IMMUNOELECTROPHORESIS

From Big Medical Encyclopedia

IMMUNOELECTROPHORESIS (Latin immunis the free, saved from something + electrophoresis) — a method of electrophoretic division of antigens (or antibodies) in gel with the subsequent their manifestation by means of precipitation the corresponding antibodies (or antigens).

And. allows to obtain various information on separate components of complex mixture of antigens without preliminary allocation of these components in pure form, including to define total number of antigens in mix; their mobility electrophoretic ratio, biochemical, or enzimol, characteristics; quantitative content of each antigen; existence in mix of required antigen; to make comparison of two or more antigenic systems, etc.

Opportunities And. in the characteristic of antibodies are limited since antibodies of various specificity, but belonging to one class of immunoglobulins have identical or very close electrophoretic mobility. With the help And. it is possible to establish to what electrophoretic fraction of immunoglobulins — gamma1 or gamma2 — the studied antibodies belong. For the rest the analysis of antibodies with the help And. has no advantages before their analysis diffusion-in-gel methods (see. Immunodiffusion ).

The most widely applied option I., offered in 1953 by Grabarom and Williams (P. G rabar, S. of Williams), the combination of two separate methods — a method of electrophoretic division of antigens in agar gel and a method of counter two-dimensional immunodiffusion represents. The principle of the first method is based on the unequal electrophoretic mobility of antigens caused by the fact that at the pH value corresponding to experimental conditions, various antigens will differ from each other in the size of a charge of their molecules. Since. The isoelectric point of the majority of antigens lies in the acid area of pH values, the electrophoresis is carried usually out in alkaline buffer systems in which antigens move with various speed to the anode and by the end of an electrophoresis hold positions at different distance from an initial position.

Fig. 1. The scheme of electrophoretic division of proteins of serum in agar gel: blood serum is placed in the hole (a black circle) which is cut out in the center of the agar plate (is limited to a frame) prepared on buffered solution; its components move to the anode (+) or to the cathode (—) depending on the size of a vector of their charge and a vector of an endosmose; at a simple electrophoresis in an agar components of serum are divided into four main fractions (antigens) — albumine (alb.) and α-globulins ((((((((((α-hl.), which move to the anode as have a high negative charge; γ-globulins ((((((((((γ-hl.) and β-globulins ((((((((((β-hl.), which move to the cathode as the positive vector of an endosmose exceeds a vector of a charge.

However during the use for an electrophoresis of agar gel the so-called electroendosmose exerts strong impact on mobility of antigens in electric field. It is caused by the fact that the agar in alkaline conditions has a negative charge owing to what near walls of capillaries of an agar sponge the positive ions of the buffer occupying by that bigger volume in capillaries accumulate than diameter of the last and, therefore, than more concentration of agar gel is less. In electric field these ions move to the cathode, entraining a liquid phase and molecules of antigens which are in it. Thus, in agar gel mobility of macromolecules decides, on the one hand, by the vector of a charge moving molecules of antigen to the anode, and on another — the vector of an electroendosmose sent to the opposite side to the cathode. The resultant of these two vectors will determine the speed and the direction of the movement of antigens in an agar. So, e.g., at an electrophoresis of serum proteins in an agar (ionic strength of the buffer 0,025, pH 8,6) the albumine and alpha globulins having a high negative charge move to the anode whereas beta and gamma-globulins, despite a negative charge of their molecules, move to the cathode since the vector of an electroendosmose exceeds a vector of a charge (fig. 1).

Fig. 2. Scheme and example of an immunoelectrophoresis: 1 — the scheme of formation of arches of precipitated calcium superphosphate at cross diffusion in an agar of antibodies and electrophoretically the divided antigens; the divided antigens (are designated by a dotted line) diffuse in an agar (the direction of diffusion is specified by shooters); a black circle — a hole with blood serum; towards to them diffuse antibodies of immune serum (the direction of diffusion is specified by shooters). poured in a trench after an electrophoresis of antigens; in meeting places of antigens and antibodies precipitated calcium superphosphate (the deposit which is dropping out in an agar as a result of compound of antigen with an antibody) in the form of arches is formed; 2 — an example of an immunoelektroforegramma (a white circle — a hole, shooters specified arches of precipitated calcium superphosphate, a white double strip — a trench).

