IMMUNODIFFUSION (Latin immunis free, saved from something + diffusion) — one of methods of immunoassay of antigens and antibodies based on a precipitation test.
Methods I. were widely adopted in biology and medicine in the analysis of antigens of fabrics and especially serum proteins, at a research of antigens of tumors and embryonal fabrics. The basis of a method is made by reaction between antigens (see) and antibodies (see), coming to gel by diffusion and forming specific complexes in a zone of an optimum ratio — strips of precipitation (see. Precipitation ). Strips of precipitation easily come to light visually or during the coloring on proteins. Feature of a method is that each couple antigen — an antibody (see. Antigen — an antibody reaction ) creates an individual strip of precipitation, and reaction does not depend on existence in the studied system of other antigens and antibodies. The method, thus, allows to find separate antigens in complex antigenic systems, napr, in biol, liquids or fabrics extracts, and specific antibodies in antiserums, without resorting to their special fractionation.
The specific precipitated calcium superphosphate forming in gel does not detain unrelated components. The location of a strip of precipitation in gel is defined by diffusion rate of antigens and antibodies and their concentration in initial solutions since formation of precipitated calcium superphosphate is possible only at an optimum quantitative ratio of reagents. As a result of independent action of these factors in the analysis by method I. complex antigenic mixtures by means of the polyvalent antiserums received to them (see. Serums ) in gel there are discrete strips of precipitation corresponding to separate couples antigen — an antibody. It allows to define a set of antigens in the studied solutions.
Reaction is carried out usually to 1 — 1,5% gel from an agar or agarose on fiziol, solution or buffer solutions with pH values at which there is no Isoelectric precipitation of proteins, i.e. in neutral or alkalescent conditions.
There are several options of a method I. both numerous and micromodifications. Method I. it is for the first time offered by Uden (J. Oudin, 1946) who developed a diffusion method of antigens in gel, antibody-containing or a method of simple diffusion. Reaction on At I will put it is carried out in test tubes in which apply antigenic solution on a column of the gel which is previously mixed with an antiserum. Antigens, diffusing with various speed in gel, react with antibodies and form the strips of precipitation which are gradually displaced in gel since at receipt in gel of new portions of antigen there is a shift of a zone of an optimum ratio of antigens and antibodies and dissolution of precipitated calcium superphosphate a lot of antigen.
A method of double diffusion according to Oakley and Fultortsu (S. of L. Oakley, A. J. Fulthorpe, 1953) in test tubes or in capillaries: gel, antibody-containing, and solution of antigens are divided by a column of pure gel. As a result of cross diffusion of antigens and antibodies of a zone of their optimum ratio in intermediate gel are not displaced, and the forming strips of precipitation in the course of reaction do not migrate. Methods I. in columns of gel apply generally to the analysis of set of antigens in complex antigenic systems and definitions of diffusion constants of antigens. At the same time they are a little suitable for comparison of antigens in different systems.
Considerably more in immunochemistry the method of double diffusion in gel on Oukhterlonya was widely used (0. Ouchterlony, 1948, 1953). In this option of immunodiffusion the principle of identification of various antigens in complex systems is combined with a possibility of direct their identification in several systems. Reaction is carried out in plates of agar gel on glass or in Petri dishes. In the stiffened gel on nek-rum distance from each other cut out holes and fill them with solutions of antigens and antiserums. At optimum picked up ratio of antigens and antibodies of a strip of precipitation form in gel between holes. Since reagents diffuse from holes concentrically, the method allows to carry out at the same time several reactions, having placed around a hole with an antiserum several holes with various drugs of antigens. Filling peripheral holes with antiserums, and central — antigenic solution, it is possible to investigate a set of antibodies in serums of a different origin. The nature of connection of the strips of precipitation formed by antigens of the compared systems in reaction with the same antiserum is ambiguous and is defined by degree of similarity or distinction of antigens. Smooth full merge or partial merge of strips of precipitation on border of adjacent reactions testifies to full identity or partial similarity of antigens whereas a crossing of the ends of strips of precipitation (i.e. formation of «spur») — about not identity of the studied antigens.
