ANTIGEN — THE ANTIBODY REACTION — formation of a complex between antigen and the antibodies directed to it. Studying Antigen antibody of reaction is of great importance for understanding of the mechanism of specific interaction of biological macromolecules and for clarification of the mechanism of serological tests.
Distinguish two phases of reaction significantly differing among themselves on the mechanism and speed of course. In the first phase of reaction there is a connection of determinant group antigen (see) or hapten (see) with groups in an active center of an antibody (see). This high-specific process proceeds in aqueous solutions with high speed. Antibodies possess at least two (antibodies of an IgG-class) and at most ten (for IgM-An-titel) active centers which are konfiguratsionno complementary to determinant group of antigen. Therefore with polyvalent antigen (i.e. with the antigen containing several determinant groups) there can be a formation of units, difficult on structure, antigen — an antibody to a molecular formula: (At) x (Ag) y , where At — an antibody and Ag — antigen. Units of this cell-bound immune complex lose solubility in isotonic solutions and drop out in a deposit. This second nonspecific phase A. — a.r. proceeds more slowly than the first — specific, and its speed depends on many external factors and first of all on composition of salt in solution of the environment. The nature of the reactions proceeding in the second phase is defined considerably by physical properties of antigen. If low-disperse antigens participate in reaction (cells, particles of the inert carrier with the antigen adsorbed on them), the phenomenon of agglutination is observed (see). Highly disperse antigens (polysaccharides, proteins and their complexes) form precipitated calcium superphosphates (flokkulyat) with antibodies. During the second phase A. — a.r. there is also an accession to a cell-bound immune complex of a complement (see. Complement , Reaction of binding complement) that also serves as the highly sensitive serological test. On the basis of data on the mechanisms which are the cornerstone of the second phase A. — a.r., it is obviously possible to approach an explanation of such important consequences of formation of a cell-bound immune complex in the immunological relation as neutralization of toxin antitoxin (see. Toxin-antitoxin reaction ), activation of system of a complement, early reaction and so forth. At the same time it must be kept in mind that it is difficult to judge a specific stage And. — a.r. on the basis of significantly other on the mechanism of the second phase of reaction. Owing to this research of thin physical and chemical mechanisms A. — a.r. are carried out preferential with use of haptens with one determinant group which are capable to form only soluble complexes with the antibodies directed to them.
In case of bivalent antibodies (e.g., antibodies of the class IgG) their reaction with monovalent hapten (G) can be written down in the form of the equation: to,
where to 1 and to 2 — according to a constant of speeds of direct and return reactions. Owing to the fact that at reaction of hapten to a specific antibody there are no visible transformations it is necessary to judge interaction by means of various physical and physical and chemical methods based on assessment of amount of the connected hapten on change of diffusion balance of the last in the presence of an antibody (dialysis equilibrium method) or on change of optical properties of hapten in a cell-bound immune complex. The size of a kinetic constant of forward reaction received in an experiment reaches 10 6 — 10 7 l • mol - 1 sec. - 1 , while back reaction much more slowly: 1 — 50 sec. - 1 .
Insufficient perfection of the measuring equipment does not allow to estimate the maximum speed of forward reaction; for theoretical reasons it could reach size 1,5•10 9 l • mol - 1 sec. - 1 .
Owing to so big speeds of forward reaction connection of an antibody with hapten comes to the end already in the course of mixing of reagents. Therefore in practice it is significantly simpler to measure a constant of affinity (to and = to 1 /к 2 ), the size a cut for a row of systems calculated with use of a dialysis equilibrium method is in limits 10 5 — 10 9 l/mol.
On the basis of data on size at various temperatures according to the van't Hoff's equation it is possible to calculate thermodynamic parameters of reaction hapten — an antibody.
Changes of free energy (deltaf) at interaction hapten — an antibody have an order of sizes from — 7 to — 12 kcal/mol. It means that even at high cultivations degree of association remains still considerable. Though in some cases essential changes of entropy during the formation of a complex hapten were not noted — an antibody (ΔS = 0), for many systems positive values ΔS, pointing to increase in entropy, but not to its reduction that should be expected owing to bigger orderliness of system at formation of a cell-bound immune complex were received. The observed effect is caused, apparently, by the fact that hapten shields a surface of an active center of an antibody with release of earlier related water molecules. In favor of it data on significant increase in partial specific volume at reaction testify in turn hapten — an antibody, received by means of a dilatometriya (see).