The method of counter immunodiffusion applied at the second stage I., it is based on formation in transparent gel of the visible precipitated calcium superphosphate formed in the course of cross diffusion of molecules of antigen and the molecules of antibodies added after an electrophoresis. For implementation of cross diffusion in an agar plate cut out a trench, edges is located parallel to the direction of the movement of antigens and on nek-rum distance (fig. 2) from them. Bring immune serum to the studied antigens then the plate is left in a moist chamber for days or more in a trench. During this time antibodies of serum diffuse from a trench, and antigens — from places which they took after an electrophoresis. In meeting places precipitated calcium superphosphate in the form of an arch is formed, the form and situation a cut will depend on a quantitative ratio of this antigen and the corresponding antibodies and on its nature. Thus, high resolving power And., distinguishing it from all other Immunochemical methods, the analysis, the antigen/antibody for all couples of studied system is explained not only by various electrophoretic mobility and a diffusion constant of antigens, but also the unequal relation. Sensitivity of the described option I. quite high also allows to define antigen at its concentration about 10 mkg/ml.

Fig. 3. The scheme of an immunoosmoforez with monospecific serum, antibody-containing to required antigen (antigen with anode mobility): the formed precipitated calcium superphosphate (in the center of the drawing) is caused by the oncoming electrophoretic traffic of antigens to the anode, and antibodies to the cathode; the method allows to receive prompt reply about availability of required antigen in a large number of the studied tests.

In laboratory practice other option I is often used., which in literature is often called immunoosmoforezy or a counter elecrophoresis. Resolving power of this method is much lower, and the main objective, about a cut it is applied, consists in receiving prompt reply about availability of required antigen in a large number of the studied tests by means of monospecific serum, antibody-containing to this antigen. When required antigen at an electrophoresis in an agar moves to the anode, all statement of an immunoosmoforez is carried out on an agar plate (fig. 3). By means of a stamp cut out two rows of holes (on number of the studied tests) to dia. 2 — 3 mm located against each other at distance of 3 — 10 mm. A number of holes from the anode fill with immune serum, and holes from the cathode — the studied tests. Owing to a weak charge of a molecule of antibodies at the expense of an electroendosmose will move to the cathode towards to the required antigen having anode mobility. In the meeting place the strip of precipitated calcium superphosphate indicating presence of antigen at the studied test is formed. The analysis of hundred and more tests of various material on presence of required antigen can be carried out by means of an immunoosmoforez less than in 1 hour.

Fig. 4. The scheme and an example of an elektroforegramma at a roket-immunoelectrophoresis (the area of peak at constant concentration of antibodies of test serum is proportional to concentration of required antigen): 1 — the scheme of an elektroforegramma, is limited to a frame the Wednesday consisting of mix of agarose with a test antiserum; black circles — holes with the tests containing required antigen in the concentration increasing (from left to right); from holes the corresponding peaks of precipitated calcium superphosphate rise; 2 — an example of a roket-immunoelektroforegramma (black circles — holes with tests from which peaks of precipitated calcium superphosphate rise).

Options I are developed., allowing to receive the quantitative characteristic for each component of the studied system. The simplest of them — a roket-immunoelectrophoresis (English rocket the rocket) and cross-иммуноэлeктрофоpeз. The principle of these methods is that the electrophoretic movement of antigens happens in the gel of agarose mixed with immune serum. Roket-immunoelektroforez allows to investigate at the same time large number of tests and gives information not only on availability of required antigen in test, but also on its quantity that gives to a method great value for a wedge, researches. For its statement on glass pour mix of agarose with immune serum (better than monospecific) in previously picked up ratio and then in gel from the cathode cut out a number of the holes filled by the studied tests. Under the conditions of an electrophoresis allowing antigen to move slowly in agarose gel with immune serum precipitated calcium superphosphate in the form of the rocket (fig. 4) forms. The area of peak at constant concentration of antibodies is proportional to amount of antigen in test. In the presence of referens-antigen the standard curve can be constructed, on a cut it is easy to determine amount of required antigen in each test by height of the created peak of precipitated calcium superphosphate.

