled with buffer solution.
Load Antibodies and Samples:
Load the specific antibodies into the wells on one side of the gel, ensuring that they are proximal to the anode. Load the biological samples containing antigens into the opposing wells, placing them proximal to the cathode.
Electrophoresis:
Run the gel at a constant voltage for a predetermined time, typically around 20-30 minutes at 100-150 V. As electrophoresis progresses, the antigens which are usually negatively charged migrate towards the anode, while the antibodies move in the opposite direction.
Precipitate Formation:
A visible precipitate line forms between the opposing wells when the antigen reacts with its specific antibody. This precipitation line indicates the presence of the specific antigen in the sample.
Result Interpretation:
Analyze the gel for the formation of precipitation arcs. The position, shape, and number of arcs provide information about the types and quantities of antigens present. Comparing the results to a standard chart can help determine the identity and characteristics of the detected antigens.
D. Rocket Immunoelectrophoresis (RIE)
Rocket immunoelectrophoresis (RIE), also known as electroimmunoassay, is a one-dimensional, quantitative method that primarily refers to the migration of antigen-antibody complexes in a single direction within the agarose gel under the influence of an electric field. RIE derives its name from the characteristic cone-shaped precipitate line formed during the assay, which resembles a rocket. This technique is a variation of traditional IEP, combining immunoprecipitation and electrophoretic separation for the analysis of antigens.
The step-by-step procedure for performing Rocket Immunoelectrophoresis (RIE) is as follows:
Prepare Agarose Gel:
Mix 1 gram of agarose with 100 ml of TAE buffer. Heat the mixture until agarose is completely dissolved. Allow the agarose solution to cool slightly, but ensure it remains in a liquid state. Add the specific antisera to the agarose solution. The antisera should be specific to the target antigen you want to analyze. Pour the mixture onto a casting tray, insert an electrophoresis comb to create wells, and allow the gel to solidify. Place the gel in an electrophoresis tank filled with buffer solution.
Load Samples:
Add 5 µl of the test sample to each well. Connect the electrodes to the electrophoresis tank, making sure that the samples are at the cathode end.
Electrophoresis Process:
Run the gel at a constant voltage for a predetermined time (usually 20-30 minutes at 100-150V). As samples migrate from the negative to the positive electrode, antigens in the sample are separated based on their charge and size within the agarose gel.
Incubation:
Keep the gel in a moist chamber or on a petri dish containing moist cotton for overnight incubation.
Result Interpretation:
In a positive reaction, a rocket-shaped precipitate line becomes visible. The height of the rocket is proportional to the amount of antigen present in the sample which can be quantified by comparing standards and the sample on the same gel.