The previous two posts were about immunoprecipition. The first post elaborated more on the immunoprecipitation in solution/ liquid, while the second post was more on the immunoprecipitation reactions in gel, especially immunodiffusion. We recommend you read the two posts before this one. In this post, we discuss the process of immunoelectrophoresis, which is a type of immunoprecipitation reaction carried out in the gel.

Immunoelectrophoresis:

As the name suggests this technique is the combination of immunoprecipitation reaction and electrophoresis. This method combines the specificity of immunochemistry and ability to separate the components of a complex antigen mix of the electrophoresis. Let us discuss the different types:

1. Classical immunoelectrophoresis:

Immunoelectrophoresis was first coined by Grabar and Williams in 1953. They separated serum proteins in an agar gel, and let them diffuse in the presence antibodies, to form precipitation arcs. It was found out by this experiment that the globulin fractions contain other important proteins like α1-antitrypsin, α2-macroglobulin, transferrin, C3, immunoglobulin G (IgG), IgA and IgM in addition to the known (at that time) α-, β- and γ-globulins. Hence this experiment redefined the field of protein identification and immunology.
This technique has specificity of the immunoprecipitation and electrophoresis aids the separation of the proteins in a complex mixture.
In this, a microscopic slide is layered with plain agar. The wells are dug into the agar for addition of antigen mix , preferably in the centre of the slide, and a trough is cut, parallel to the direction of the antigen run, for the addition of the antibody (as shown in the fig 1A). The slide is placed into the electrophoretic apparatus, and connected using wet filter paper wicks to the electrode wells and a direct electric current is passed for around 1-2 hours.

First the antigen mixture is added to the well and the electrophoresis is carried out. Under the electric field the charged antigens get separated based on their size and charge (fig 1B).

Fig 1: Different steps of separation of antigens and their detection with help of classical immunoelectrophoresis.

Following the separation of the antigens, the antisera is added to a trough cut in the agar and the slide is incubated overnight at room temperature in humid chamber. During the incubation, the antigen and the antibody diffuse into the gel (fig 1C) and the immunoprecipitation is formed wherever the equivalent concentration is reached.

The immunoprecipitation is observed in the form of an arc due to the radially diffusing antigen and the laterally diffusing antibody from the trough (fig 1D). The pattern of the immunoprecipitation arc formed depends on the antigen concentration.

Advantages:
The technique can be used to analyse simple to complex mixture of antigens.
The complex mixture of antigens are separated and the presence of the individual component of the given complex mixture can be analyzed in a single experiment.

2. Counter Current Immunoelectrophoresis:

This technique is also known as cross over immunoelectrophoresis. The gamma globulin (IgG) is positively charged (cation) at the high pH of around 8.0. However, at such alkaline condition, most of the proteins possess net negative charge (anion, in fig 2).

Fig 2: The charges on the proteins in different pH conditions.

Therefore, at alkaline conditions, the gamma globulin moves towards cathode (-ve electrode) owing to their net positive charge while most proteins move towards the anode (+ve electrode) under the electric field.
In cross over immunoelectrophoresis, this difference in the charges of the IgG and the antigens, is taken advantage of. The IgG are loaded towards anode (+ve electrode) and antigen towards cathode (-ve electrode) and the electric field is applied. As the current is passed, the IgG and the antigen mixture move towards each other and the immunoprecipitation occurs at the zone of equivalence (fig 3).

Fig 3: Counter current electrophoresis.

Advantage:
As the antigen and antibody move towards each other under the electric field, this technique is much faster (15-20 mins) than the normal immunodiffusion techniques, where the diffusion is solely based on the concentration.

Applications:

Counter-current immunoelectrophoresis has been used for the detection of microbial antigens in body fluid including blood, sputum, tissue and cell extracts, urine, etc.

(Just for info: Read this old but simple paper on detection of malarial antibodies by counter current immunoelectrophoresis.

3. Laurell’s rocket electrophoresis:

This is a quantitative one–dimensional immunoelectrophoresis, as this technique can be used to quantify the antigen concentration. It is also known as electroimmunoassay. This method was designed for the first time by Laurell in 1966, to compare sample of unknown concentration with a series of dilutions of a known concentration of the protein.

Fig 4: Rocket immunoelectrophoresis.

In this the antibody is added and fixed in the agar. The wells are cut into the agar, to which antigens are added. The slide is kept in the electrophoretic apparatus and the electric field is applied.

