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][3] A solution containing the antigen is then placed in a well that is punched into the gel.[2][3][4] The slide or container is then covered or closed to prevent evaporation.[2][3][4]

The antigen diffuses radially into the medium, forming a circle of precipitin that marks the boundary between the antibody and the antigen.[1] 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] The antigen is quantitated by measuring the diameter of the precipitin circle and comparing it with the diameters of precipitin circles formed by known quantities or concentrations of the antigen.[1][2][3][6]

Antigen-antibody complexes are small and soluble when in antigen excess. Therefore, precipitation near the center of the circle is usually less dense than it is near the circle's outer edge, where antigen is less concentrated.[1][2]

Expansion of the circle reaches an end point and stops when free antigen is depleted and when antigen and antibody reach equivalence.[1][2][5] However, the clarity and density of the circle's outer edge may continue to increase after the circle stops expanding.[1]

For most antigens, the area and the square of the diameter of the circle at the circle's end point are directly proportional to the quantity of antigen and are inversely proportional to the concentration of antibody.[1][2][5][6] Therefore, a graph that compares the quantities or concentrations of antigen in the original samples with the areas or the squares of the diameters of the precipitin circles on linear scales will usually be a straight line when all circles have reached their end points (equivalence method).[1][3][5][6]

Circles that small quantities of antigen create reach their end points before circles that large quantities create.[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 that contains the smaller quantities or concentrations of antigen and will be curved in the portion that contains the larger quantities or concentrations.[1][5]

While circles are still expanding, a graph that compares the quantities or concentrations of the antigen on a logarithmic scale with the diameters or areas of the circles on a linear scale may be a straight line (kinetic method).[1][2][4][5][6][7] However, circles of the precipitate are smaller and less distinct during expansion than they are after expansion has ended.[1][5] Further, temperature affects the rate of expansion, but does not affect the size of a circle at its end point.[1] In addition, the range of circle diameters for the same quantities or concentrations of antigen is smaller while some circles are enlarging than they are after all circles have reached their end points.[1][5]

The quantity and concentration of insoluble antigen-antibody complexes at the outer edge of the circle increase with time.[1] The clarity and density of the circle's outer edge therefore also increase with time.[1] As a result, measurements of the sizes of circles and graphs produced from these measurements are often more accurate after circles have stopped expanding than they are when circles are still enlarging.[1] For those reasons, it is often more desirable to take measurements after all circles have reached their end points than it is to take measurements while some or all circles are still enlarging.[1]

Measurements of large circles are more accurate than are those of small circles.[1] It is therefore often desirable to adjust the concentration of antibody and the quantity of antigen to assure that precipitin rings will be large.[1]