HEMAGGLUTINATION

From Big Medical Encyclopedia

HEMAGGLUTINATION (grech, haima blood + lat. agglutinatio pasting) — a phenomenon of pasting of erythrocytes. There can be a straight line, i.e. occur due to immediate effect of these or those agents on erythrocytes, and indirect (passive) when processed by antigen (or antibodies) erythrocytes are agglutinated respectively by immune serum (or antigen).

Anti-erythrocyte serums, extracts from fabrics of saliva, serum of the person and animals, and also some bacteria (staphylococcus, colibacillus, typroid, paratyphoid, dysenteric microbes) and many viruses can cause direct G. Agglutination of erythrocytes normal serums is divided into isohemagglutination if serum and erythrocytes belong to individuals of one look, and heteroagglutination when there is a pasting of alien erythrocytes.

Serum can gain ability to G. at some diseases. So, e.g., serum of patients with an infectious mononucleosis agglutinates erythrocytes of a ram (see. Paul-Bunnell reaction ).

G. caused by viruses has great theoretical and practical value. It was described for the first time in 1941 Mr. Hurst (G. To. Hirst), Mac-Klillend and Hare (L. MacClelland, R. Hare). They established that the influenza virus agglutinates erythrocytes of hens on the basis of what hemagglutination reaction (RGA) was developed. Afterwards the hemagglutinating properties were found in many viruses. With the phenomenon G. connected also hemadsorption, i.e. ability of the cells infected with the nek-ry hemagglutinating viruses to adsorb erythrocytes on the surface (see. Hemadsorption ). Ability of viruses to cause G. is suppressed with the corresponding antiviral serums that is used in reaction of braking (repayment) of hemagglutination (RTGA).

RGA and RTGA are widely used both at theoretical researches in the field of virology, and at diagnosis of viral infections for indication, identification and classification of viruses, and also for identification of antiviral antibodies (anti-hemagglutinins) in blood serum of patients. So, at allocation of influenza viruses and parotitis the indicator is agglutination of chicken erythrocytes an allantoisny and amniotic fluid of the infected chicken embryos.

For identification selectivity of some viruses is used to agglutinate a certain type of erythrocytes. The virus of measles, e.g., agglutinates only erythrocytes of monkeys, and a virus of an entsefalomiokardit of mice — erythrocytes of a ram.

At the majority of viruses hemagglutinin (the substrate responsible for G.) is a structural component of virion.

At viruses which capsid is dressed by an outside lipoprotein cover (influenza viruses, a parainfluenza, the majority of an arbovirus) hemagglutinin is in this cover and is structurally connected with so-called fibers. By the chemical nature Hemagglutinins of these viruses are gliko-or lipoproteids. So, hemagglutinin of an influenza virus represents the tetramer consisting of two couples of glycoproteins with the general a pier. weighing 150 000. The hemagglutinating glycoprotein of a cover of an arbovirus of group B has a pier. weight 50 000.

At the viruses which do not have an external cover, hemagglutinin is connected with structures of a capsid. So, at adenoviruses the fibrilla which is coming out vertex capsomeres has the hemagglutinating activity.

Hemagglutinin of smallpox viruses is a lipoproteid and represents one of products of their reproduction, but, apparently, virion as the cleared virus particles of G. do not cause is not included.

Can cause both infectious virus particles, and inactivated therefore the Hemagglutinating titre of a virus does not reflect its infectious activity. In some cases hemagglutinin can separate from a virus particle (e.g., at adenoviruses). Some viruses (flu, measles, ECHO) can create the empty, deprived of RNA virions which also have the hemagglutinating activity in the course of the reproduction.

The mechanism G. was studied by hl. obr. in experiences with an influenza virus. Its interaction with erythrocytes takes place two phases — adsorption and the subsequent elution (see). The first stage of adsorption of viruses on erythrocytes represents physical. process is also defined by a difference of charges and an intermolecular attraction (Van der Waals's forces). The second stage is chemical interaction of a virus with receptors of an erythrocyte.

