As seen in the last post, immunoprecipitation can be carried out in solution/liquid to determine the presence of antibodies, antigens or haptens. The reaction in liquid requires proper ratio of antigen to antibody to be in the region of equivalence, or else false negative result is achieved. The technique may hence become complicated and may require large quantities of antisera or antigens. Hence, the precipitation reactions in gel are preferred.

• Precipitation reaction in gels:

In the immunoprecipitation reactions in gels, the gel is usually semisolid or soft gel made of 1% agar or 1% agarose. These antibody and antigens can move around in the gel, and when the zone of equivalence is reached, the white precipitation can be observed. The major types of immunoprecipitation reaction in gel are the immunodiffusion and immunoelectrophoresis. Immunoelectrophoresis can be considered to be a modification of immunodiffusion. Let’s see different techniques of immunodiffusion in this post.

A. Immunodiffusion:

Immunodiffusion is the movement of the antigen and/or the antibody in the gel. The reactants are added to the wells, from which they diffuse to the region of lower/no concentration.  As the reactants diffuse into the gel, a gradient of its concentration is formed. The immunoprecipitation occurs in region of equivalent concentration of the antibody and the antigen, which is in form of bands.

These bands can be seen by naked eyes directly or after staining. If the reactants are radioactively labelled, autoradiography can be used.

The immunodiffusion assays in gels are of two major types:

1. Single immunodiffusion

2. Double immunodiffusion

Let’s read about these in more detail:

1. Single / Simple immunodiffusion:

In single/simple immunodiffusion, only one of the reactants (antigen) diffuses while the other reactant (antibody) is added to and fixed in the gel. The different types of assays under single immunodiffusion are;

a. Single diffusion in one dimension

b. Single diffusion in two dimensions

Let’s have a look at these subtypes;

a. Single diffusion in one dimension:

In these assays, the diffusing reactant, diffuses only in a single direction. This includes oudin tube method.

– Oudin tube method:

In this test, the solution or gel containing antigens is layered onto a layer of agar containing antiserum in a tube. Antigen diffuses into the antibody containing agar gel region due to gravity/ concentration gradient. Initially, as the antigen moves the antibody concentration is higher. At one point the antigen is equal in concentration to antibody or the equivalence concentration is reached, and the precipitation occurs (fig 1). The precipitation allows the detection of the presence or absence of antigen, i.e. qualitative analysis.Single Diffusion in one dimension

Fig 1: The single diffusion in one dimension gel method or the Oudin tune method.

b. Single diffusion in two dimensions:

This includes the method called single radial immunodiffusion (SRID), also known as Mancini method. In this method the antibody against a known antigen is incorporated into the gel. Wells are dug into these gel layer, to which antigen of interest is added. The antigen diffuses out radially from the well, again forming a concentration gradient. At the point where point of equivalence is reached, the precipitation occurs in form of a ring around the well.

Single Radial Immuno Diffusion
Fig 2: The radial diffusion of antigen from the well dug in the gel containing antibody and the formation of precipitation ring in the zone of equivalence.

SRID is used to quantitate the amount of antigen. Wells are dug into the antibody- incorporated gel and the antigen of different concentrations are are added to each of the well, each antigen concentration will have its zone of equivalence at different distance (fig 3a).

Fig 3: Single radial immunodiffusion (SRID). 3a: SRID with antigen solutions of different concentration. 3b: Plotting of graph and quantification of antigen concentration in unknown sample.

The calibration curve is obtained by plotting a graph of diameter or the ring area against the antigen concentration. The unknown concentration of the antigen sample can be found using its diameter or ring area and plotting it in the graph with the calibration curve (fig 3b).

Fig 4: Example of Single radial immunodiffusion (SRID): Gel containing antisera with dilutions of reference antigen (Column 1) and dilutions of influenza vaccine candidate (Abhg, Column 2 and 3) in the wells. Gel is stained with Coomassie brilliant blue. (Cropped image from Vodeiko and Weir, 2011).

(Just for info: Read this paper titled ‘Determination of H5N1 vaccine potency using reference antisera from heterologous strains of influenza’, which involves use of SRID.)

2. Double diffusion:

As opposed to the single diffusion method, where only one of the reactants diffuses, the double diffusion techniques involves the diffusion of both the reactants towards each other. The different methods using this technique are:

a. Double diffusion in one dimension:

This is similar to the single diffusion in one dimension, except that, there is a layer of only agar placed between the layers of antigen and antibody. As a result both the reactants diffuse towards each other and the precipitation ring is formed in the middle layer of agar (fig 5). Each of the reactants diffuse in one direction either upwards or downwards due to the concentration gradient, hence the diffusion in one dimensional.

