Radial Immunodiffusion

Radial Immunodiffusion


Radial Immunodiffusion Protocol

Capable of detecting and quantifying antigens, the radial immunodiffusion is a technique in which antibody is incorporated into an agar gel, followed by the addition of antigen into formed wells of the antibody-containing agar. After incubation, diffusion proceeds and the antigen which has been allowed to diffuse into the agar reacts with specific antibody, produces a ring of precipitation that will form at the point where the antigen and antibody have reached equivalence. However, as diffusion proceeds radially from the well, an excess of antigen develops in the area of the precipitate causing it to dissolve only to form once again a greater distance from the site of origin. Precipitate will occur only at the zone of equivalence. The greater the concentration of the antigen in the well, the faster precipitation will take place. Diffusion of antigen will proceed from the well with a build-up of precipitate at the outer edge of the ring, where the antigen will be encountering additional antibody. The system is initially in a dynamic state, as the rings increase with time. A static state of precipitation is reached when all the antigen has diffused into the gel and precipitation is complete. (2,3)

The Precipitation Ring

The precipitation ring surrounds an area proportional to the concentration of antigen measured 48 to 72 hours following diffusion, with antibody concentration kept constant. The diameter of the precipitin ring can be used to quantify the antigen concentration through comparison with antigen standards. Standard curves can be employed using these known antigen standards. The antigen concentration is easily determined through measuring the diameter of the precipitation ring. This technique provides sensitivity in detecting an antigen to 1 to 3 micrograms/mL antigen. For greater sensitivity, ELISA assays should however be used. (2,3)

Standard Calibration Curve

In a simple experiment, numerous known BSA concentrations and a single known sample, can be placed into individual wells within an anti-BSA agar plate. The diameters of the precipitin discs can then be measured and plotted on semi-logarithm graph paper. The standard calibration curve can then be plotted as the BSA concentration versus the diameter of the precipitin discs. The curve allows for the determination of the unknown sample concentration. The standards from each formed a gradient of precipitin ring in direct relation to their antigen concentration. Slight procedural differences, such as poor well filling and disc measurements, can lead to slight deviations of a few standard points,.

The Static Stage

At the static stage, the circumscribed area of the ring is proportional to the antigen concentration in the well. Area is proportional to the square of the radius of the ring, and therefore diameter squared is proportional to the concentration of the antigen. Plotting diameter squared versus concentration will produce a straight line, as will using semilog paper and graphing diameter of ring versus antigen concentration. Therefore, unknown concentrations may be discovered through measuring the ring precipitation around the unknown well and finding its' corresponding concentration from the standardized curve. But, extrapolation from the standard curve is not possible. Determination of random unknown concentrations either above or below the available standard curve values cannot be used, because the calibration curve alters from a linear form, into a non-linear form at the upper regions of the curve, while at the curves lower end, precipitation diameters become difficult to measure accurately. Setting up additional standards at the higher end of a curve, above any unknown precipitation rings. This would allow the curve to include additional diameters above the last highest value, thereby passing the unknown diameter value, and therefore having a concentration value for its' diameter. Moreover, additional agar plates with higher antibody concentrations may also be setup for additional standard curves, compared to setting up higher antigen concentration standards. In addition, the procedure could be performed with the unknown sample diluted with normal saline. Adversely, for unknown precipitation rings below the standard curve lowest extreme, the above mentioned procedures could come into affect, however, with the adverse steps. Fro example, to obtain a stadard curve with lower values (diameter), lower standard concentrations would be used. Decreasing antibody concentration within the agar plate could also be used. (2,3,4)

Monoclonals

The use of a monoclonal antibody against BSA, which is a population of clones all specific for a unique epitope, instead of an anti-BSA serum, would prevent crosslinking, which is essential for precipitation, if each antigen had only one epitope. Monoclonals recognize only one epitope on the antigen, thereby not allowing for the crosslinking of immune complexes to occur. The use of antiserum, which contains populations of various antibodies that differ in their specificities towards the antigen, polyclonal antibodies, allows for the antibodies to bind similar but different specific antigen epitopes, and thus crosslinking the immune complexes into a lattice required for precipitation. (2,3,4)

 

References:

1. Janeway, C.A., Travers, P., Walport, M., and Capra, J.D. 1999. Immunobiology: The immune system in health and disease. Garland Publishing, 4th ed., New York, USA.

2. Delves, P., and Roitt, I. 1999. Encyclopedia of Immunology. Academic Press Inc., 2nd ed., San Diego, USA.

3. Cruse, J. and Lewis, R. 1995. Illustrated Dictionary of Immunology. CRC Press Inc., USA.

4. Bryant, N. 1986. Laboratory Immunology and Serology. B. Venable, W.B. Saunders Company, 1st ed., Philadelphia, USA.

 

IHC