Radial immunodiffusion

A solution containing antibody is added to a heated medium such as agar or agarose dissolved in buffered normal saline.

The molten medium is then poured onto a microscope slide or into an open container, such as a Petri dish, and allowed to cool and form a gel.

[1][2] The diameter of the circle increases with time as the antigen diffuses into the medium, reacts with the antibody, and forms insoluble precipitin complexes.

[1][2][5] Therefore, if areas or diameters of circles are measured while some, but not all, circles have stopped expanding, such a graph will be straight in the portion whose wells initially contained the smaller quantities or concentrations of antigen and will be curved in the portion whose wells contained the larger quantities or concentrations.

[1][5] The quantity and concentration of insoluble antigen-antibody complexes at the outer edge of the circle increase with time.

[1][9] It is therefore often desirable to adjust the concentration of antibody and the initial quantities of antigen to assure that precipitin rings will be large.

Best-fit line plots. RID often produces similar plots when the horizontal abcissa (X-axis) contains the initial antigen concentrations, the vertical ordinate (Y-axis) contains the squares of the precipitin ring diameters, and each plot is a measurement time. The entirely curved purple plot (bottom) appears when all rings are expanding. The entirely straight red plot (top) appears when all rings have reached their endpoints and have stopped expanding. Intermediate plots are partially straight and partially curved (see [ 1 ] and [ 7 ] ).
Straight line on a best-fit semi-logarithmic plot. RID often produces a similar plot while all precipitin circles are expanding, as in the kinetic method (see [ 1 ] and [ 10 ] ).