Radial immunodiffusion


Radial immunodiffusion

Radial immunodiffusion (or Mancini method, Mancini immunodiffusion, single radial immunodiffusion assay) is an immunodiffusion technique used in immunology to determine the quantity of an antigen by measuring the diameters of circles of precipitin complexes surrounding samples of the antigen that mark the boundary between the antigen and an antibody suspended in a medium, such as an agar gel. The diameters of the circles increase with time as the antigen diffuses into the medium, reacts with the antibody, and forms insoluble precipitin complexes.

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.

The quantity and concentration of insoluble antigen-antibody complexes at the outer edge of the circle increases with time. Therefore, the clarity and density of the outer edge increases with time.

Expansion of the circle reaches an end point and stops when antigen and antibody reach equivalence. However, the clarity and density of the outer edge may continue to increase after the circle stops expanding.

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. 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. Circles created by small quantities of antigen reach their end points before large quantities do. 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.

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. However, circles of the precipitate are smaller and less distinct during expansion than they are after expansion has ended. Further, temperature affects the rate of expansion, but does not affect the size of a circle at its end point. 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. Therefore, measurements of the sizes of circles and of graphs produced from such measurements are often less accurate when circles are expanding than they are after expansion has ended. For that reason, 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 expanding.

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

References

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