Statement of reaction by method of double diffusion on Oukhterlonya is technically not difficult. Prepare 1 — 1,5% gel from an agar, in flux spill an agar in Petri dishes or on glass plates a layer in 1,5 — 3 mm. In the stiffened gel holes, number, a form which sizes and an arrangement depend on specific goals of experience cut out. The linear arrangement of holes by several rows, an arrangement on corners of an equilateral triangle, according to the «square» scheme offered by G. I. Abelev (1960) for the analysis of two full systems antigen — an antibody, on a circle with an antiserum well in the center or located excentricly and other options is applied. Plates after filling of holes with reagents place in a moist chamber. Reaction develops at the room temperature within several hours or days. For registration of results of a plate photograph; strips of precipitation on the washed and dried up plates paint dyes on protein, carrying out gistokhy, reactions for definition of enzymatic activity of antigens is in certain cases possible.
Work with polyvalent antiserums makes one of the first stages of immunodiffusion analysis. Often there is a need to investigate not all range of the antigens revealed by a polyvalent antiserum, and the certain antigens interesting the researcher. For blocking of «unnecessary» reactions gel is previously saturated with the corresponding antigens of heterologous system.
Quantitative assessment of content of antigens in drugs by means of methods I. it can be received in the presence of monospecific antiserums. The analysis is carried out by titration of antigenic drugs and definition of their final cultivations giving positive reaction in comparison with a standard sample or test system. A quantitative method of determination of content of antigens is also statement of reactions by a method of radial immunodiffusion of Mancini (G. Mancini, 1965). At the same time use the principle of diffusion of antigens in gel, antibody-containing to the studied antigen. Determining radiuses of ring zones of precipitation, according to previously constructed graph them from concentration of this antigen calculate the content of antigen in drug.
Use in immunodiffusion analysis is radioactive marked antigens and antibodies led to development of the methods of an immunoautoradiography opening essentially new opportunities in studying of antigens.
One of methods of an immunoautoradiography is intended for a research of biosynthesis of antigens [Perelmann and Haltin (P. Perelmann, T. Hultin), 1958; G. I. Abelev, R. D. Bakirov, 1968]. The method consists of the following stages: a) inclusion marked isotopes of predecessors of antigens (e.g., 14C-amino acids for proteins) in in vivo or in vitro fabric; b) carrying out reactions And. with extracts of fabrics and specific antiserums to the studied antigens; c) autoradiography of reactions And. For receiving the author of adiografichesky prints the washed from not reacted substances and dried-up plates of agar gel cover with a photoemulsion or impose a highly sensitive film. Exposure time is established by practical consideration. Comparison of results of immunodiffusion analysis at usual registration and autoradiography (see) allows to judge existence and synthesis of antigens in the studied fabrics. The method is widely applied also to the analysis of secretion of antigens. For this purpose pieces of fabrics or the dissociated cells incubate in the small volume of the environment containing is radioactive marked predecessors, and in an immunoautoradiography investigate the concentrated culture medium.
Sensitivity of methods I. at visual registration of strips of precipitation it is equal to 1 — 4 mkg/ml. The method of an indirect immunoautoradiography developed for increase in sensitivity of immunodiffusion of Rowe (D. S. Rowe, 1969). The principle of a method is similar to the principle of «sandwich» used in an indirect immunofluorescence (see. Immunofluorescence ), also consists in identification of invisible precipitated calcium superphosphate in agar gel with the help it is radioactive marked immunoglobulins to the antibodies which are contained in precipitated calcium superphosphate. For this purpose the washed agar plates with reactions process solution of marked 131 I or 125 The I immunoglobulins to antibodies of that species of animals, from to-rogo immune serum to the studied antigen was received. An autoradiography of the reactions processed in such a way And. allows to find individual antigens in concentration of 0,1 — 0,05 m of kg/ml.
The method of an indirect immunoautoradiography is developed as for radial (Rowe, 1969), and for double diffusion on Oukhterlonya (D. A. Elgort, G. I. Abelev, 1971). In addition to high sensitivity, the method has that undoubted advantage that with its help it is possible to check easily participation of antibodies of immune serum in formation of precipitated calcium superphosphate and thus to exclude various artifacts which can distort results of a research.
Methods I. allow to receive well reproducible results and to reveal antigens in complex systems. However use of methods I. it is limited since they can be applied to the analysis only of soluble antigens and antibodies at which interaction precipitated calcium superphosphates are formed.
Bibliography: Immune marker analysis, under the editorship of L. A. Zilber, M., 1968; Methods of developmental biology, under the editorship of B. L. Ayetaurov, page 434, M., 1974, bibliogr.; About u with h-t e of 1 about and O. have Handbook of immunodiffusion and immunoelectropboresis, Ann Arbor, 1968.
V. M. Barabanov.