The antigens move towards the anode and the immunoprecipitation is formed in zone of equivalence. Due to movement of the antigens, the immunoprecipitation is formed in a shape of a rocket. Most of the antibody-antigen precipitate is concentrated at the tip of the rocket, but the fine precipitation lines form the sides of the rockets by a small amount of antigen diffusing sideways. This small amount of antigen react equally small equivalent amount of antibody and precipitate. The precipitation arc becomes stationary after around 1 to 10hrs.

Fig 5: Transferrin patterns in hemodialysed patients. (I–II) Standard solutions (controls); (1–6) sera samples from selected from patients at the beginning, (6–12) sera samples from selected patients after 1 year of the study (Formanowicz & Formanowicz, 2011)

The height of the rocket is directly proportional to the antigen concentration.
The rocket height is plotted on one axis and the concentration on the other. The heights of rockets formed by the antigen samples with known concentration are plotted in the graph to obtain the calibration curve and the concentration of the unknown antigen sample can be determined with help of the calibration curve (as seen in fig 6).

Fig 6: Plotting of calibration curve and determination of unknown.

Advantages:

This method is simple, quick and reproducible and can be used for determining the concentration of a (single) specific protein in a protein mixture.

Rocket electrophoresis can be a done using highly complex mixture of proteins (e.g., serum sample, tissue extract, urine sample, cerebrospinal fluid, etc) to study the component of interest.

Several unknown samples can be analysed on a single plate.

(Just for info: Read this paper, titled ‘Transferrin changes in haemodialysed patients’ by Formanowicz & Formanowicz, 2011)

4. Two Dimensional Immunoelectrophoresis:

This method was first described by Ressler in 1960, and was improved by Laurell, Clarke & Freeman, and later Weeke. This technique is also known as crossed immunoelectrophoresis.

It allows the quantitation of one or more proteins in a mixture of proteins. It is used for the quick analysis of the constituents of protein mixtures, as opposed to single protein quantification by the rocket electrophoresis.

This assay involves two different electrophoresis steps: a normal electrophoresis and rocket electrophoresis, hence is a modification of rocket electrophoresis. The first electrophoresis step separates the antigen mix into its individual components and in the the second step, i.e. in the rocket electrophoresis step, the antigens react with antibody and are specifically detected by the immunoprecipitation.

Fig 7: Two dimensional immunoelectrophoresis A: Separation of antigens by electrophoresis. B: Immunoelectrophoresis of separated antigen components in the slice of gel in perpendicular direction.

Here, first the antigens in the mixture is separated using the electrophoresis. A section of gel is selected, sliced and placed on a glass plate. A layer of agar with antiserum is poured onto the glass plate adjoining the slice of gel at one end.

The gel slice end is placed towards the cathode and the rocket electrophoresis is performed at the perpendicular direction as the first run. As the electrophoresis advances, the antigens from the gel slice enters into the antisera containing agar and the precipitin arcs are formed at zone of equivalence.
The height of the arcs of standards can be used to find the amount of individual antigen present in the sample, similar to Laurell’s rocket electrophoresis.

Advantages:

Crossed immunoelectrophoresis can be used for quantification of different components of proteins.

This method is very sensitive, as well as specific, due to use of antibody-antigens interactions.

(Just for info: Read this paper from 1974 titled ‘Rapid Two-Dimensional lmmunoelectrophoresisof Human Serum Proteins’ to understand more about crossed immunoelectrophoresis. )

—-

This is all for this post. Hope u like this post, if yes please comment, like and share!!

Also follow us on Facebook, Twitter, Instagram or mail us (thebiotechnotes@gmail.com).

Have a nice day!

Thank you!!

—-

Read other posts by The Biotech Notes:

Micropropagation.

Bacterial Transformation..

Biofilm: and the Immune System..

—-

References:

Formanowicz and Formanowicz (2012) Transferrin changes in haemodialysed patients. Int Urol Nephrol. 44(3): 907–919.

Laurell (1973) Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies. Scand J Clin Investig. 124: 21–28.

Walker (1996) Rocket Immunoelectrophoresis. In: Walker J.M. (eds) The Protein Protocols Handbook. Humana Press.

Laurell (1965) Antigen-antibody cross-electrophoresis. Anal. Biochem. 10: 358.

Laine (1992) Crossed immunoelectrophoresis. Methods Mol Biol 80:207-13.