Immunodiffusion Assays
Immunodiffusion assays are a type of serological test that can be used to determine the species origin of a biological sample. In this test, antibodies from various species are allowed to diffuse through a gel medium and react with antigens in the sample. The resulting visible precipitin lines can be used to identify the species of origin. There are different types of immunodiffusion assays, including Single immunodiffusion (Radial Immunodiffusion) and Double immunodiffusion (Ring assay and Ouchterlony assay), discussed in detail below:
🤷🏻♀️❓Difference between Single and Double Immunodiffusion
In single immunodiffusion, a concentration gradient is established for either the antigen or the antibody. The antigen or antibody diffuses from a single well into the surrounding gel, where it encounters the other component (antibody or antigen, respectively) and forms a visible line of precipitation. Single immunodiffusion is relatively simple and easy to perform, but it is less sensitive and less specific than double immunodiffusion.
In double immunodiffusion, a concentration gradient is established for both the antigen and the antibody. Two wells are cut into the gel matrix: one containing the antigen and the other containing the antibody. As the antigen and antibody diffuse through the gel, they encounter each other and form a visible line of precipitation.
SINGLE IMMUNODIFFUSION
Single immunodiffusion test is based on the principle of diffusion of either antigen or antibody through a semi-solid medium (such as agarose gel) and the formation of a visible line of precipitation where the antigen-antibody complex is formed.
A. Radial Immunodiffusion
Radial immunodiffusion, also known as Mancini immunodiffusion, is a common single immunodiffusion test in which a concentration gradient is established for an antigen, while the antibody is uniformly distributed in the gel matrix. This means that the concentration of the antigen decreases as it moves away from the sample well, while the concentration of the antibody remains constant throughout the gel.
Figure: Procedure for Radial Immunodiffusion Assay
The steps involved in performing a radial immunodiffusion assay are as follows:
Preparation of the agarose gel: Prepare 1-2% Agarose gel solution by dissolving agarose in a buffer solution and heating it until it becomes homogeneous. Pour the gel on a plastic plate and allow it to solidify.
Addition of the antibody: Add the antibody specific to the antigen of interest to the surface of the gel and allow it to diffuse into the gel. Note: The antibody can also be added directly to the agarose gel solution and then allowed to solidify.
Cutting of the wells: Cut circular wells into the gel using a cork borer or a similar instrument.
Addition of the sample, standards, and controls: Prepare a series of dilutions of known concentration of antigen ‘standards’ (such as 10ng/mL, 20ng/mL, 40ng/mL, and 80ng/nL) and add the dilutions, the sample to be tested (such as blood, saliva, or other bodily fluid), and negative control (such as distilled water) to one well each.
Incubation: Incubate the gel at an appropriate temperature to allow it to diffuse and the reaction to occur.
Interpretation: After the incubation period, examine the gel for the presence of a line of precipitation. The line of precipitation will be visible where the antigen and antibody bind to each other forming an antigen-antibody complex, indicating the presence of the antigen in the sample. The resulting visible line of precipitation is proportional to the concentration of the antigen in the sample.
Measure the signal: Measure the diameter of the ring of precipitation for each standard dilution and the unknown sample.
Plot a standard curve: Plot the diameter of the ring of precipitation for each standard dilution on a graph with concentration on the x-axis and diameter on the y-axis. A standard curve will be obtained with a linear relationship between concentration and diameter.
Determine the concentration of the unknown sample: Finally, use the standard curve to determine the concentration of the antigen in the unknown sample by measuring the diameter of its ring of precipitation and interpolating the value on the standard curve.
DOUBLE IMMUNODIFFUSION
The second type of assay is double diffusion in which a concentration gradient is established for both an antigen and an antibody. The most common examples are the ring assay and the Ouchterlony assay.
A. Ring Assay
In the ring assay, the antigen and antibody are allowed to diffuse toward each other and form a precipitation ring at the interface between the two solutions. The steps for performing a ring assay are as follows:
Prepare the test tube: Label three test tubes as follows- 'A' for the test sample, 'B' for positive control, and 'C' for negative control.
Add the antiserum: Add the antiserum (containing antibodies for the antigen of interest) to all three test tubes.
Layer the sample solution: Carefully layer an equal amount of the sample solution to be tested on top of the antibody solution in test tube 'A', without mixing. Similarly, add an equal amount of a known sample containing the antigen of interest to test tube 'B', and distilled water to test tube 'C', to act as positive and negative controls, respectively.
Incubate: Leave the test tube undisturbed for 5 minutes and allow the antigens present in the sample, and the antibodies to diffuse toward each other.
Observe for a ring of precipitation: After incubation, observe the test tube for the presence of a ring of precipitation at the interface of the two solutions. A positive reaction is indicated by the presence of a precipitation ring, and a negative reaction is indicated by the absence of a precipitation ring.
Figure: Ring Precipitation Assay
B. Ouchterlony Assay
The Ouchterlony assay was developed by a Swedish immunologist, Örjan Ouchterlony, in 1949. The assay involves the diffusion of antigens and antibodies from two different sources onto a gel matrix, which results in the formation of a visible ring of precipitation at the point of equivalence where the concentration of antigen and antibody are balanced.
The steps involved in performing the Ouchterlony assay are as follows:
Preparation of the agarose gel: Prepare 1-2% Agarose gel solution by dissolving agarose in a buffer solution and heating it until it becomes homogeneous. Pour the gel on a plastic plate and allow it to solidify.
Cutting of the wells: Cut circular wells into the gel at desired locations. Usually, a pattern with five wells surrounding a well in the centre is used.
Addition of the antibody, sample and controls: The questioned samples and the controls are loaded in the surrounding wells, while the antibody is loaded in the central well. For example: In the figure below, the central well contains human antiserum, while the surrounding wells (A, B, C, D, and E) contain blood samples retrieved from the knife, cloth, floor, door, and table at the crime scene, respectively.
Incubation: Incubate the gel at an appropriate temperature to allow the double diffusion of the antigen and the antibody from the wells towards each other.
Interpretation: Examine the gel for the presence of a lines of precipitation. The line of precipitation will be visible where the antigen and antibody bind to each other forming a antigen-antibody complex, indicating the presence of the antigen in the sample. The resulting visible line of precipitation is proportional to the concentration of the antigen in the sample.
Based on the results presented in the figure below, it can be inferred that the blood samples obtained from the knife (A), floor (C), and door (D) were of human origin, while those from the cloth (B) and table (E) were not.
Figure: Ouchterlony Assay