The mechanism of the process of pasting of erythrocytes is not absolutely clear. Change of an electrostatic charge of erythrocytes after adsorption of viruses on them can matter.

Perhaps also education by virus particles of «bridges» between separate erythrocytes.

The conjunction of an influenza virus and some paramyxoviruses with a surface of erythrocytes are the receptors of the last representing disaccharide 6 (N-atsetilneyraminil) alpha D - N-atsetilgalaktozamin. Under the influence of virus enzyme of a neuraminidase receptors of erythrocytes are split on N-atsetilgalaktozamin and N-atse-tilneyraminovuyu to - that.

At t ° 37 ° in several hours there is an elution of an influenza virus from erythrocytes. In hypertensive solution of sodium chloride this process proceeds (quicker. Owing to destruction of receptors erythrocytes lose ability to be agglutinated repeatedly by the same virus though can stick together under the influence of some other viruses.

Glikoproteidny receptors of erythrocytes can be destroyed also by the periodate, trypsin and a filtrate of cholera vibrioes containing a neuraminidase.

The majority of other viruses (smallpox, an arbovirus, etc.) does not destroy receptors of erythrocytes. Their elution happens not spontaneously, and at influence of immune serum, change of electrolytic structure of the environment, its pH, etc.

G. depends on properties both a virus, and erythrocytes (tab).


THE VIRUSES CAPABLE TO CAUSE AGGLUTINATION OF ERYTHROCYTES OF SOME VERTEBRATA


The hemagglutinating activity is various both at members of one classification group, and at different strains of one virus and even at separate clones of one strain. E.g., shtammovy distinctions are expressed at enteroviruses of some serotypes. In population of a virus of Koksaki of A-21 were found as the hemagglutinating particles and deprived of this property.

For visible G.'s receiving virus suspension shall contain not less than 105 — 106 virus particles in 1 ml.

Can increase the hemagglutinating activity of some viruses (e.g., measles, parotitis, a rubella) by processing of a virus suspension the twin-80 and ether probably owing to disintegration of an outside viral envelope.

Depends also on the environment of cultivation of a virus and on availability of the inhibitors blocking this process.

E.g., at cultivation of a virus of Koksaki of A-21 in the intertwined cells of a malignant origin only not hemagglutinating particles are produced. Source of receiving the hemagglutinating antigens of an arbovirus is the hl. obr. a brain of the infected mice suckers containing many Inhibitors. Therefore apply extraction of brain fabric to preparation of these antigens borate and salt solution with pH 9,0, cleaning with freon, precipitation with acetone.

The unblocking of hemagglutinin with big efficiency can achieve also by after-treatment of a suspension the twin-80 and ether, ultrasound and trypsin in small concentration.

At some viruses ability to cause G. depends on number of passages in this or that substrate. Here adaptation of a virus to culture conditions plays a role and increases in activity of its reproduction to level when concentration of virus particles becomes sufficient for G. Inogd's appearance inverse relation is observed: with number of passages the Hemagglutinating activity of a virus decreases up to total disappearance. Perhaps, these phenomena selection (during passages) is the cornerstone of the hemagglutinating or not hemagglutinating particles.

From among the factors characterizing erythrocytes, their specific accessory has special value.

Ability of erythrocytes to be agglutinated by this or that virus is established empirically. Usually the viruses relating to one classification group agglutinate the same types of erythrocytes. At the same time also individual properties of the donor matter.

Also the age and a sex of the donor influences G. E.g., the virus of a variolovaccine agglutinates erythrocytes of adult hens, than chickens more actively.

Erythrocytes of young birds are preferable to work with an arbovirus. Besides, it is recommended to use erythrocytes of ganders, but not gooses as hormonal shifts in the period of a yaytsekladka and an incubation of eggs change properties of a surface of erythrocytes therefore they can have a refrakternost to action of a virus or tendency to spontaneous agglutination.

