EPHE METHOD (N. To. Jerne) — the method of local hemolysis in an agar allowing to define quantity of antiteloprodutsent in the general population of lymphoid cells. It is more correct to call this method method Erne and A. A. Nordin since his first description is made by both of these authors in 1963. In general E. the m is quite simple, highly sensitive and easily reproduced. It expanded opportunities immunol, the analysis and promoted interpretation of many immunol, phenomena. E. the m finds application in many sections for both theoretical, and practical immunology.
For statement of E. m of laboratory animals immunize heterological erythrocytes, is more often than a ram. In various terms after immunization allocate lymphoid bodies and prepare from them a suspension of cells. Further this suspension and erythrocytes of a ram on 0,1 ml add the agar melted 0,7% prepared on salt solution or a medium to 2 ml. Mix is mixed and poured out in Petri dishes which after hardening of an agar place for 60 min. in the thermostat at t ° 37 °. After an incubation add 2 — 3 ml of a complement to cups (cultivation 1:5 — 1:10). Cups incubate 30 min. at t ° 37 ° again and after that carry out the accounting of result of reaction. At survey of cups in a transmitted light on a pink background small ochazhka — sites of a lysis of erythrocytes («direct plaques») with the naked eye are visible. Depending on experimental conditions their diameter can fluctuate. At microscopy with a lens 8 in the center of an active area one cell is visible.
Preliminary warming up of a cellular suspension at t ° 56 ° (30 min.), cultivation of cups with Wednesday at t ° 17 °, addition to an agar of inhibitors of cellular respiration (sodium azide, potassium cyanide, an antimitsin And etc.) sharply suppress formation of active areas.
In most cases the cell which is in the center of a plaque is a producer of antibodies hemolysins. Having counted number of active areas and knowing quantity of the lymphoid cells added to an agar it is possible to calculate how many antiteloprodutsent are in initial suspension. Finally do recalculation of their quantity on 10^6 lymphoid cells or on all body.
Outcome E. the m depends on culture conditions of cells. In experience it is necessary to use agarose or an agar, and at use of agarose the best results turn out. Suitability of a colloid bearer is defined by lack of anticomplementary activity. Besides, the best results turn out during the use of mediums No. 199 or Eagle (in comparison with salt solutions). In the first case of an active area have the bigger size and over time increase. Perhaps, on Wednesdays without amino acids of a cell only release to the environment of an antibody, the formed in vivo, and synthesis of new molecules does not happen. At the same time presence at an agar of normal serum does not lead to significant improvement of results. Tenfold fluctuations of concentration of erythrocytes (from 0,5 to 5%) do not exert impact on identification of antiteloprodutsent. The complement can be added to an agar not only after an hour incubation, but also to later terms. According to Uertis (H. H. Wortis et al., 1968), during a five-hour incubation approximately identical number of antiteloprodutsent comes to light. Before statement of experience the emitted suspensions of cells can be stored at least within 120 min. at t ° 4 ° without reduction in the amount of antiteloprodutsent.
Not all active areas are caused by the cells cosecreting antibodies. In some cases they are formed by the elements which are passively occluding on all surface of an antibody. The last, unlike true antiteloprodutsent, are insensitive to inhibitors of cellular respiration [Rouzmen (J. M of Roseman) et al., 1969].
By means of E. m generally come to light the cells synthesizing IgM-antibodies. It is connected with the fact that in reaction of an immune lysis they are much more effective than IgG-antibodies.
In 1965 Mr. Shtertsl and Rich (J. Sterzl, J. Riha) both Uertis and Dresser (H. H. Wortis, D. M of Dresser) found that addition in the corresponding antiserum dilutions against IgG of the animal who is the donor of lymphoid cells increases number of active areas in comparison with control. These additional zones of a lysis are called indirect plaques. Their considerable part is caused by the cells synthesizing IgG-antibodies. It turned out that under the influence of an antiserum against IgG hemolitic activity of IgG-antibodies sharply increases, and a komplementzavisimy lysis of erythrocytes the same as in case of IgM-antibodies, can arise from one molecule IgG.
