PRECIPITATION (Latin. praecipitatio rapid falling) — immunological precipitation reaction from solution of a complex antigen — an antibody, the soluble antigen (precipitinogen) which is formed as a result of connection with specific antibodies (pretsipitina).
P.'s reaction is widely used for identification and quantitative definition of the most various antigens and antibodies (see. Immunodiagnosis ), at a serodiagnosis inf. diseases (see. Serological researches ), for detection of impurity in foodstuff, during the studying of evolutionary interrelations in an animal and flora, at a research of structure various biol, connections, in forensic medicine for definition of specific accessory of spots of blood and others biol, liquids.
For the item it is open in 1897 t. R. Kraus observing breaking (precipitated calcium superphosphate) during the mixing of acellular transparent filtrates of bouillon cultures of bacteria of plague, cholera, typhus with homologous immune serums. In 1899 F. Ya. Chistovich, immunizing rabbits serum of an eel, received precipitant antibodies and by that for the first time showed specific specificity of serum proteins. P.'s use in court. - it was offered to medical examination for definition of specific accessory of blood in 1901 P. Ulengutom. Reaction received the name of reaction of Chistovich — Ulenguta. Afterwards it was shown that precipitant antibodies (see) are formed at representatives of different types of vertebrata to any alien high-molecular substances (see. Antigens ). Precipitant antibodies belong to immunoglobulins of the classes G and M (see. Immunoglobulins ). Speed and intensity of biosynthesis of precipitant antibodies are defined by a number of factors: dose and in the way of administration of antigen, scheme of immunization, features of chemical structure of antigen and genetic features of an immuniziruyemy organism.
For receiving precipitant serums use various schemes of immunization. Good results are yielded by schemes from several cycles of immunization, each of which includes several intravenous or intramuscular injections of antigen in the increasing quantities. In 1915 M. I. Raysky offered the scheme consisting of primary immunization and the remote booster shot. Receiving precipitant serums of a high caption is based on this principle. Primary immunization can be carried out by antigen to mixes with any depositing substance (lanolin, mineral oil, aluminum potassium alum, etc.) strengthening an immune response, and the remote booster shot — only antigen. Widely apply as the depositing substance adjuvant (amplifier) of Freynd consisting of mix of mineral oils and the killed mycobacteria of tuberculosis (see. Adjuvants ).
The solution of antigen emulsified in the equal volume of a Freund's adjuvant is entered an experimental animal subcutaneously or intramusculary into several points of a back or into small pillows of back pads or into subnodal limf. nodes of back extremities. In some schemes use combinations of the listed ways of introduction. In a month an animal enter solution of antigen intravenously or intramusculary. If necessary before a booster shot carry out desensitization across Bezredka (see. Bezredki methods ). At an insignificant consumption of antigen (1 — 3 mg for proteinaceous antigens on a course of immunization) the quantity of the formed antibodies reaches several milligrams in 1 ml of immune serum.
High specificity is characteristic of a precipitation test. In a series of works of K. Landshteyner with antiserums to the conjugated antigens as which determinant groups various organic radicals acted it was shown that in P.'s reaction it is possible to differentiate stereoisomers of organic compounds. Force of the observed cross-reactions is defined by proximity of chemical structure of determinant groups of immunoantigens and test antigens. Antigens and antibodies, specific to them, are a part of precipitated calcium superphosphate and practically other serum proteins, except a complement do not join.
The item — highly sensitive reaction. With its help the tenth shares of microgram of antigen can be found. During the definition of antibodies limit of sensibility of reaction makes apprx. 20 mkg of protein. Sensitivity of reaction considerably increases if apply antigens or antibodies, marked radioisotopes (see).
Statement of reaction
At statement of a precipitation test it is necessary to consider its zone character which is expressed that the molecular structure and amount of the formed precipitated calcium superphosphate are defined by a ratio entered into reaction of antigen and antibodies (see. Antigen — an antibody reaction ). During the use of constant quantity of an antiserum and the increasing amounts of antigen the amount of precipitated calcium superphosphate among test tubes increases in the beginning, reaches a maximum, and then decreases up to total disappearance. In nadosadochny liquid of the first test tubes find free antibodies (a zone of surplus of antibodies), liquid over the maximum precipitated calcium superphosphate contains neither free antibodies, nor free antigen (equivalence point), in nadosadochny liquid of the last test tubes find soluble cell-bound immune complexes and free antigen (a zone of excess of antigen). Formation of soluble cell-bound immune complexes with a small molecular weight in a zone of excess of antigen is characteristic of all precipitant systems in which antibodies belong to IgG. This zone of reaction is called therefore a zone of a delay, or a post-zone. It should be noted that cell-bound immune complexes of antigens with IgM-antibodies are insoluble in very big excess of antigen in tens of times exceeding its quantity sufficient for formation of soluble cell-bound immune complexes with IgG-antibodies.
