Ouchterlony double immunodiffusion
The Ouchterlony double immunodiffusion is a simple and proven method for the detection of special antigens (ENAs, Extractable Nuclear Antigens). The method (like the simple immunodiffusion test Ouchterlony test ) was named after its inventor Örjan Ouchterlony .
Action
Several holes are placed in a body of agar gel . Exemplary use: an extract of human cells obtained from almond tissue is pipetted into the centrally located hole. This extract contains a mixture of natural human antigens that are sought after. The patient's blood serum is then placed in the outer holes, and the test vehicle is then left to rest for 48 hours. During this time, the antigens from the middle hole with the extract diffuse outwards, while the antibodies move out of the holes with the blood serum towards the middle. When the two substances meet (roughly in the middle), an antigen-antibody reaction occurs, provided that there were antibodies in the blood that match the antigens . The reaction partners form an immune complex . This immune complex becomes visible as a fine white line (precipitation line) in the gel. If more than one hole has been used, it is possible to compare the concentration of substances in different samples, depending on how antigens and antibodies react. An antigen-antibody equilibrium is shown by a circular precipitate line.
theory
The Ouchterlony technique can be applied to many different questions. Precipitation, i.e. the complexes becoming visible in the agar gel, take place with many antigens if the antigen being examined can bind to the various antibodies used. Antibodies have at least two antigen contact points (antigen binding region), which is why large complexes of antigens and antibodies sometimes form. If, as an experiment, a larger amount of antigens is added to a constant amount of antibodies, all the antigens are initially bound in the precipitate if the antigen concentration is low. This phase is called the antibody excess phase. If the antigen concentration increases further, more and more antigens are bound until the ratio is finally balanced (compensation phase). If the amount of antigens then exceeds the antibodies, the precipitate will decrease again (antigen excess phase).