Immunoelectrophoresis Test Principle, Applications, Procedure, Results, Advantages And Disadvantages.

Immunoelectrophoresis Definition

Immunoelectrophoresis is a powerful qualitative technique for the characterization of an antibody.

The term “immunoelectrophoresis” was first coined by Grabar and Williams in 1953.
Immunoelectrophoresis is a process of a combination of immuno-diffusion and electrophoresis.

In this method one antigen mixture is electrophoresed in an agarose gel that allows the separation of its different components based on their charge along the gel slide, followed by the lateral diffusion of the serum or monoclonal antibody within the gel.
Antibodies specific to the antigens form white precipitation arcs which can be seen against a dark background.

Objective

To learn the technique of Immunoelectrophoresis.

Principle of Immunoelectrophoresis

In immunoelectrophoresis, the antigen mixture is first electrophoresed to separate its constituents by charge. The antiserum containing the antibodies added into the troughs diffuses with a plane front to react with the antigens. Due to diffusion, density gradients of the antigen and antibody are obtained and at a specific antigen/antibody ratio (equivalence point) huge macromolecules are formed.

They form a visible white complex that precipitates as arcs in the gel. The arc is closer to the trough at the point where the antigen is in the highest concentration. The method is very specific and highly sensitive because distinct zones are formed. In this method, it is important that the ratio between the quantities of antigen and antibody be proper (antibody titer).

Material Required

Glass wares: Conical flask, Measuring cylinder, Beaker

Reagents: Distilled water, alcohol
Other requirements: Incubator (37oC), Microwave or Bunsen burner, Electrophoresis unit, Vortex mixer, spatula, Micropipettes, Tips, Gel cutter, Moist chamber (box with wet cotton)

Procedure of Immunoelectrophoresis

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

Mark the side of the glass plate that will be towards negative electrode during 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 a well with the help of the gel puncher corresponding to the markings on the template at the negative end. Use gentle suction to avoid forming rugged wells.
Cut two troughs with the help of the gel cutter, but do not remove the gel from the troughs.
Add 10 l of the antigen to the well and place the glass plate in the electrophoresis tank such that the antigen well is at the cathode/negative electrode.

Pour 1X Electrophoresis buffer into the electrophoresis tank such that it just covers the gel.
Electrophorese at 80-120 volts and 60-70 mA, until the blue dye travels 3-4 cms from the well. Do not electrophorese beyond 3 hours, as it is likely to generate heat.
After electrophoresis, remove the gel from both the troughs and keep the plate at room temperature for 15min. Add 80 l of antiserum A in one of the trough and antiserum B in the other.

Place the glass plate in a moist chamber and incubate overnight at 37oC.

Observation and Result

Observe for precipitin lines between antiserum troughs and the antigen well.

Note : For better precipitin lines incubate for longer period at 37oC

Advantages of Immunoelectrophoresis

It is an important analytical procedure with high resolving power as it connects the departure of antigens by electrophoresis with immunodiffusion against an antiserum.
The main benefit of immunoelectrophoresis is that a number of antigens can be recognized in serum.

Disadvantages of Immunoelectrophoresis

It is a slower, less sensitive process, and more challenging to perform than Immunofixation electrophoresis.

It is unable to detect some minute monoclonal M-proteins because the most rapidly emigrating immunoglobulins present in the highest concentrations may obscure the presence of small M-proteins.
In food, analysis the use of immunoelectrophoresis is limited by the availability of specific antibodies.

Application of Immunoelectrophoresis

This technique is useful in determining the blood levels of three major immunoglobulins: IgM, IgG and IgA. The process combines the antigen separation technique of electrophoresis and immunodiffusion of the separated antigen against an antibody.

It is used extensively to check the presence, specificity and homogeneity of the antibodies and can detect relatively high antibody concentrations.
In the clinical laboratory, immunoelectrophoresis is used diagnostically.
It is utilized in examining certain serum abnormalities, especially those involving immunoglobulins, urine protein, cerebrospinal fluid, pleural fluids and other body fluids.

In research, this procedure may be used to monitor antigen and/or antibody purifications, to detect impurities, analyze soluble antigens from plant and animal tissues, and microbial extracts.

Interpretation

The formation of the precipitin line indicates the presence of antibodies specific to the antigen.

Homogeneity of the antiserum to the antigen is denoted by the presence of a single continuous precipitin line

Heterogeneity of the antiserum to the antigen is denoted by presence of more than one precipitin line which not only gives an indication of the number of immunodominant epitopes, but also the non identical nature of such epitopes.

Samples should be loaded directly into the well and troughs without spilling to the sides otherwise it will lead to the Inadequate filling of the well and troughs as a result No precipitin lines will be observed.
Ensure that the moist chamber has enough moist cotton to avoid drying of the gel otherwise, it will lead to the drying of the agarose gel during incubation.

Ensure that the antigen travels at least 3/4th of the gel otherwise, it will lead to insufficient electrophoresis of the antigen.
Samples should be loaded directly into the well and troughs without spilling to the sides otherwise, the Samples will not be loaded properly into the well and troughs.
Place the glass plate on a flat surface while pouring the gel. Do not disturb the plate once the gel is poured otherwise, it will lead to the uneven pouring of gel.

Precautions of Immunoelectrophoresis

Before starting the experiment the entire procedure has to be read carefully.
Always wear gloves while performing the experiment.
Preparation of 1X TAE: 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 Electrophoresis Buffer, 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.

Add the antiserum to agarose only after it cools to 55oC as a higher temperature inactivates the antibody.
Ensure that the moist chamber has enough wet cotton to keep the atmosphere humid.

References

https://microbenotes.com/immunoelectrophoresis-principle-procedure-results-and-applications-advantages-and-limitations/

https://en.wikipedia.org/wiki/Immunoelectrophoresis
https://himedialabs.com/TD/HTI005.pdf
http://lnmuacin.in/studentnotice/2020/Immunoelectrophoresis.pdf

http://www.ndvsu.org/images/StudyMaterials/Micro/Immunoelectrophoresis-test.pdf