REACTION OF BINDING COMPLEMENT (PCK; synonym: reaction of consumption, reaction of fixing, complement deviation test, alexin fixation test, activation of system of a complement, Borde's reaction — Zhangu) — the immunological reaction of definition of antibodies on the known antigen or antigens on the known antibody based on ability of the antibodies which are a part of cell-bound immune complexes to connect a complement. RSK is for the first time described in 1901 by Zh. Borde and Zhangu (O. Gengou).
RSK found preferential application at a serodiagnosis of bacterial and viral infections, and also in court. - medical examination. The RSK quantitative methods apply in scientific research to studying of reaction antigen — an antibody (see. Antigen — an antibody reaction ).
According to modern representations complement (see) — the multi-component biological system which is in blood sera in an inactive state. Antibodies of IgG and IgM (see. Antibodies ), being included a cell-bound immune complex, gain ability to connect Clq — a subcomponent of the first component of a complement that causes the consecutive activation of other components of a complement which is coming to the end with formation of lytic unit (C5 — C9). Effects of fixation of the complement, and on modern terminology of activation of system of a complement a cell-bound immune complex, are defined by the nature of the antigen entering it. So, lytic unit of a complement causes a lysis of erythrocytes — hemolysis (see) and a lysis of nek-ry gram-negative bacteria — bacteriolysis (see), sensibilized homologous antibodies. According to Carpenter (Ph. Carpenter, 1975), on the contrary, fixation of the complement cell-bound immune complexes, antigen in to-rykh is presented by soluble compounds, and also gram-positive bacteria, does not lead to visible effects. However as a result of activation components of a complement undergo irreversible changes and lose initial properties, in particular ability to lyse sensibilized erythrocytes. RSK is based on these patterns of complement activation.
RSK — two-phase reaction. In the first phase of reaction required antigen (or an antibody) is mixed with diagnostic blood serum (or antigen diagnosticum) in the presence of a complement and mix maintain a nek-swarm time for completion of reaction. In the second (indicator) phase of reaction mix incubate with the hemolitic system consisting of erythrocytes of a ram, sensibilized anti-erythrocyte (hemolitic) serum. If in the first phase of reaction the cell-bound immune complex is formed, i.e. required antigen or an antibody are present at mix, then there is fixation of the complement and sensibilized erythrocytes added to mix in the second phase of reaction do not lyse. If the cell-bound immune complex was not formed, then the complement which remained free causes a lysis of sensibilized erythrocytes. Thus, hemolysis in the second phase of reaction testifies to absence required antigen or an antibody.
RSK has advantages before other serological tests. With its help it is possible to register formation of a cell-bound immune complex when visible manifestations of reaction antigen — an antibody, such as precipitation (see), agglutination (see) or flocculation (see), no. In RSK can be used both soluble, and insoluble antigens (see). Thanks to the high sensitivity, by 100 — 200 times exceeding sensitivity of a precipitation test in RSK analyze systems with the low content of antigens and antibodies.
Five components participate in RSK: antigen (unknown or diagnostic), serum (ispytuyemy or diagnostic), complement, erythrocytes, hemolitic serum. The volume, concentration and a ratio of these components have significant effect on fixation of the complement in the first phase RSK and on its hemolitic activity in the second phase of reaction. Functional activity of a complement is defined environmental factors also nearby: temperature, concentration of hydrogen ions in solution, contents in it one - and bivalent cations and anions. Optimal conditions for carrying out RSK are established: the isotonic solution of sodium chloride brought to pH 7,4 by veronal or three-etilenaminom, with ionic strength 0,147, containing 0,15 mmol of calcium chloride and 0,5 mmol of magnesium chloride; it is shown that fullestly the complement is fixed by cell-bound immune complexes at t ° apprx. 4 ° within 18 — 20 hour. This reception is widely used in scientific research and at a serodiagnosis of bacterial and viral infections.
There are numerous modifications of RSK. The main methodical requirement during the use of each of techniques is exact observance of the chosen reaction conditions, standardization of components of reaction in the offered conventional units and observance of certain quantitative relations between components of reaction.
Components of reaction of binding complement and principles of their standardization
Usually use erythrocytes of a ram. For their receiving blood of a ram is taken sterilely in the equal volume of the modified Olsver's solution or preservative of blood 7B. In day of experience the portion of erythrocytes which is selected sterilely from preservative and washed by the buffer is used for preparation of standard suspension. Kabat and Meyer (E. A. Kabat, M. M. Mauyeg, 1968) suggest to use as standard the suspension containing in 1 ml 1*10 9 erythrocytes. The optical density (OD) of the lysate prepared by way of cultivation to 15 times of standard suspension of 0,1% Na solution 2 CO 3 , defined on the spectrophotometer at the wavelength of 541 nanometer in a ditch with the optical course of 1 cm, it is equal 0,700. For bringing the suspension of erythrocytes which is in advance prepared for 5% to standard concentration carry out calculation for a formula:
V2 = V1(ОD/0,7)
where OD — the optical density of a lysate of the suspension prepared for 5%, V1 — the volume of the suspension prepared for 5%, V2 — volume, shall be brought to Krom by 5% suspension for obtaining standard concentration of erythrocytes.