Double Diffusion in one dimension
Fig 5: Double diffusion in one dimension.

b. Double diffusion in two dimensions:

This assay is also known as Ouchterlony technique and is the most widely used immunochemical technique. It is particularly used to detect if two antigens are similar or not. Ouchterlony technique can be performed on petri-dish or even a microscopic slide, overlaid with agar. The wells are cut in different positions in the agar and the antigen and antibody solutions are added. This can be used for both  qualitative as well as quantitative analysis.

b.1. Qualitative Ouchterlony technique:

The qualitative Ouchterlony technique is used to compare antigens for identical or cross-reacting determinants. In this test, the two solutions of antigen are placed in two adjacent wells and the homologous antibody is placed in the center well.

The precipitation line formed by the antigens in the two wells fuse (fig 6A) due to equivalent antigen concentration reached in those region, the antigens are said to be identical. The reaction is known as a reaction of identity and the pattern is known as pattern of identity.

Fig 6: Qualitative Ouchterlony Technique. A- Identical antigens give fused precipitation line; B- Non identical antigens give crosses lines; C: Similar antigens result in a spur formation.

If two unrelated antigens are added to the two wells and a centre well is filled with antibodies for each of the antigens, the immunoprecipitations of the antigens occur independently with the specific antibodies. The two precipitation lines will cross each other following their own zone of equivalence and form a cross shaped structure (fig 6B). This reaction is known as a reaction of non-identity and pattern is known as pattern of non-identity.

When two related antigens are added to the wells with the antisera in the centre well, a spur is produced. The spur indicates that one of the antigen determinant is present in both the samples and one is only present in one of the sample. The spur is the precipitation line of the additional  antigen which projects toward the antigen sample with only the common determinant (fig 6C). This is called a reaction of partial identity.

(Just for info: Read this paper titled ‘Comparative Phytochrome Immunochemistry as Assayed by Antisera against Both Monocotyledonous and Dicotyledonous Phytochrome’ and observe the different patterns of Ouchterlony assay.)

b.2. Quantitative Ouchterlony technique:

This is similar to SRID, wherein the relative position of precipitation line allows a semi-quantitative estimate of antigen concentration. The more the concentration of antigen, farther would be the precipitation line and and lesser the concentration, the nearer it would be (fig 8).

Fig 8: Quantitative Ouchterlony technique: The antigen solution with different concentration will give precipitation line at different distance.

Hence, the distance of the precipitation line will depend on the concentration of antigen solution added to the well. The graph of distance to the concentration can be plotted and a calibration curve can be obtained (similar to SRID, see fig 3b). Using the calibration curve the unknown concentration of antigen samples can be found.

Fig 9: Semiquantitative determination of antibody titer to α-fetoprotein (AFP) in hyperimmune rabbit serum by Ouchterlony method. AFP was applied into the central well and total rabbit hyperimmune serum (dilutions) were added to surrounding wells. K is the negative control (Khramtsov, 2014).

(Just for info: Read this paper by Khramtsov (2014) which involves use of Ouchterlony to quantitate the antibody titre to α-fetoprotein (AFP) in hyperimmune rabbit serum. Here AFP (antigen) was added to the central well and diluted solutions of antiserum in the surrounding wells.)

B. Immunoelectrophoresis:

Immunoelectrophoresis is also an immunoprecipitation technique carried out in gel. It is a combination of electrophoresis and immunodiffusion in gels. We shall discuss about the immunoelectrophoresis and it’s different types in the next post.

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Read other posts by The Biotech Notes:

Let’s go FISH’ ing

DNA Replication: Prokaryotes.

Neurons: Introduction

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References:

– Hull (2014) Chapter 13 – Assay, Detection, and Diagnosis of Plant Viruses. Plant Virology (Fifth Edition):755-808.

– Khramtsov (2014) Application of Diagnosticum Based on Functionalized Carbon Nanoparticles for Monitoring of Immunoglobulins Affinity Purification. Applied Biochemistry and Microbiology 50(6): 683–688.

– Vodeiko and Weir (2011) Determination of H5N1 vaccine potency using reference antisera from heterologous strains of influenza. Influenza and Other Respiratory Viruses 6(3):176–187.