Erythrocytes of some animal species (rabbits, rats, mice) quite often give spontaneous agglutination that needs to be considered during the development of reference conditions of G. with each virus. Erythrocytes of birds it is more preferable than erythrocytes of mammals as they quickly settle, give a clear picture and are a little subject to spontaneous agglutination. At statement of RGA with nek-ry viruses, napr, an influenza virus, can be used both fresh erythrocytes, and preserved by means of 25% of formalin.

Depends on electrolytic structure of the environment, concentration of hydrogen ions and temperature. In the environment without electrolytes of agglutination of erythrocytes viruses does not occur.

There is a certain optimum of electrolytic structure of the environment; e.g., adsorption of hemagglutinins of a virus of a variolovaccine on chicken erythrocytes is maximum at 0,45 — 1,8% of sodium chloride.

Statement of RGA is carried out at t ° 4; 20 — 25 or 37 °. The influenza virus, e.g., best of all agglutinates erythrocytes at t ° 4 °, a virus of a variolovaccine — at t ° 37 °, and for G. an arbovirus temperature does not matter.

Requirements of different viruses to concentration of hydrogen ions are also not identical. Most of them causes G. at pH 6,0 — 8,5. Therefore as Wednesday most often use isotonic solution of sodium chloride, to Krom sometimes add 0,014 M the phosphatic buffer with pH 7,2 (at G. with influenza viruses, measles, a variolovaccine, etc.).

Arbovirus which ability to G. very weak, demand strictly certain concentration in are portly also ions: the deviation from pH, optimum for each virus, is allowed no more, than on 0,3 — 0,4 pieces.

As Hemagglutinins of these viruses are stable only in alkaline condition (at pH 9,0), and for statement of RGA the zone with pH 5,6 — 7,0 is optimum, necessary concentration of hydrogen ions is created at the time of compound of antigen with erythrocytes, adding the erythrocytes which are in acid buffer solution to an alkaline suspension of a virus.

The composition of buffer solutions can influence a specific range of sensitivity of erythrocytes. If, e.g., in the environment of ordinary structure the virus of a rubella agglutinates erythrocytes of chickens, pigeons and geese, then during the use of 0,025 M of HEPES-bu-fera (N-2-hydroxyethylpiperazine — N12-ethanesulfouic acid) pH 6,2 with addition of 0,4 M of NaCl, 0,001 M of CaCl2, 1% of albumine of serum of cattle and 0,00025% gelatin it agglutinates also erythrocytes of adult hens, the person (blood 0 of group), monkeys, sheep, pigs, cats, rabbits, rats, hamsters and mice.

For confirmation of specificity of virus G., and also for identification in serums of virus anti-hemagglutinins at serol, researches RTGA serves. Its specificity is not identical to different groups of viruses. For an arbovirus of a sort alpha and flavivirus of RTGA, is group-specific, i.e. reveals antigenic bonds between members of this group. It complicates assessment of results serol, researches at existence in any area of several viruses of one group. At adeno-and reoviruses by means of RTGA type-specific features come to light, and at influenza viruses even subtle differences between strains of one look are caught.

For RTGA it is desirable to use highly active antigens. Antigens with low activity quite often contain many not hemagglutinating virus particles which can connect to antibodies and interfere with their identification. During the use as a source of hemagglutinin of the infected cellular cultures exclude serum from structure of the environment or previously delete from it Inhibitors.

The serumal inhibitors blocking G. on chemical structure are generally beta lipoproteids, and by the size of molecules are close to 198 antibodies. The serums investigated in RTGA exempt from inhibitors by heating at t ° 56 or 62 ° within 30 min., processing by a filtrate of cholera vibrioes or a neuraminidase, trypsin, adsorptions of inhibitors a kaolin, precipitations of antibodies acetone, processings by magnesium chloride and heparin, processings by a sulphatic dextran and calcium chloride, processings by Rivanolum. Efficiency of separate methods concerning removal of various inhibitors is not identical. The first three methods are sufficient for removal of inhibitors G., caused by influenza viruses and a parainfluenza. Processing of serums a kaolin and acetone is used during the work with an arbovirus, Rivanolum, during the studying of enteroviral infections. At identification of antibodies apply processing to a virus of a rubella a kaolin, magnesium chloride and heparin or sulfate of a dextran and calcium chloride.