Fig. 5. The scheme and example of an elektroforegramma at a cross-immunoelectrophoresis: 1 — the scheme: the plate of agarose with electrophoretically the divided antigens is connected to a plate from the agarose containing an antiserum; as a result of reaction of immunoprecipitation at the second stage of an electrophoresis the corresponding peaks of precipitated calcium superphosphates giving the chance to define amount of any antigen which is a part of the studied test turn out; a black circle — a hole with the studied substance; 2 — an example of the created precipitated calcium superphosphates at a cross-immunoelectrophoresis.

The cross-immunoelectrophoresis differs from roket - an immunoelectrophorus - behind the fact that allows to carry out the accounting of the same characteristics at the same time on several components of the studied test if it represents complex antigenic mixture. The studied test is subjected at first to a simple electrophoresis in agarose gel without serum. After that the strip of gel containing the antigens divided by an electrophoresis is cut out and placed on the edge of wider glass plate, filling the remained area of this plate with mix of agarose with an antiserum. Glass with gel is placed in an electrophoresis apparatus so that the direction of current was perpendicular to the direction of the first electrophoresis, and the antigens concluded in agarose were located from the cathode. Antigens, moving in agarose with an antiserum, create precipitated calcium superphosphate also in the form of peak, but with wider basis at the expense of the bigger space occupied by each antigen in agarose after the first simple electrophoresis (fig. 5). Since various components of the studied test, having differential electrophoretic mobility, took unequal positions of rather initial point after the first electrophoresis, and peaks of precipitated calcium superphosphate formed afterwards never completely are imposed at each other and, therefore, can be accurately differentiated. It is promoted by also various height of peaks caused by unequal concentration of the antigens which are a part of test. In comparison with other methods of immunochemistry the cross-immunoelectrophoresis has the greatest resolving power. The quantitative characteristic of each antigen is also determined by the standard curve constructed on referens-antigen, however definition goes not on height of peak, and on the space occupied by it. Sensitivity of both options considered here quantitative And. for the majority of antigens makes 0,5 — 1 mkg/ml. When the roket-immunoelectrophoresis or a cross-immunoelectrophoresis are used for the analysis of antigens with mobility of gamma-globulins or for studying of immunoglobulins, apply the preliminary methods of processing of these macromolecules allowing to change their isoelectric point by increase in a charge of molecules. The reaction of a karbamilirovaniye reducing an isoelectric point of immunoglobulins to pH 5,0 is widely applied to this purpose.

The main types I sketched here. do not exhaust all variety of the options of this method which are found in literature. Possibilities of these options at their correct use are extraordinary wide and allow to obtain rich information on the studied antigens on material, insignificant by quantity. Those options I are more and more improved., where the isotope tag is used. These new methods allow to carry out the analysis of material of several microlitres and to quantitatively define required antigen even if its contents in test makes 50 — 100 pikogramm.


Bibliography: Grabar P. and Burten Item. The immunoelectrophoretic analysis, the lane with fr., M., 1963; Immune marker analysis, under the editorship of L. A. Zilber, M., 1968; The Guide to a quantitative immunoelectrophoresis (methods and use), under the editorship of N. Akselsen, etc., the lane with English, M., 1977, bibliogr.; L a lire 11 S. V. of Electroimmuno assay, Scand. J. clin. Lab. Invest., Suppl. 29, v. 124, p. 21, 1972, bibliogr.

H. A. Dorfman, G. I. Abelev.

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