Hemolitic serum (hemolysin, the amboceptor) — blood serum of rabbits, immunizirovanny suspension of erythrocytes or a stroma of erythrocytes of a ram. Before the use hemolitic serum for an inactivation of the complement which is contained in it shall be heated-up at f 56 ° within 30 min. Take its greatest cultivation giving full hemolysis of a certain dose of standard suspension of erythrocytes in the presence of constant quantity of a complement in reference conditions of experience for a caption of serum.
The hemolitic system is prepared, flowing to standard suspension of erythrocytes the equal volume of hemolitic serum in the cultivation exceeding its caption by three-four times. The hemolitic system is used in day of experience.
Complement. Usually blood serum of Guinea pigs is a source of a complement. For prevention of an inactivation of a complement freshly cooked blood serum should be lyophilized or stored at t ° below — 50 °. Activity of a complement remains within several weeks at t ° 3 — 5 ° in the presence of boric to - you and sodium sulfate.
In RSK use the dosed quantity of a complement, a cut it is accepted to measure in conditional 100% or 50% hemolitic units (SN 100 , CH 50 ), equal to the quantity of a complement causing respectively 100% or 50% a lysis of a certain portion of standard hemolitic system in reference conditions of experience. Dependence of hemolysis on quantity of the complement entered into reaction graphically is expressed to a S-shaped curve. At the same time 100% hemolysis are in a zone where sensibilized erythrocytes are insufficiently sensitive to addition of new portions of a complement; 50% hemolysis correspond to a middle part of a curve where extent of lysis of sensibilized erythrocytes sharply changes at change of quantity of the complement used in reaction. On the basis of these data for quantitative expression of hemolitic activity of a complement it is reasonable to use SN 50 . Activity of a complement in SN 50 it can be calculated but one experimental point in a zone of partial hemolysis by means of the table made with use of a formula of Kroga. The formula of Kroga reflects dependence between quantity of the complement (X) fixed in experience and percent of hemolysis (at):
X = (y / [1-y]) 0,2
Percent of hemolysis determines by the scale of hemolysis prepared from a lysate of standard suspension of erythrocytes lizirovanny by triple water volume.
Antigens. The antigens used in RSK shall be checked for lack of an anti-complementarity and haemo toxicity, i.e. shall not connect a complement for lack of antibodies and lyse erythrocytes.
Antiserums before the use shall be heated-up at t 56 ° within 30 min. for an inactivation of the complement which is contained in them and are occluded by erythrocytes for removal of natural hemolysins.
Methods of definition of antigens and antibodies
Methods of definition of antigens and antibodies can be conditionally divided into two basic groups — serological and quantitative. Serological methods of RSK use for diagnosis of various infectious diseases. For diagnosis in the studied blood sera it is possible to define antigen by diagnostic serum. However is more often in blood serum of patients or convalescents define specific antibodies, using antigens diagnosticums (see. Diagnosticums ).
At statement of RSK for the purpose of standardization of reaction conditions use so-called working doses of a complement and antigen, to-rye select just before statement of the main experience. As a working dose of a complement use usually 1,25 — 1,5 SN 100 or 3 — 5 SN 50 complement. As a working dose of antigen in nek-ry techniques use its maximum quantity, a cut the anti-computer-lementarnostyyu does not possess. A number of researchers revealed zone character of RSK and established that the caption of an antiserum depends on the amount of antigen taken in reaction. Its surplus, as well as a shortcoming, leads to understating of a caption of an antiserum. Therefore for each serological system it is necessary to define an optimum dose (or the range of doses) antigen, at a cut reaction with the studied serum reveals its maximum caption. Usually it is selected, titrating cultivations of antigen with constant amount of obviously positive serum and a working dose of a complement. The maximum cultivation of antigen connecting a complement is called antigenic unit. As a working dose at titration of serums use 2 — 4 antigenic units.
At statement of the main experience equal volumes of the increasing cultivations of titrable blood serum mix with working doses of antigen and a complement. Along with pilot tests put control of antigen and serum on an antikomilementarnost and control of a complement. Experienced and checks incubate at t ° 37 ° within 60 min. or at t ° 4 ° — 18 — 20 hours then they to all tests flow identical quantity of hemolitic system and test again incubate at t ° 37 ° within 60 min. In the presence of specific antibodies in the studied blood serum in pilot tests in comparison with control the delay of hemolysis is observed. Its greatest cultivation is accepted to a caption of the studied blood serum, at Krom the delay of hemolysis is observed. During the definition in RSK of antigen a series of its cultivations is mixed with a diagnostic antiserum (not divorced or divorced in insignificant degree) and a working dose of a complement. The working dose of a diagnostic syvorokhka is previously not selected since it is shown that the caption of antigen is not defined by the used cultivation of diagnostic serum.