Formation of soluble cell-bound immune complexes and in a zone of surplus of antibodies, i.e. formation of a pro-zone is characteristic of horse antiproteinaceous serums (see. Neyssera-Veksberg phenomenon ). This feature of reaction G. Ramone in system for the first time found diphtheritic toxin — anti-toxic horse serum (see. Flocculation ). Dissolution of cell-bound immune complexes in a zone of surplus of antibodies was observed afterwards at P.'s carrying out with rabbit and dog blood sera against a bull seralbumin, with human blood serum against thyreoglobulin, a sheep antiserum against synthetic polypeptides.
The molecular composition of precipitated calcium superphosphate is defined also a pier. it is powerful (mass) of antigen. For ovalbumin, a pier. weight to-rogo 42 000 distance tone, in an equivalence point is the share of one molecule of antigen on average 2,5 molecules of antibodies. With increase a pier. the weight of antigen the number of molecules of the antibodies connected by one molecule of antigen increases.
Items use for qualitative and quantitative test of antigens and antibodies. The Bystry, simple and sensitive qualitative method P. — the interfacia offered in 1902 Mr. of Askoli. The interfacia is applied to identification of soluble antigens of microorganisms. Reaction is carried out in narrow test tubes or capillaries, carefully layering solution of antigen on immune serum. At positive reaction on border of two liquids the ring of precipitation appears. The result of reaction is not influenced by excess of antigen thanks to gradual diffusion of reagents to border of liquids. If as antigens use the prokipyachenny and filtered water extracts of bodies or fabrics, then reaction carries the name «thermoprecipitation» (see. Askoli reaction ). By means of thermoprecipitation find thermostable bacterial antigens (coctoantigens) in fabrics and bodies of the dead of animals at diagnosis of plague, cholera, a malignant anthrax. The interfacia and thermoprecipitation are carried out with antiserums of a high caption.
Methods of assessment of force of serums and amount of antigens on their limit cultivation giving still visible P. with standard antigen or an antiserum and methods of optimum proportions can be carried to semi-quantitative methods P.
At titration of serums it is necessary to select such amount of antigen for limit cultivation not to get to a zone of a delay. Therefore previously define the smallest cultivation of test antigen, at Krom there is a reaction to obviously positive serum. This working cultivation (dose) of antigen is used for definition of limit cultivation (caption) of examinees of serums. Comparative titration of antigen by method of limit cultivations can be carried out without preliminary selection of a working dose of serum if it contains antibodies of the precipitant, but not flocculating type.
The method of optimum proportions is based on definition of a point of equivalence serol. systems on initial And. and on that observation that a point of equivalence in each serol. to system arises at a certain relation of an antibody to antigen. Therefore at titration of serums, having determined the amount of standard antigen corresponding to a point of equivalence by P.'s speed it is possible to express its activity in any conditional biol. units if in preliminary titration with serum of the known force it is established to how many its units standard antigen is equivalent. Similar calculations carry out at titration of antigen with standard serum. The method of optimum proportions can be executed in the a-option offered by Dean and Webb (H. Dean, R. Webb, 1928) — with a constant volume of serum and the increasing cultivations of antigen and in ß option offered G. Ramonom (1922) — with a constant volume of antigen and the increasing cultivations of serum.
The quantitative method of definition of antibodies in weight units offered in 1933 to Geydelbergerom (M. Heidelberger) and Kendall (F. E. Kendall), is founded that in an equivalence point practically all antigen and all antibodies drop out of solution in a deposit. Having determined by any chemical method amount of protein of precipitated calcium superphosphate in this point and having subtracted from it amount of the antigen added in test, calculate the amount of protein in draft falling to the share of antibodies.
At P.'s statement any of the described methods should work with well ottsentrifugirovanny solutions of antigens and serums. Reaction shall be followed by control: immune serum + isotonic solution of sodium chloride, normal serum + antigen, heterologous serum + antigen. It is necessary to prevent a possibility of bacterial pollution, carrying out P. in sterile conditions or applying preservatives like mertiolat, sodium amide. Reaction is carried out at fiziol. concentration of salt (0,15 M solution of sodium chloride), in the range of pH of 6,5 — 8,0.