Until recently many questions concerning the mechanism A. — and. rubles, remain open. Between determinant group of antigen and groups in an active center of an antibody it is possible to judge the nature of bonds on the basis of optical properties of the connected hapten.
So, by means of a spektrofotometriya and a spektrofluorimetriya it was established for a number of the haptens (representing aromatic compounds) that at linkng with a specific antibody they appear in an unpolar microenvironment and, therefore, are capable to be fixed in an active center due to hydrophobic interactions. In case of antibodies to nitrophenylic derivatives education in an active center of a complex with transfer of an electron between the rest of tryptophane and gaptenny group was shown. At last, the possibility of education between the amino-acid remains in an active center of an antibody and determinant groups of antigens of hydrogen bindings and dalnodeystvuyushchy electrostatic interactions does not raise doubts. All this, however, does not explain with sufficient clarity the mechanism of a specific phase of reaction antigen — an antibody. Judging by the data obtained by means of physical other methods at the time of binding conformational reorganization of actually active center of an antibody and located out of its sites of a molecule arises an antibody of determinant group of antigen. At the same time the molecule of an antibody becomes steadier against various denaturant influences, as well as to hydrolysis by proteolytic enzymes. Obviously, in the course of binding of determinant group of antigen there is an adaptation to it of an active center of an antibody; this process can be similar to reorganization of an active center of enzyme during the binding of substrate.
Interaction of a molecule of antigen with an antibody or its active Fab-fragment is followed by changes of spatial structure of a molecule of antigen. So, the myoglobin turns in apomioglobin at interaction with the antibodies directed to an apomioglobin, and the inactive, received from the corresponding mutants ß galactosidase turns into active enzyme after reaction with antibodies to the active ß galactosidase form. In experiments with the synthetic polypeptides used as antigens clearly it was shown that antibodies to a α-spiral form of polypeptide are capable to stabilize this structure and, moreover, to provide structural transition of peptides from a form of the disorder ball in a α-spiral. It is necessary to emphasize that the structural transitions observed under the influence of antibodies in a molecule of antigen happen in the course of the first phase of reaction, but not due to the effects arising at aggregation of cell-bound immune complexes. It is undoubted that restructurings in antigen cause the monovalent active Fab-fragments of antibodies incapable to provide aggregation of antigen.
Owing to structural heterogeneity of antibodies the constants of affinity found in an experiment of this hapten to antigen represent the average size of many constants reflecting features of binding of hapten various molecules of antibodies. Distribution of antibodies on constants of affinity is described by Gauss's curve. In view of the fact that as a part of population of molecules of antibodies are always present such which differ in rather low degree of affinity, the number of the molecules of hapten connected on mol of a bivalent antibody reaches size 2 only at noticeable excess of hapten. By precipitation of antibodies to dinitrophenylic group the increasing quantities of a specific antigen succeeded to receive several fractions of the antibodies differing at the same animal on affinity to hapten on four orders (from 1,0•10 5 to 1,1•10 9 ). At reimmunizations size for more late antibodies increases especially considerably in that case when small amounts of antigen are applied to immunization. Obviously, antigen connects in an organism first of all to receptors of predecessors of the antiteloobrazuyushchy cells differing in the greatest affinity to antigen. At immunization by high doses of antigen heterogeneity of antibodies in size increases due to involvement in an immune response of cells with the antitelopodobny receptors which are characterized by low affinity to antigen and having ability to synthesize only antibodies with low degree of affinity.
At assessment of kinetic parameters A. — and. rubles often resort to definition of an equilibrium constant (k), to-ruyu calculate in an experiment on inhibition And. — and. river by means of specific hapten on the basis of the equation:
[Agate] = to [GHATS] [Ag] / 
where each size in brackets means molecular concentration of substance. Size (k) is generally close to size to and . The method of inhibition of a precipitation test found the broadest application for assessment of structure of determinant groups of environmental antigens — proteins and polysaccharides. In this case as haptens use the oligosaccharides and peptides emitted from natural compounds or received in the synthetic way.