Introduction of SN 50 complement allowed to develop the RSK quantitative methods which on accuracy are not inferior to a method of quantitative precipitation. The reception of back titration of a complement offered independently by Meyer et al. in 1948 and A. Konikov in 1953 is the cornerstone of quantitative modification of RSK. Its essence is that at statement of RSK experienced and checks bring identical obviously excess quantity of a complement in all. After end of a phase of fixation of the complement (60 min. at t ° 37 ° pl of 18 — 20 hours at t ° 4 °) in experienced and checks titrate the complement which remained untied, expressing its quantity in SN 50 . The quantity of the complement which is specifically connected by serological system is determined by a difference between a free complement in control and pilot tests. Reception of back titration allows to work with anticomplementary drugs of antigens and immune serums provided that their anti-computer-lementarnost it will be blocked by surplus of the complement entered into reaction. At constant quantity of antibodies the dependence of fixation of the complement on amount of the antigen entered into reaction expressed graphically reminds a curve of formation of specific precipitated calcium superphosphate. The maximum of fixation of the complement, as well as a maximum of formation of specific precipitated calcium superphosphate, lies in the area of the optimum relations of an antibody and antigen. In the field of excess of antigen the delay of fixation of the complement is observed. The relation of antibodies to antigen in a point of the maximum fixation of the complement — a constant for each serological system. On the basis of this pattern of I. A. Tarkhanova, And. P. Konikov and V. V. Akimova (1957) offered a quantitative method of titration of antigens in RSK on a point of the maximum fixation of the complement, similar to methods of optimum proportions for titration of antigens and antibodies in precipitation tests and flocculation.
In the field of big surplus of antibodies direct dependence between quantity of the complement connected by system and amount of the antigen participating in reaction is found that was used by a number of researchers for quantitative definition of nek-ry antigens in RSK.
Uodsvort and Molteyner (E. A. Wadsworth, F. Maltaner), etc. in 1938 and Rice (S. E. Rice) in 1942 reported about detection of linear relation between quantity of the connected complement and the maintenance of antibodies in titrable serum in the presence of optimum amount of antigen. On this basis it was offered to express a caption of antiserums the relation of quantity of a complement necessary for 50% of hemolysis in the presence of immune serum and optimum amount of antigen, to quantity of a complement, a cut it is necessary for 50% of hemolysis in the presence of only immune serum. However in later works clear heterogeneity of antibodies of the same specificity concerning ability to connect a complement therefore the quantity of the complement connected by immune serum cannot serve as rather exact measure of maintenance of antibodies in it was revealed.
One of modifications of RSK is indirect RSK, or the reaction of a delay of fixation of the complement offered in 1948 by Rice for detection of antibodies in blood sera of birds and nek-ry mammals. Blood sera of these animals after warming up do not connect a complement in usual RSK though, according to other serological tests, contain the antibodies connecting with Goma logical antigen. The principle of indirect RSK consists in competitive binding of antigen the antibodies which do not have complement-linked activity that interferes with its subsequent binding by fixators of the same specificity, but from other look. Indirect RSK is applied in veterinary science to definition of an ornithosis at birds and a foot-and-mouth disease at cattle.
Other modification of RSK are the RSK active methods, in to-rykh use the complement and natural hemolysins which are contained in the studied blood sera. Advantage of active methods before usual RSK — titration of not heated-up immune serums. Active methods are intended for definition of antibodies, credits to-rykh are so low that they are not defined after a temperature inactivation of a complement in the studied serums. For the first time such method was described in 1908 by N. A. Chernogubov. It is possible to rank as active methods also reaction of consumption of a complement, in a cut use a complement of titrable serum, but in an indicator phase use usual hemolitic system. Active methods apply at diagnosis of syphilis, definition auto-and gomoantitet.
Bibliography: Immune marker analysis, under the editorship of L. A. Zilber, page 79, M., 1968, bibliogr.; Kcal and N and N V. S. and Ginzburg S. I. Modification of reaction of binding complement and its use, M., 1946; Konikov A. P. Modification of reaction of binding complement for quantitative definition of antigens and antibodies, Zhurn. mikr., epid. and immun., No. 1, page 57, 1953; To e about t E. and Meyer M. Experimental immunochemistry, the lane with English, M., 1968; Reznikova L. S. A complement and its value in immunological reactions, M., 1967, bibliogr.; The guide to immunology, under the editorship of O. E. Vyazov and Sh. of X. Hod-zhayeva, page 316, M., 1973; Bordet J. e. Gengou O. Sur I'existence de substances sensibilisatrices dans la plupart de serums antimicrobiens, Ann. Inst. Pasteur, p. 289, 1901; Rice of Page E. Some factors influencing selection of complement-fixa-tion method, J. Immunol., v. 59, p. 95, 1948; Wallace A. L., Osier A. G. a. Mayer M. M. Quantitative studies of complement-fixation, estimation of complement-fixing potency of immune sera and its relation to antibody-nitrogen content, ibid., v. 65, p. 661, 1950.
I. A. Tarkhanova.