AGGLUTINATION (late lat. agglutinatiq — pasting) — pasting and settling-out of corpuscular particles — bacteria, erythrocytes, leukocytes, thrombocytes, cells of fabrics, corpuscular reactive particles with adsorbed by a pas them antigens or antibodies weighed in the environment of electrolytes.
The antibodies causing reaction And., call agglutinins, and the antigens participating in reaction — agglutinogens. Agglutinins are called usually according to cells which are taken in reaction. So, e.g., agglutinins against erythrocytes — hemagglutinins, against leukocytes — leykoagglyutininam etc.
Distinguish agglutinins full and incomplete. Reaction And. allows to find antibodies in blood serum, extracts of fabrics, secrets of an organism at infectious diseases, at auto-, from - and heteroimmunizations (see. Blood groups , Immunohematology ), allergic states.
Reaction And. can be specific, nonspecific and spontaneous. Specific reaction And. arises under the influence of serums of animals or the person (see. Antibodies ), immunizirovanny various antigens (see. Antigens , Antigen — an antibody reaction ), or normal serums of the person. Nonspecific reaction And. arises when aggregation and settling-out of corpuscular particles happens under the influence of change of environmental factors (pH of the environment, salt content, increase or fall of temperature, etc.).
Reaction can be referred to a nonspecific agglutination test And. erythrocytes of the person and animals (see. Hemagglutination ) under the influence of water extracts from seeds and fruits of some species of plants.
Among plants, hl. obr. bean, the hemagglutinins possessing and specific ability to agglutinate erythrocytes of the person only of certain serological types were found. The properties which are observed at agglutinins of the specific serums received from immunizirovanny animals are inherent in the antibodies which are contained in salt extracts of fruits and seeds of many species of plants (phytohemagglutinins). These phytohemagglutinins received the name lectins (see).
Spontaneous reaction And. it is observed in cases when bacteria at reproduction are not divided into separate cells, and remain connected among themselves in chains or clusters. Such suspensions not homogens. A part of culture always is in draft. Under the influence of bacteria and viruses erythrocytes can give spontaneous
A. V to infectious immunology reaction specific And. it is applied to diagnosis of various infectious diseases, to identification of the microorganisms allocated from patients and bacillicarriers for studying of an immune response of an organism at an infection and preventive immunization.
For the first time reaction And. bacteria it was described by Sharren and Roget (A. Charrin, G. N of Roger, 1890) and I. I. Mechnikov (1891). Practical application of reaction And. it was offered Gruber, Durham and Vidal (M. to Gruber, N. E. to Durham, F. Widal, 1896) for diagnosis of a typhoid (see Vidal reaction).
Reaction And. bacteria it is carried out in test tubes of 1 ml (the macroscopic developed reaction And.). In a support a number of test tubes with numbers is established. In all test tubes, except the first, pour isotonic solution of sodium chloride, then bring 1 ml of immune serum in cultivation in the first and second test tubes 1: 100. After mixing from the second test tube take away 1 ml of serum in cultivation 1: 200 also transfer I the third test tube etc. Pour out of a penultimate test tube of 1 ml of divorced serum. In the last test tube serum is not brought. In each test tube add on two a drop of a dense suspension studied a bacterium (2 billion microbic bodies to 1 ml according to the optical standard) and vigorously stir up. The last test tube is control of homogeneity of culture of the studied bacteria. The amount of antigen in each test tube is identical. The first phase of reaction proceeds at t ° 37 ° in the thermostat during 2 hours, then 18 hours at the room temperature. Consider indications of reaction And., as a rule, with the naked eye. In the last, control, a test tube there has to be a uniform suspension of bacteria.
Reaction And. it is estimated as follows:
++++ full agglutination, liquid is transparent, culture in draft;
+++ incomplete agglutination, liquid is not completely transparent, there is a deposit;
++ weak agglutination, liquid is not transparent, there is a deposit;
+ traces of agglutination, liquid it is not transparent, a small deposit;
— negative reaction, in all test tubes a suspension evenly muddy, is not present a deposit.