By means of E. to m it is established that the cells producing antibodies to erythrocytes of a ram are present before immunization practically at all laboratory animals. At intact animals background an antibody producers generally are in a spleen. In limf, their nodes it is less, and in a thymus gland they are practically absent.
After immunization by erythrocytes of a ram the number of plaque-forming cells sharply increases. Antiteloprodutsenta in the quantity exceeding background level appear for the 2nd day after immunization, reach a maximum for the 4th day then their quantity sharply falls. Use of highly sensitive modification (see below) allows to reveal plaque-forming cells in 24 hours after immunization and the number them by 4 times exceeds quantity of background.
IgG-antiteloprodutsenta appear for the 4th day, reach the maximum sizes for the 6th day, further their number gradually goes down. At reimmunization shorter incubation interval is observed, the peak of IgM-and IgG-antiteloprodutsentov forms at the same time, on the 5th days after immunization, and the second sharply prevail over the first. On a maximum of primary and secondary response the number of IgM-and IgG - anti-teloprodutsentov makes 1100 and 500, 200 and 1500 respectively. At immunization of animals the soluble antigens received from erythrocytes of a ram at primary answer population IgG-antiteloprodutsentov generally forms. Immunization of rabbits S. enteritidis O-antigen causes preferential emergence of the cells synthesizing IgM-antibodies. Using highly sensitive modification E. m and postponing by means of the micromanipulator antiteloobrazuyushchy cells to Wednesdays with various antiserums, G. J. V. Nossal et al. (1971) showed that population of IgM - anti-teloprodutsentov is heterogeneous in the functional relation.
In addition to classical E. m, for studying of an immune response to heterologous erythrocytes, are widely used its numerous modifications. Cunningham's method is quite often applied (And. J. Cunningham, 1965), consisting in cultivation in fluid medium in the form of a monolayer of mix from lymphoid cells, erythrocytes and a complement. Active areas consider microscopically. Test-sensitivity is 3 times higher than classical.
Almost along with Erne and Nordin (1963) Ingrekhem and Basserd (J. S. Ingraham, A. Bussard, 1964) developed a method of identification of antiteloprodutsent in a monolayer from KM cellulose, erythrocytes, lymphoid cells, a complement and a medium. G. J. V. Nossal with sotr. (1971) considerably improved this technique, sharply increased its sensitivity (by 10 — 40 times) and used the micromanipulator to extraction from an active area of antiteloprodutsent.
Kennedy and Akselrad (J. The village of Kennedy, M. of A. Axelrad, 1971) suggested to use instead of an agar poly-L - the lysine having strong positive charge and owing to this fact connecting erythrocytes that considerably facilitates morfol. and autoradio graphic researches of antiteloprodutsent.
E. the m can be used not only for identification of the cells producing antibodies to heterological erythrocytes, but also to a number of soluble antigens.
Landi (M. Landy) et al. (1965), V. V. Solovyov et al. (1966), A. A. Korukov (1971) was shown that at cultivation in a semi-fluid agar of the erythrocytes of a ram loaded with bacterial polysaccharide (About - or Vi - an anti-genome), and lymphocytes of animals, immunizirovanny this polysaccharide, in the presence of a complement active areas form (passive local hemolysis in an agar). It is possible to load erythrocytes with also proteinaceous antigens (a bull seralbumin, ovalbumin etc.) and haptens (dinitrophenyl, penicillin etc.) during the processing by their carbodiimide, glutaraldehyde, periyodaty [Kaplan and Freeman (A. M. Kaplan, M. J. Freeman), 1968; E. S. Golub et al., 1968]. For identification of the cells synthesizing antibodies to bacteria (a cholera vibrio, colibacillus) in E. the m instead of indicator erythrocytes can use these microorganisms [Shvartts, Brown (S. And. Schwartz, W. Braun), 1965; R. McAlack, 1971].
Morphologically the cells forming zones of a lysis are characterized by big heterogeneity. Big and average lymphocytes, blasts, plasmablasts, unripe and mature plasmocytes, basphilic and not basphilic reticulocytes and other types of cells with dominance at primary answer of basphilic lymphocytes concern to them [I. Kiyohiro et al., 1969]. At a combination E. m, to an autoradiography and a submicroscopy it was shown that at early stages of an immunogenesis lymphocytes and blasts really prevail, and at later stages plasmocytes begin to appear [F. G. Gudat et al., 1971]. Antiteloobrazuyushchy lymphocytes differ from usual in existence of well developed Golgi's device and an endoplasmic reticulum. Background antiteloprodutsenta are presented only by small and medium lymphocytes.
