Ouchterlony double immuno diffusion

Ouchterlony double immuno diffusion (or agar gel immunodiffusion) 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.

Precipitation occurs with most antigens because the antigen is multivalent i,e., has several antigenic determinants per molecule to which antibodies can bind. Antibodies have at least two antigen binding sites, thus large aggregates or lattices of antigen and antibody are formed. Experimentally, an increasing amount of antigen is added to a constant amount of antibody in solution, initially at low antigen concentration, all of the antigen is contained in the precipitate. this is called the antibody-excess zone (Prozone phenomenon). As more antigen is added, the amount protein precipitated increases until the antigen/antibody molecules are at an optimal ratio. This is known as the equivalence zone or equivalence point. When the amount of antigen in solution exceeds the amount of antibody, the amount of precipitation will decrease. This is known as the antigen excess zone.

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.

History
It was developed by Örjan Ouchterlony.