Definition of the individual antigens which are in mix with other substances is possible in P.'s reaction only during the use of monospecific serums. Specific antibodies in serums can be identified if P. carry out with individual antigens. For the analysis, characteristics and comparisons of multi-component systems antigen — an antibody without their preliminary fractionation use the methods based on P.'s carrying out in gel, in particular a method of double immunodiffusion on Oukhterlonya (see. Immunodiffusion ).
The item — two-phase reaction. Phases of reaction differ on the mechanism and speed of course (see. Antigen antibody reaction ). It is necessary to consider that a number of nonspecific factors exerts impact on the second phase of reaction — actually formation of precipitated calcium superphosphate —: concentration in solution of salts and hydrogen ions, temperature, volume of reagents. At increase in salt content is higher fiziol, values (0,15 M) the amount of the formed precipitated calcium superphosphate decreases. In 15% solution of sodium chloride the precipitated calcium superphosphates formed by polisakharidny antigens dissociate. Change of concentration of hydrogen ions in fiziol. limits of pH (from 6,5 to 8,0) considerably does not influence formation of precipitated calcium superphosphate. At decrease in pH of solution to 5,0 or increase to 9,0 the amount of the formed precipitated calcium superphosphate significantly decreases, and at pH lower than 3,0 and higher than 11,0 earlier formed precipitated calcium superphosphates dissociate. Methods of allocation of pure antibodies and antigens from specific precipitated calcium superphosphates are based on property of precipitated calcium superphosphates to dissociate in strong salt solutions and at extreme pH values. The most used dissociating agents — the concentrated solutions of neutral salts diluted to - you and alkalis, the concentrated solutions of amides, polyanions.
Precipitation in the medicolegal relation
In forensic medicine of P. apply to differentiation of blood of the person and animals (see. Blood ). The greatest distribution was gained by an interfacia, but it is not suitable for a research of muddy solutions of antigen and is subject to nonspecific influences of pollution of an object of examination. These shortcomings P. in agar gel is deprived, however she demands long terms of observation and is less sensitive. Implement in practice electroprecipitation, or the passer immunoelectrophoresis (see), P. combining advantages in an agar with high sensitivity and speed of carrying out reaction. All options P. carry out with immune serums (see), pretsipit ruyushchy proteins of the person, dog, horse, etc. They shall be active and specific, i.e. cause P. of homologous antigen (e.g., the corresponding normal blood serum of the person of a pla of an animal) and not to form precipitated calcium superphosphate with heterological (alien) antigens.
From the studied spots of blood prepare extracts and part them to necessary concentration of protein. For P. in an agar it is possible to take cuttings (extract) from spots and to carry out reaction with several precipitant serums. In parallel test control sites of a subject — the carrier of spots which shall not cause the Item. At a positive take with a spot of blood and precipitant serum draw a conclusion about specific accessory of blood, e.g. blood of the person, a dog, etc. At the same time it is impossible to establish precisely an origin of blood if it belongs to closely related animals (e.g., blood of a dog or wolf). The negative take in the presence in an extract of protein testifies to accessory of blood to an animal, protein to-rogo does not come to light by means of a usual set of precipitant serums. If in an extract protein is not established, then take only a positive take since lack of precipitated calcium superphosphate can be explained with insufficient amount of protein in an extract into account.
Bibliography: Boyd U. Fundamentals of immunology, the lane with English, page 314, M., 1969; To the z-boat E. and Meyer M. Experimental immunochemistry, the lane with English, page 8, etc., M., 1968; Paradise y M. Bystroye receiving strong pretsipitin, Harkovsk. medical zhurn., t. 20, No. 8, page 135, 1915; it, Reimmunization as method of receiving precipitant serums, in the same place, page 142; it As in blood of an immunizirovanny animal strong pretsipitina, in the same place, No. 9, page 161 long remain; it As it is necessary to immunize that the animal is steady and long kept in blood strong pretsipitina, in the same place, of page 169; Tumanov A. K. Bases of forensic medical examination material доказательств^ page 57, M., 1975; H and r N y y V. I. Establishment of specific specificity of blood proteins, M., 1976; Chistovich F. Ya. Changes of properties of blood at injection of foreign serum and blood, in connection with the theory of immunity of Ehrlich'a, Russian arkh. patol., wedge, medical and bakt., t. 8, century 1, page 21, 1899; With and-r of enter Ph. L. Immunology and serology, Philadelphia, 1975; Methods in immunology and immunochemistry, ed. by C. A. Williams a. M. W. Chase, v. 3, N. Y. — L., 1971.
I. A. Tarkhanova; V. I. Charny (court.).