The considerable percent of plaque-forming cells synthesizes DNA. Claflin and Smitiz (A. J. Claflin, O. of Smithies, 1967) directly observed cell division, forming an active area. Owing to mobility of daughter cells which continued to cosecrete antibodies the zone of a lysis takes the asymmetric form. If after division to transfer daughter cells by means of the micro manipulator to the new environment of cultivation, then after identical stage of latency they create identical by the sizes and outlines of a plaque [Nossel and Lewis (N. Lewis), 1971], i.e. are absolutely equivalent on functional activity.
Rappoport (I. Rappoport, 1973) offered option E. m, on Krom lymphoid cells it is long cultivated on the milliporovy filter and periodically did them prints on an agar with erythrocytes of a ram and a complement. It allows to watch dynamics of secretion of antibodies separate cells.
E. the m is applied to studying of ability of cells to synthesize antibodies of various spetsifichnost. At immunization of mice erythrocytes of a ram and camel any did not form two types of antibodies of more than 16 thousand studied antiteloprodutsent. Rabbits were immunized a human seralbumin, to Krom fixed two various haptens. Then each of haptens was attached separately to erythrocytes of a ram and used in E. m. The research showed that any of nearly 28 thousand cells-antiteloprodutsentov does not synthesize antibodies of two spetsifichnost [H. Gershon et al., 1968]. By means of modified E. to m it is shown also that antibody formation to various antigenic determinants of a bull seralbumin occurs in various cells [Benjamin, Veygl (D. Page of Benjamin, W. Lake of Weigle), 1970]. The replica technique allowed to establish that at immunization by two various haptens the vast majority of immunocytes specializes in synthesis of antibodies of one specificity, however occasionally (no more than 1,4%) the cells synthesizing antibodies against two haptens meet [Hanna, Merchent (E. E. Hanna, V. Merchant), 1971].
The question is important whether one cell can produce various classes of antibodies of one specificity. The replica technique when cells of immunizirovanny animals place between two layers of an agar is for this purpose applied. One agar layer contains erythrocytes of a ram and a complement, to other layer, in addition to these ingredients, the antiserum against IgG is added. Thus, two remarks with action of the same cells turn out. It turned out that the vast majority of immunocytes synthesizes antibodies only of one class or a subclass. But, according to various authors, from 1,5 to 3,5% of cells produce at the same time IgM-and IgG-antibodies. Such «double» cells come to light on 7 more often — the 9th day after immunization and, perhaps, are connected with switching of synthesis of IgM-to synthesis of IgG-antibodies (Nossel et al., 1971).
Studying of allotypic markers of immunoglobulins by means of antiallotipichesky serums showed that at heterozygous individuals in a mature plasmocyte the immunoglobulin bearing only one of two allelic markers is synthesized [Pernis (V. G. Pernis) et al., 1963]. If v4/v5 to proimmunizirovat rabbits with a genotype erythrocytes of a ram, then each separate plaque-forming cell produces antibodies only of one allotype [Z. Brahmi, Ingrekhy, 1970]. The phenomenon of an allelic exception is characteristic of the cells forming active areas as well as of all immunocytes cosecreting immunoglobulins.
Bibliography: Babichev V. A., Uteshev B. S. and Pinegin B. V. Kinetika of formation of population of antiteloobrazuyushchy cells at the immunological answer, Usp. sovr, biol., t. 78, No. 4, page 122, 1974, bibliogr.; Font of ying L. N. of l. Immune responsiveness of lymphoid bodies and cells, M., 1967; J erne N. To. a. N about of d i n A. A. Plaque, formation in agar by single antibody-producing cells, Science, v. 140, p. 405, 1963; Sell S., Park A. B. a. N about r-d i n A. A. Immunoglobulin classes of antibody-forming cells in mice, J. Immunol., v. 104, p. 483, 1970, bibliogr.
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