An agglutination reaction (RA) is the adhesion and precipitation of microbes or other cells under the influence of antibodies in the presence of an electrolyte. The resulting precipitate is called an agglutinate.
RA is used:
1. To detect antibodies in the patient’s blood serum (serodiagnosis).
2. To determine the type and serovar of a pure culture of pathogenic microorganisms isolated from a patient (serotyping).
The agglutination reaction is used to determine antibodies in the blood serum of patients, for example, with typhoid fever and paratyphoid fever (Vidal reaction), brucellosis (Wright, Heddleson reaction), tularemia, leptospirosis and others infectious diseases, as well as to determine the pathogen isolated from a patient (intestinal infections, whooping cough, etc.). RA is used to determine blood groups, Rh factor, etc.
The reaction requires the following components:
1. The antigen (agglutinogen) must be corpuscular, that is, it is a suspension of living or killed microorganisms (diagnosis m), erythrocytes or other cells. Typically, a daily culture of microorganisms grown on agar slants is used. The culture is washed off with 3 - 4 ml of isotonic solution, transferred to a sterile tube, and the density is determined. The suspension must be homogeneous and contain up to 3 billion microbial cells per 1 ml. The use of a suspension of killed microbes - diagnosticums - facilitates the work (prepared in production).
2. Antibodies (agglutinins) are found in the patient’s serum (during serodiagnosis) or in agglutinating serum (during serotyping). Agglutinating sera are obtained by immunizing rabbits with killed bacteria.
Agglutinating titer serum is called its highest dilution, in which it reacts with the corresponding antigen under certain experimental conditions.
Agglutinating sera can be native (non-adsorbed) and adsorbed. Native sera in small dilutions interact not only with the type of microorganisms with which the animal was immunized to obtain the serum, but also with related types of microorganisms, since they contain group antibodies (antibodies to microorganisms that have common antigens). Native sera are used for a detailed agglutination reaction (for serodiagnosis), which takes into account not only the presence of the reaction, but also the dynamics of the increase in antibody titer.
If group antibodies are extracted (adsorbed) from native serum by interaction with related bacteria that have group antigens, adsorbed sera are obtained. Adsorbed sera can be monoreceptor (or type-specific), containing antibodies to only one antigen receptor. Polyvalent sera consist of a mixture of several adsorbed or non-adsorbed sera. Adsorbed sera are used for the glass agglutination reaction.
When animals are immunized with motile bacteria with the H-antigen, H-agglutinating sera containing H-antibodies are obtained (for example, Salmonella monoreceptor H-agglutinating serum). By immunization with O-antigen, O-agglutinating sera containing O-antibodies are obtained (for example, Salmonella group adsorbed O-agglutinating serum, anticholera O-agglutinating serum). By immunization with H- and O-antigens, sera with H- and O-antibodies are obtained.
Moreover, O-agglutinins produce a fine-grained agglutinate, and H-agglutinins produce a coarse-grained sediment.
3. Electrolyte - isotonic NaCl solution (0.9% sodium chloride solution prepared in distilled water).
There are two main methods for performing an agglutination reaction: a reaction on glass (sometimes called an indicative or plate reaction) and a detailed reaction (in test tubes)
Setting up an agglutination reaction on glass. Two drops of serum and a drop of isotonic sodium chloride solution are applied to a fat-free glass slide. Diagnostic agglutinating serum is taken in one dilution, which, depending on its titer, is 1:10, 1:25, 1:50 or 1:100. The culture of the microorganism under study is added into one drop of serum and a drop of isotonic solution using a loop and mixed thoroughly. A drop of sodium chloride with microorganisms is antigen control, a drop of serum without microorganisms is serum control. You cannot transfer the culture from a drop with serum to a drop with NaCl. The reaction takes place at room temperature for 1-3 minutes. If the serum control remains clear, uniform turbidity is observed in the antigen control, and agglutinate flakes appear in the drop where the culture is mixed with serum, then the result is considered positive. If there is uniform turbidity in the drop with serum and antigen, then this is a negative result. The reaction is more clearly visible against a dark background.