Its cultivation which gave reaction is accepted to a caption of serum And. on + + at total absence And. in a control test tube.
The deposit which is dropping out at positive reaction And., can have an appearance of large friable flakes of a pla of fine compact grains. The Krupnokhlopchaty deposit is formed by the bacteria having two antigens: flagellar — N-antigen and somatic — O-antigen (see. Bacteria , antigens of bacteria). Large flakes at full And. drop out in a deposit within 2 — 4 hour. The fine-grained deposit is formed by the bacteria which do not have flagellums. These bacteria have only one somatic antigen. In process And. bodies of bacteria stick together and slowly (during 18 hours) drop out in a deposit.
For the purpose of more exact definition of extent of manifestation of reaction And. use various devices: agglyutinoskop, photo-electric densitometer, photo-electric colorimeter, spectrophotometer.
In practical work the drop method of statement of reaction is applied And. in test tubes of l on a slide plate. Several modifications of a drop method of statement of reaction are offered And., beginning from mixing of not divorced specific serum with equal amount of the studied bacterial culture before cultivation of serum 1: 400.
Results of reaction And. on a slide plate consider in 5 min. at constant rocking of a slide plate or in 30 min. if a slide plate is in a moist chamber. Reaction And. on a slide plate it is more often used as approximate (see. Noblya reaction ). It is possible to remove a half of colony of the studied culture of bacteria in solid nutrient medium and in reaction And. on a slide plate to define their look. Reaction And. on a slide plate completely satisfies research objectives if the adsorbed monoreceptor serums are applied.
Often specific serums received from immunizirovanny animals or sick people give positive reaction And. not with one estimated infestant, and and with other species of microorganisms. So, e.g., serum of patients with a typhoid can agglutinate in high credits not only a typroid stick, but also the bacteria causing a paratyphoid And yes In (group And.). It indicates a community of antigens of these bacteria. It is considered to be that the disease was caused by a microorganism which is agglutinated by serum of the patient in higher caption, than other species of bacteria. On distinction of credits in reaction And. quite often happens insignificant, and in these cases apply a method of adsorption of agglutinins (see Castellani a method). It is based that specific serum is sated with several cultures which are agglutinated by this serum. In a test tube where the specific culture is added, all agglutinins are adsorbed and where the nonspecific culture is added, only group agglutinins are adsorbed (specific remain free).
For sensitization of reaction And. passive reaction of A. Sushchnost of this reaction is often applied consists that specific antigens and antibodies interact on a surface of inert particles which at positive reaction drop out in a deposit. As corpuscular particles for adsorption of antigens and antibodies various adsorbents were offered, but only those which have high adsorbability are acceptable for practical application, uniformity of particles and the smallest tendency to spontaneous A. K to such adsorbents erythrocytes of the person and animals, particles of latex, a collodion, bentonite, etc. belong.
Reaction passive the diagnostic purpose was offered to Ampere-second by A. T. Kravchenko (1943) and M. I. Sokolov (1945). Erythrocytes of the person were loaded with the haptens prepared from bacterial cells of causative agents of infectious diseases. These erythrocytes gained ability to be agglutinated under the influence of specific serum.
Several modifications of passive reaction are offered And. the loaded native erythrocytes and reaction of braking passive And. the loaded erythrocytes. Eritro Zita of the person and the animals who are previously processed tannin to - that, gain ability to more adsorb proteinaceous antigens on the surface (see Voyden reaction). Processing of erythrocytes tannin to - that pulls together their properties with indifferent particles.
Erythrocyte diagnosticums — the erythrocytes loaded with antigens of different types of bacteria or antibodies were for practical purposes offered. Erythrocyte diagnosticums prepare for diagnosis of a typhoid, dysentery, cholera and others went. - kish. diseases, and also plague, whooping cough, typhus. Besides, erythrocyte diagnosticums are applied at a research of the nature of allergic reactions.
Practical application was found by so-called latex test. Particles of latex are an intermediate product of synthesis of rubber. The suspension of particles of latex of 0,79 in size — 0,81 microns is issued especially for serological a research. The initial suspension of latex is filtered for removal of particles of the bigger size. The suspension of particles of latex gets divorced concerning 1: 10th borate or glycine buffer (pH = 8,2). The prepared suspension is «loaded» with antigen or antibodies in the ratio 1:10 and maintained at 37 ° 2 hours.
