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Radial immunodifusion tests for the presence/absence of viral antigens in a sample. Antigen diffuses into the agar which contains specific antibody and a ring of precipitate is formed when antigen-antibody interactions occur. The diameter of the ring is directly proportional to the concentration of the antigen and can thereby be used to quantitate the amount of antigen. A reverse radial immunodiffusion test, in which antigen is incorporated in the agar, can be used to quantitate the amount of antibody in a sample.

Capable of detecting and quantifying antigens, the radial immunodiffusion is a technique in which antibody is incorporated into an agar gel, followed by the addition of antigen into formed wells of the antibody-containing agar. After incubation, diffusion proceeds and the antigen which has been allowed to diffuse into the agar reacts with specific antibody, produces a ring of precipitation that will form at the point where the antigen and antibody have reached equivalence. However, as diffusion proceeds radially from the well, an excess of antigen develops in the area of the precipitate causing it to dissolve only to form once again a greater distance from the site of origin. Precipitate will occur only at the zone of equivalence. The greater the concentration of the antigen in the well, the faster precipitation will take place. Diffusion of antigen will proceed from the well with a build-up of precipitate at the outer edge of the ring, where the antigen will be encountering additional antibody. The system is initially in a dynamic state, as the rings increase with time. A static state of precipitation is reached when all the antigen has diffused into the gel and precipitation is complete.

The precipitation ring surrounds an area proportional to the concentration of antigen measured 48 to 72 hours following diffusion, with antibody concentration kept constant. The diameter of the precipitin ring can be used to quantify the antigen concentration through comparison with antigen standards. Standard curves can be employed using these known antigen standards. The antigen concentration is easily determined through measuring the diameter of the precipitation ring. This technique provides sensitivity in detecting an antigen to 1 to 3 micrograms/mL antigen. For greater sensitivity, ELISA assays should however be used. (2,3)

Standard Calibration CurveIn a simple experiment, numerous known BSA concentrations and a single known sample, can be placed into individual wells within an anti-BSA agar plate. The diameters of the precipitin discs can then be measured and plotted on semi-logarithm graph paper. The standard calibration curve can then be plotted as the BSA concentration versus the diameter of the precipitin discs. The curve allows for the determination of the unknown sample concentration. The standards from each formed a gradient of precipitin ring in direct relation to their antigen concentration. Slight procedural differences, such as poor well filling and disc measurements, can lead to slight deviations of a few standard points
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Q: What is Radial immunodiffusion test?
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Related questions

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