Ouchterlony Double-Radial Immunodiffusion

If an antigen-antibody complex has to be precipitated by a secondary antibody (e.g., antigen bound by mAb precipitated by goat anti-(mouse-IgG) antibody), this range of equivalence must be known. For estimation of the range of equivalence the precipitin assay is used. 

Prepare a series of 1 1 or 1 2 dilutions of the first component, e.g., rabbit immunoglobulin or serum, in PBS. Pipet 0.5 ml of each dilution into a 2 ml tube. Add 0.5 ml of a dilution of the second component, e.g., goat anti-(rabbit IgG) antiserum 1 100 diluted in PBS, to each of the first dilutions. Vortex and incubate at 37 °C for 1 h or at 4 °C overnight. Enhance precipitation by addition of 0.25 ml of Soln. A. 

Spin at 8000 x g for 20 min and wash the pellet twice with PBS. If unlabeled components were used, dissolve the pellet with 0.1 ml Soln. B and determine the protein content. If a radioactive labeled compound was involved, count for radioactivity. 

Plot the amount of protein and radioactivity, respectively, against dilution. The maximum of the obtained Heidelberger curve indicates the range of equivalence. 

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Ouchterlony technique. Double-radial immunodiffusion for the detection of precipitating autoantibodies against extractable nuclear antigens . Method of high diagnostic specificity but low sensitivity for diagnosis of autoimmune rheumatic diseases. 

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