The particles of latex loaded with antigen or antibodies can be used in reaction of definition of unknown antigen on the known serum or determine unknown serum by the known antigen. Two drops of the unknown serum divorced the buffer which is previously warmed up at 56 ° within 30 min. mix on a slide plate with one drop of the particles of latex loaded with antigen. Reaction comes, as a rule, quickly (2 — 3 min.) and only in certain cases it is required to sustain 30 min. at t°37 °. The come reaction And. particles of latex it is well visible with the naked eye on speed a background or under small increase in a microscope.
Reaction And. it is widely used for detection of anti-erythrocyte antibodies, and also antigens in erythrocytes (see. Blood groups ).
Importance, especially recently, reaction got And. leukocytes (leukoagglutinating). By means of reaction of leukoagglutinating typing of the leukocytic antigens which are of great importance in a problem is made transplantations (see).
For statement of reaction of leukoagglutinating use syvorot ki, received from the multigiving birth women, the serums of patients after repeated hemotransfusions containing antileukocytic antibodies. Reaction And. leukocytes it is made with the leukocytes received from blood or limf. nodes (lymphocytes), ex tempore since the leukocytes allocated from blood keep viability only the 6th hour.
At allocation of leukocytes from blood it is necessary to take all precautionary measures, avoiding damage of cells. Methods of receiving leukocytes from blood for reaction of L. are based on different rate of sedimentation of leukocytes and erythrocytes from defibrirovanny blood. 10 ml of isotonic solution of the sodium chloride containing 2,5 g zhelatinf, 3 g of sodium citrate and 3 g of sucrose during the mixing with blood in the ratio 10: 3 besiege erythrocytes completely within 30 min. In nadosadochny liquid there are leukocytes and thrombocytes. Careful centrifuging of nadosadochny liquid at 800 rpm within 4 min. allows to receive leukocytes in draft, and thrombocytes in nadosadochny liquid. The deposit consisting of leukocytes is washed solution still twice.
Receiving leukocytes shall be made at a low temperature and in the conditions of sterility since bacterial pollution of a suspension of leukocytes involves spontaneous And.
For leukoagglutinating it is recommended to use the suspension of leukocytes containing from 3000 to 5000 leukocytes in 1 mkl (calculation in Goryaev's camera). Serum before statement of reaction of leukoagglutinating is warmed up at t ° 56 ° within 30 min.
Reaction is carried out in test tubes, on glass with holes or on plates of organic glass with holes. The reacting liquids undertake in the ratio 0,1 ml of divorced serum on 0,05 ml of a suspension of leukocytes.
The volume of reaction mixture can be varied. Mix is maintained at t ° 37 ° within 1 hour. Then add to each test tube on one drop 3% of solution acetic to - you for removal of the erythrocytes which remained during the washing. Reaction of leukoagglutinating is considered under small increase in a microscope. Assessment of the indication of reaction is begun with a control test tube. Positive reaction is estimated as usual reaction And. any suspended particles, on four-point system.
For studying of antigenic structure thrombocytes (see), and also detection of antithrombocytic antibodies in serum of patients after many multiple hemotransfusions apply reaction And. thrombocytes. Complexity of statement of reaction And. thrombocytes consists in receiving a suspension of thrombocytes without signs spontaneous And. The choice is important for receiving such suspension of thrombocytes anticoagulant (see), and also the special processing of ware excluding damage of thrombocytes, their sticking to a surface of ware and spontaneous And.
For reaction it is recommended to take a suspension of thrombocytes in concentration of 150 000 — 250 000 cells in 1 mkl.
The ready suspension of thrombocytes (before mixing with serum) is upheld within 30 min. by Ispytuyemaya and control serums get warm previously at t ° 56 ° within 30 min. Reaction mixture is placed in the thermostat on 1 hour at 37 °. Assessment of reaction is carried out under a microscope at safe control on four-point system. However more accurate results on studying of antigenic structure of thrombocytes receive during the use reactions of binding complement (see).