Along with studying of the mechanism of the first phase A. — and. the river for many years goes intensive studying of the second phase of reaction and hl. obr. precipitation tests (see). Now does not raise doubts that the phenomenon of precipitation is connected with formation of lattices thanks to the polyvalent nature of antigen and an antibody. The theory of «lattice» which is most accurately formulated for the first time by J. R. Marrack finds confirmation in the numerous facts, the basic of which following: 1) monovalent haptens do not give a precipitation test while in the presence in a molecule of hapten of two and more determinant groups precipitated calcium superphosphate in the presence of an antibody is, as a rule, formed; 2) the monovalent Fab-fragments received as a result of splitting of a molecule of an antibody do not pretsipitirut polyvalent antigen, though connect to it; 3) the hybrid artificially received bivalent F(ab) 2 - fragments of antibodies, each of monovalent fragments of which belongs to molecules of antibodies, various on specificity, pretsipitirut only in the presence of both antigens, to the Crimea they are directed, and 4) bivalent haptens, containing two various determinant groups on structure, are capable to form precipitated calcium superphosphate only in the presence of antibodies to both determinant groups. The direct evidence of formation of lattices during the formation of precipitated calcium superphosphate was obtained by means of a submicroscopy. Schematically these structures are presented in fig. 1.
Existence of at least two valencies at antigen and an antibody does not exhaust the requirements necessary for formation of immune precipitated calcium superphosphates, and also for implementation of other processes proceeding in the second phase of reaction antigen — an antibody. So, though the antibodies belonging to the classes IgA and IgE contain two active centers, they are in essence deprived of ability to participate in precipitation tests and agglutination and to connect a complement in the presence of a specific antigen. Apparently, the main distinctions between precipitant and not precipitant antibodies are covered in ability of the first to form bridges between molecules of antigen or owing to beforehand the long distance set by features of structure between active centers (as it takes place in case of antibodies of an IgM-class), or on condition of sufficient flexibility of a molecule of the antibody providing a possibility of increase in distance between active centers in the presence of antigen. The last is inherent in antibodies of an IgG-class thanks to rotation of the Fab-fragments bearing active centers concerning a Fc-fragment (see. Antibodies ). So, according to data of a submicroscopy in the presence of the low-molecular bivalent hapten interacting with both active centers of an antibody, the corner between Fab-fragments makes 10 °, but can increase to 180 ° at a ratio hapten — an antibody (or antigen — an antibody), providing formation of large units which part 4 and more molecules of antibodies are. The specified changes of conformation of a molecule of an antibody of an IgG-class are presented in fig. 2. After interaction with antigen the molecule of an antibody turns from Y-shaped in sterzhneobrazny with the active centers which are most removed from each other located on the distal ends of Fab-fragments.
Need of conformational reorganization of antibodies of IgG and IgM of classes with the participation of antigen is obvious also to implementation of other processes proceeding in the second phase A. — and. river, and, in particular, for activation of system of a complement. These conformational changes affect the area of a molecule supporting the specialized center for binding of the first component of a complement (see. Complement , Reaction of binding complement ). As a result of restructuring of this center located in a Fc-fragment (fig. 2), apparently, its affinity to the first component of a complement increases that is necessary for transformation of the last into active enzyme — CI esterase. Undoubted proofs of change of tertiary structure of a molecule of an antibody at formation of complexes by them with antigen in an equivalence point (i.e. at a ratio of the reagents providing the maximum precipitation and fixation of the complement) were received by method of optical rotatory dispersion. It is necessary to emphasize that increase in negative left rotation of antibodies was observed as a part of the specified complexes and had no place in the complexes formed in excess supply antigen. It shows that conformational changes in immune units, big by the size, are connected with structural changes of the sites of a molecule of an antibody located out of area of their active centers. Thus, it is necessary to state emergence of two types of conformational changes of a molecule of an antibody after se interactions with antigen. The first of them as it was stated above, arise directly in the field of an active center of an antibody and in its immediate environment during the first phase of reaction regardless of degree of an agregirovannost of a cell-bound immune complex. The second arise directly in the second phase of reaction as a result of formation of the units including not less than two — four molecules of antibodies, and are accompanied by restructuring of areas of a molecule of an antibody, remote from an active center, in particular its Fc-fragment.