The serums investigated in RTGA exempt also from agglutinins of that type of erythrocytes which is used at statement reaction. It is carried out by adsorption of agglutinins by the concentrated suspension of these erythrocytes.

Technology of statement of RGA and RTGA

Reactions put in test tubes or on plates from organic glass with deepenings. Broad application is found by a micromethod with use of the microtitrator of Takachi which represents a set of small plates with U-shaped deepenings, a set of droppers and dilyutor. The volume of reaction mixture at a macromethod makes 0,8 ml, and at a micromethod — 0,1 ml. Erythrocytes use from 0,25 to 1% in concentration.» For statement of RGA connect 0,2 (0,025) ml of antigen, 0,2 (0,025) ml of salt solution and 0,4 (0,05) ml of a suspension of erythrocytes. In RTGA the same ratios of ingredients remain, but instead of salt solution the studied serum is brought. In RGA define a caption of hemagglutinin, i.e. the greatest cultivation, a cut gives accurate G. Kolichestvo of hemagglutinin, contained in 0,2 ml of this cultivation, will make one hemagglutinating unit (HU). For titration of serums in RTGA depending on features of a virus use 4 — 8 agglutinating units (AU).

the Scheme of hemagglutination reaction for detection and titration of a virus in the infected cultures and hemagglutination-inhibition reaction for titration of immune serums on their ability to prevent hemagglutinations), caused by a virus (on the example of an influenza virus). In an upper number of the drawing — titration of hemagglutinin. Define a working dose of hemagglutinin which shall make 4 hemagglutinating units (THOSE) and corresponds to cultivation of a virus 1: 40; one of E corresponds to cultivation 1: 160 (the maximum cultivation of viruses capable to cause hemagglutination). In the lower number of the drawing — titration of serum from 4 G E hemagglutinins. Hemagglutinin in a dose of 4 G of E at connection with serum before cultivation 1: 160 does not cause agglutination of erythrocytes (stopping of reaction). Smaller amounts of serum (cultivation 1: 320 and above) agglutinations do not interfere. The agglutinated erythrocytes form bottom sediment of a test tube (small points), not agglutinated erythrocytes look in the form of a compact disk (a black circle).

Results of reaction, i.e. existence or G.'s absence, estimate on character of a deposit of erythrocytes (fig). The agglutinated erythrocytes settle in the form of a film, sometimes deckle-edged that reminds the overturned umbrella. In the absence of agglutination erythrocytes accumulate in the center of deepening in the form of a compact disk. Time necessary for sedimentation of erythrocytes varies of 45 min. to 2 hours depending on specific accessory of erythrocytes, volume of reaction mixture and temperature. Settle the «heavy», containing kernels erythrocytes of birds quicker. At more high temperature of G. occurs quicker, than at lower.

Reaction of indirect (passive) hemagglutination (RNGA or RPGA) has two main versions: a) agglutination of erythrocytes, sensibilized antigen, immune serum; b) agglutination sensibilized antibodies of erythrocytes in the presence of antigen. Distinguish two phases of reaction. During the first change of surface characteristics of erythrocytes results from adsorption of antigens on them (or antibodies). In the second phase on sensibilized erythrocytes antibodies are adsorbed (or antigens) and there is a formation of conglomerates.

For the diagnostic purposes with bacterial antigens of RNGA it was used by A. T. Kravchenko and M. I. Sokolov in 1946. In alkaline condition removed polisakharidny antigen from bacterial cells, adsorbed on erythrocytes of the person of group 0 and immediately connected to diagnostic serum. The method does not demand release of pure growths of bacteria since adsorption of antigen can be carried out directly from patol, material. By means of this technique it was possible to find such amount of antigen in 1 ml of salt solution, a cut there correspond 50 — 100 million microbic bodies determined by the optical standard.

RNGA across Kravchenko and Sokolov and her modification found application in bacteriology, but opportunities it were limited to the fact that on native erythrocytes it is possible to adsorb only polisakharidny antigens, but not proteins. But in 1951 Mr. S. V. Boyden showed that the erythrocytes pickled tannin to - that, gain ability to adsorb on the surface and proteins (see. Boydena reaction ).

