Single Radial Immunodiffusion

Abstract

Single radial immunodiffusion is used extensively for the quantitative estimation of antigens (1). In this method, the antigen-antibody precipitation is made more sensitive than in double immunodiffusion (see Chapter 135) by the incorporation of the antiserum in the agar solution before the gel is made (2). Thus, the antiserum is uniformly distributed throughout the agar gel. Antigen is then allowed to diffuse from wells cut into the agar gel. This is an example of single (simple) immunodiffusion. Initially, as the antigen diffuses out of the well, its concentration is relatively high and it forms relatively soluble antigen—antibody adducts. However, as it diffuses further and further from the well, its concentration decreases. When its concentration becomes equivalent to that of the antibody in the gel, a disk of antigen—antibody precipitate (precipitin) is formed. The greater the initial concentration of antigen in the well, the greater the diameter of the precipitin disk. Thus, by running a range of known antigen concentrations on the gel and by measuring the diameters of their precipitin disks, a calibration graph can be constructed. The antigen concentrations of unknown samples run on the same gel can then be found by simple interpolation having measured the diameters of the respective precipitin disks.