Reaction And. has two phases. The first phase — specific, comes down to compound of antigens of bacteria, erythrocytes and other cells, and also the antigens adsorbed on a surface of indifferent particles with the agglutinating antibodies. The second phase — nonspecific which is shown And. erythrocytes, bacteria, leukocytes, indifferent particles with the complex loaded on them antigen — an antibody.
Reaction And., unlike a precipitation test, can be positive at very big cultivations of serum. The exact quantity of antibodies necessary to cause reaction And., it is not known. It is shown that the total amount of reaction product A. always exceeds the initial volume of bacteria, but extent of increase in volume is always various and depends on many difficult considered reasons. Positive reaction And. the quantity of antibodies capable to be adsorbed by antigens on a surface of cells, bacteria or particles can be caused by quantity of antibodies, many times over smaller, than. In certain cases at statement of reaction And. zones of a delay can be observed. Absence And. at big cultivations of serum is called a post-zone, and at small cultivations — a pro-zone. It was established that a delay of reaction And. at the maximum quantity of specific antibodies is caused by inhibitors which reduce forces of adhesion of bacteria. Neag glyutiniruyushchiyesya bacteria after their washing become agglyutinabelny again at big cultivations of the same serum. Nadosadochny liquid contains inhibitors which can reduce coupling of the bacteria processed by serums. The inhibitors causing a pro-zone in reaction And., have looking alike blocking antibodies (see. Antibodies ).
Use in scientific research on immunology of various absorbents gave the chance to model processes of interaction of antigens and antibodies on the surface of adsorbents. One of convenient models for studying of patterns of reaction And. the erythrocytes loaded with various antigens are.
It was established that interaction of native erythrocytes with polysaccharides of colibacillus, the causative agent of cholera, plague and others a bacterium submits to patterns of chemical reaction.
In a crust. time is not present the theory of the mechanism of reaction And., well explaining all known experimental data (see. Antigen — an antibody reaction ). In literature the theory of adsorption of Borde, the theory of a lattice of Marrak are known. theory of occlusion of Boyd, etc. The greatest distribution was gained by the theory of a lattice, according to a cut it is supposed that antibodies have not less than two active centers capable to connect to determinant groups of antigens. A molecule of a specific antibody, connecting to determinants of two or more antigens, forms a lattice — the unit which in the presence in the environment of electrolytes drops out in a deposit. stock A. does not happen to incomplete aptitela. Considered that monovalent antibodies have only one active center, therefore, they can only unite to antigen, but cannot form units which drop out in a deposit. It is suggested that monovalent antibodies too bivalent, but their active centers are located so closely to each other that cannot connect to two molecules of antigen at the same time. Besides, it is known that processing of monovalent antibodies or erythrocytes trypsin involves normal manifestation of the second phase of reaction And. Detection of monovalent antibodies is possible by means of reaction And.: erythrocytes, sensibilized monovalent antibodies, can be used as antigen if on them to work specific antiglobulinovy serum (see. Koombs reaction ).
Agglutination in the medicolegal relation — see. Blood groups .
See also Serological researches .
Bibliography: Adamov A. K. Principles of bystry detection of pathogenic microbes and viruses, Saratov, 1964, bibliogr.; Boyd U. Fundamentals of immunology, the lane with English, page 274, M., 1969; Govallo V. I. The reactions based on a phenomenon of agglutination in book: Laboratory methods of a research in neinfsktsionny immunology, under the editorship of O. K. Vyazov, page 73, M., 1967; Gostev V. S. Reaction antigen — an antibody. Mnogotomn. the management on mikrobiol., wedge, and epidemiol. infekts. diseases, under the editorship of N. N. Zhukov-Verezhnikov of t. 3, page 117, M., 1964, bibliogr.; P. N. Immunologiya's jambs of isoantigens and isoantibodies, M., 1965, bibliogr.; Meyer M. An experimental zhkhmunokhimiya, the lane with English, page 106, M., 1968;
A. T. Kravchenko.