Ouchterlony Double Immuno Diffusion

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Ouchterlony double immuno diffusion is a simple, rather dated method which is still considered to be the gold standard for detection of ENAs (Extractable Nuclear Antigens). A gel plate is cut to form a series of holes in the gel (The geometry is critical) . An extract of human cells ( harvested from tonsil tissue ) is placed in the centre well. This extract contains a cocktail of natural human antigens that we wish to look for. The patients serum is then placed in one of the outer wells and the plate left for 48 hours to develop. During this time the antigens in the tonsil extract migrate in a radial fashion out of the centre well and the antibodies in the patients serum migrate in a radial fashion out of the outer well. When the two meet if there are antibodies in the patients serum to any of the antigens in the tonsil extract the antibodies will bind to the antigens and form what is known as an immune complex. This immune complex precipitates in the gel to give a thin white line.

The line will give a full identity (continuous line), partial identity (continuous line with a spur coming off one end - like a branch off the main line) or a non identity where the two lines cross completely.

The type of line will tell us that the patient does have some form of antibody likely to be associated with a connective tissue disease.

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