Radial immunodiffusion and Ouchterlony double diffusion

Principle

• Both are precipitation tests in agarose gel; visible precipitin lines or rings form where antigen and antibody meet at equivalence.

Radial immunodiffusion (RID)

• Single diffusion: specific antibody is mixed evenly into the agarose; antigen (patient serum) is placed in wells and diffuses outward.
• A precipitin ring forms; its size relates to antigen concentration.
• Mancini (end-point) method: after complete diffusion (about 48–72 hours), ring diameter squared is directly proportional to antigen concentration.
• Fahey–McKelvey (kinetic) method: read at a fixed earlier time (about 18 hours); diameter is proportional to the log of concentration.
• Standards of known concentration are run on the same plate to construct a calibration curve.
• Uses: IgG, IgA, IgM, C3, C4, transferrin and other proteins when automated immunoassays are unavailable.

Ouchterlony double diffusion

• Antigen and antibody are placed in separate wells cut in plain agarose; both diffuse toward each other and form precipitin lines.
• Identity: lines fuse into a smooth arc (same antigenic determinants).
• Non-identity: lines cross (unrelated antigens).
• Partial identity: lines fuse with a spur (antigens share some but not all determinants).
• Qualitative or semi-quantitative (by titrating antibody).

Procedure

• Fill wells with exact volumes; incubate in a humid chamber at room temperature for 24–72 hours; read with oblique light; may stain with Coomassie blue for clarity.

Quality control and pitfalls

• Drying, uneven gel thickness, temperature changes and inaccurate well volumes affect results.
• RID: antigen excess or reading before diffusion is complete gives errors; monoclonal or low molecular weight proteins (e.g. 7S IgM) diffuse differently and give inaccurate values.
• Slow and less sensitive than nephelometry or ELISA.

Clinical use

• RID: quantitation of immunoglobulins and complement. Ouchterlony: detection of antibodies to extractable nuclear antigens (historic), fungal precipitins (Aspergillus, Histoplasma H and M bands), and the Elek test for diphtheria toxin (a modified immunodiffusion).