Ouchterlony double immunodiffusion

The Ouchterlony double immunodiffusion is a simple and proven method for the detection of specific antigens ( ENA, extractable nuclear antigen ). The method was named ( as the simple immunodiffusion test Ouchterlony test) after its inventor Örjan Ouchterlony.

Procedure

In a body made of a plurality of holes are placed agar gel. Exemplary Uses: An extract of human cells, which was recovered from tonsil tissue is pipetted into the central hole. In this case, this extract contains a mixture of natural human antigens, which are searched for. Thereafter blood serum of the patient is placed in the outer holes, and then the test carrier rest 48 hours. During this time the antigens from the central hole to diffuse with the extract to the outside, at the same time, the antibody of the holes with the blood serum to move towards the center. If the two substances (approximately in the center) meet, there is, unless because antibodies were in the blood which match the antigen to an antigen- antibody reaction. The reactants form an immune complex. This immune complex is visible as fine white line ( precipitin ) in the gel. If there is used more than one hole, comparisons of the concentration of substances in different samples will be available, depending on such as antigens and antibodies react. An antigen -antibody equilibrium is visible on an annular precipitation line.

Theory

The Ouchterlony technique is applicable to many different topics. Rainfall, so the appearance of the complexes in agar gel, take place in many antigens, if the under- studied antigen can bind to the different antibodies used. Antibodies have at least two antigen contact points ( antigen binding region ), therefore partially form large complexes of antigens and antibodies. Are you piloted a larger amount of antigens to a steady constant flow of antibodies, all antigens are initially, at low antigen concentration, bound in the precipitate. This phase is called antibody - excess phase. Finally, the antigen concentration increases further, more and more antigens are attached, until finally the ratio is balanced ( balancing phase ). Then, when the quantity of the antigens, the antibodies, the precipitate is again decrease ( antigen excess phase).