Radial immunodiffusion
Encyclopedia
Radial immunodiffusion 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.
Immunodiffusion
Immunodiffusion is a diagnostic test which involves diffusion through a substance such as agar.Two commonly known forms are Ouchterlony double immunodiffusion and radial immunodiffusion....
technique used in immunology
Immunology
Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. It deals with the physiological functioning of the immune system in states of both health and diseases; malfunctions of the immune system in immunological disorders ; the...
to determine the quantity of an antigen
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
by measuring the diameters of circles of precipitin complexes
Precipitin
A precipitin is an antibody which can precipitate out of a solution upon antigen binding.-Precipitin reaction:The precipitin reaction provided the first quantitative assay for antibody, which has since been rendered obsolete by current diagnostic techniques such as ELISA and RIA...
surrounding samples of the antigen that mark the boundary between the antigen and an antibody
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
suspended in a medium, such as an agar
Agar
Agar or agar-agar is a gelatinous substance derived from a polysaccharide that accumulates in the cell walls of agarophyte red algae. Throughout history into modern times, agar has been chiefly used as an ingredient in desserts throughout Asia and also as a solid substrate to contain culture medium...
gel
Gel
A gel is a solid, jelly-like material that can have properties ranging from soft and weak to hard and tough. Gels are defined as a substantially dilute cross-linked system, which exhibits no flow when in the steady-state...
. 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.