The question of dissociation of a complex antigen — an antibody which includes a problem of reversibility of the conformational changes of a molecule of an antibody accompanying process of its interaction with antigen is of considerable interest. It was already noted above that back reaction antigen — an antibody proceeds much more slowly than a straight line. In an equivalence point are involved in structure of a cell-bound immune complex entirely both antigen, and an antibody owing to what almost notable dissociation at the same time is not observed. However dissociation of a cell-bound immune complex in rather soft conditions nevertheless is possible already at least because between partners of reaction there are only not covalent interactions. It is possible to allocate two main ways by means of which can be partially or the antigen which is already created a complex — an antibody is completely divided. The first consists in replacement of antibodies by excess of antigen (hapten), and the second — in impact on a cell-bound immune complex of the external factors leading to bond breaking (reduction of affinity) between antigen and an antibody.
Already during the washing of immune precipitated calcium superphosphate it is possible to mark out with normal saline solution from its structure constantly decreasing quantities of antibodies and only trace amounts of antigen. Process of dissociation can be written down in the form of the equation:
where n and m correspond to number of molecules of Ag and At in a complex and to number of free molecules of At.
Efficiency of dissociation of a cell-bound immune complex significantly increases in presence of big excess of antigen (hapten). In practice for this purpose use the monovalent haptens which are characterized by rather small degree of affinity to antibodies and capable to force out from a complex first of all antibodies with rather low affinity to antigen. Naturally, antibodies are eluated in the form of a complex with hapten. The last owing to rather low affinity to an antibody can be removed or long dialysis, or using ion-exchange chromatography. Allocation of antibodies to dinitrophenylic group with use as hapten of dinitrophenol can be an example of such isolation method of antibodies.
Partial dissociation of a complex antigen — an antibody can be reached generally at temperature increase. That And. — and. the river is exothermic, is quite natural that the majority of complexes antigen — the antibody, created at t ° 0 ° can be partially dissociated at least at increase in t ° to 40 ° above. Extent of thermal dissociation significantly varies depending on the nature of an antibody. Especially effectively dissociate at t ° 37 — 40 ° cell-bound immune complexes, educated so-called. The cold-reactive antibodies directed, as a rule, to antigens of a membrane of erythrocytes.
As the most universal way of dissociation of the cell-bound immune complexes formed by the most various antibodies serves their processing by diluted acids and alkalis, and also strong solutions of amides (urea, muriatic guanidine). Increase in a dissociation constant of a complex antigen — an antibody in these conditions is connected, obviously, with a rupture of the noncovalent bonds and change of conformation of a molecule of an antibody (antigen) which are followed, in particular, by reorganization of an active center of an antibody and determinant group of antigen. Among dissociating a complex antigen — an antibody of agents also polyanions (polymethacrylate and polystyrene sulphonate) and strong solutions of neutral salts were used.
Reversibility of conformational changes of an antibody after its extraction from a cell-bound immune complex needs even further studying. At elution of antibodies acid or alkali (especially the last) naturally note decrease in their precipitant activity though ability to connect antigen remains, apparently, without essential changes. Slightly affinity of antibodies and after their elution by the urea concentrated (8M) changes. Judging by these data, the structure of an active center of the antibody taken from a cell-bound immune complex is returned in general to initial. Irreversible changes, at least at a part of molecules, undergo the structures responsible for ability of antibodies to participate in the second phase A. — and. river. In the light of modern ideas of the mechanism A. — and. the river and the mechanism of a precipitation test it is possible to assume that at the antibodies taken from a cell-bound immune complex by alkali or acid freedom of rotary motions of Fab-fragments of a molecule though it is impossible to exclude also other damages decreases.
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