In 1956 Rytsay (T. Rycaj) modified Boyden's technique: erythrocytes sensibilize antibodies and use for detection of various antigens. For adsorption on erythrocytes use immunoglobulin of immune serums. RNGA across Rytsayu can be applied not only to indication of antigens, but also to titration of serums, using a phenomenon of clearing, or braking, RNGA. In this case the studied serum in the corresponding cultivations is connected to antigen, against to-rogo assume to find antibodies, and then add sensibilized erythrocytes. In the presence of antibodies antigen communicates them and agglutination does not happen. At a research of serums both on an original technique of Boyden, and on Rytsayu previously it is necessary to remove inhibitors and heterohemagglutinins from serum.

The RNGA mechanism is studied insufficiently; in this regard at selection of conditions of a sensitization of erythrocytes different antigens and antibodies, and also at the choice of a type of erythrocytes use generally empirical approach.

The adsorptive activity of native erythrocytes is small, but she manages to be raised, processing erythrocytes tannin, acrolein, glutaric dialdehyde, bidiazotirovanny connections (unit hemagglutination).

For creation of stable drugs developments of methods of chemical accession of antigen or antibodies to erythrocytes, in particular by creation of diazobonds are conducted. For this purpose use, e.g., diazotized benzidine, toluflax-2,4-diisocyanate, water soluble karbodimid, diftorodinitrobenzen. Use of a borftorid 4,4 for for accession of the polycondensed antibodies to erythrocytes of a ram for indication of activators of tick-borne rickettsioses is described.

RNGA is widely applied in bacteriology. At plague, cholera, a brucellosis and a tularemia use both kinds of reaction, at scarlet fever, diphtheria and dysentery — only antigenic option, for detection of botulinum toxin erythrocytes, sensibilized antibodies serve.

In virusol. researches RNGA it was for the first time carried out with viruses of parotitis and the Newcastle disease in 1946 — 1948, then after almost ten-year break messages on reproduction of this reaction with adenoviruses, a virus of herpes, myxoviruses, a virus of a variolovaccine, an arbovirus, cytomegaloviruses, a virus of a foot-and-mouth disease, leukoses of hens, etc. followed. Optimal conditions of reaction for different viruses select individually.

For detection of a virus of a tick-borne encephalitis reaction in modification of Rytsaya is described. Erythrocytes, sensibilized immunoglobulin from serum of a horse, immune to a tick-borne encephalitis, use for indication of viruses of vernal and Scottish encephalitis in culture of VNK-21. For this purpose virussoderzhashchy liquid is dissolved in 1% solution of normal horse serum with coefficient 2. Add 1 — 2 drop of sensibilized erythrocytes to 0,5 ml of antigen of each cultivation. Reaction is considered in 1 — 2 hour. RNGA can be applied to detection of a virus of a variolovaccine and natural smallpox both in laboratory cultures, and in patol, material from patients (a detritis and crusts).

See also Agglutination , Virologic researches .


Bibliography: Gaydamovich S. Ya. also J Showed. Comparative study of the hemagglutinating arboviral antigens prepared from fabric cultures and from a brain of mice, Vopr, virusol., No. 2, page 238, 1968; L of e in and M. I. and Basov H. H. Erythrocyte diagnosticums and their use in a serology, Probl, special and dangerous infekts., century 2, page 207, Saratov, 1970; Socks F. Page, etc. Use of reaction of indirect hemagglutination for laboratory diagnosis of natural smallpox, Vopr, virusol., No. 3, page 347, 1972; Remezov P. I., about l ub e in D. B. isinitsynv. A. Hemagglutination reaction, L., 1964, bibliogr.; P y c and y T. Detection of botulinum toxin of type A in foodstuff by method of specific hemagglutination, Bulletin Polsk., academician of sciences, t. 4, JsTs 9, page 341, 1956.

S. Ya. Gaydamovich.

Яндекс.Метрика