Serum
1. antigen control
2. serum control
13.1. Antigen-antibody reactions and their applications
When an antigen is introduced, antibodies are formed in the body. Antibodies are complementary to the antigen that caused their synthesis and are able to bind to it. The binding of antigens to antibodies consists of two phases. The first phase is specific, in which rapid binding of the antigenic determinant to the active center of the Fab fragment of antibodies occurs. It should be noted that the binding is due to van der Waals forces, hydrogen and hydrophobic interactions. The strength of the bond is determined by the degree of spatial correspondence between the active site of the antibody and the epitope of the antigen. After the specific phase, a slower phase begins - nonspecific, which is manifested by a visible physical phenomenon (for example, the formation of flakes during agglutination, etc.).
Immune reactions are the interaction between antibodies and antigens, and these reactions are specific and have high sensitivity. They are widely used in medical practice. With the help of immune reactions, the following problems can be solved:
Determination of unknown antibodies by known antigens (antigenic diagnosticum). This task occurs when it is necessary to determine antibodies to a pathogen in the patient’s blood serum (serodiagnosis). Finding antibodies allows you to confirm the diagnosis;
Determination of unknown antigens using known antibodies (diagnostic serum). This study is carried out when identifying a pathogen culture isolated from a patient’s material (serotyping), as well as when detecting
antigens of microbes and their toxins in the blood and others biological fluids. There are many types of immune reactions, differing in the technique of staging and the recorded effect. These are agglutination reactions (RA), precipitation reactions (RP), reactions involving complement (RSC), reactions using labeled components (RIF, ELISA, RIA).
13.2. Agglutination reaction
Agglutination reaction (RA) is immune reaction interaction of antigen with antibodies in the presence of electrolytes, and the antigen is in a corpuscular state (erythrocytes, bacteria, latex particles with adsorbed antigens). During agglutination, corpuscular antigens are glued together by antibodies, which is manifested by the formation of a flocculent precipitate. The formation of flakes occurs due to the fact that antibodies have two active centers, and antigens are polyvalent, i.e. have several antigenic determinants. RA is used to identify the pathogen isolated from the patient’s material, as well as to detect antibodies to the pathogen in the patient’s blood serum (for example, the Wright and Heddleson reactions for brucellosis, the Widal reaction for typhoid fever and paratyphoid fever).
The simplest way to diagnose RA is the reaction on glass; this is an approximate RA, which is used to determine the pathogen isolated from the patient. When a reaction is established, diagnostic agglutinating serum is applied to a glass slide (at a dilution of 1:10 or 1:20), then a culture from the patient is added. The reaction is positive if a flocculent sediment appears in the drop. A control is placed nearby: instead of serum, a drop of sodium chloride solution is applied.
If the diagnostic agglutinating serum is not adsorbed 1, then it is diluted (to the titer - the dilution to which agglutination should occur), i.e. put expanded RA in test tubes with increasing1 Unadsorbed agglutinating serum can agglutinate related bacteria that have common (cross-reacting) antigens. Therefore they use adsorbed agglutinating sera,
dilutions of agglutinating serum, to which 2-3 drops of a suspension of the pathogen isolated from the patient are added. Agglutination is taken into account by the amount of sediment and the degree of clearing of the liquid in the test tubes. The reaction is considered positive if agglutination is observed in a dilution close to the titer of the diagnostic serum. The reaction is accompanied by controls: the serum diluted with isotonic sodium chloride solution should be transparent, the suspension of microbes in the same solution should be uniformly cloudy, without sediment.
To determine antibodies to the pathogen in the patient's blood serum, full-scale RA is used. When setting it up, the patient’s blood serum is diluted in test tubes and an equal amount of diagnosticum suspension (suspension of killed microbes) is added to the test tubes. After incubation, the highest serum dilution at which agglutination occurred is determined, i.e. a precipitate (serum titer) has formed. In this case, the agglutination reaction with O-diagnosticum (bacteria killed by heating, retaining the thermostable O-antigen) occurs in the form of fine-grained agglutination. The agglutination reaction with H-diagnosticum (bacteria killed by formaldehyde, retaining the thermolabile flagellar H-antigen) is coarse and proceeds faster.
Indirect (passive) hemagglutination reaction(RNGA or RPGA) is a type of RA. This method is highly sensitive. With the help of RNGA, two problems can be solved: to determine antibodies in the patient’s blood serum, to which is added an antigenic erythrocyte diagnosticum, which is erythrocytes on which known antigens are adsorbed; determine the presence of antigens in the test material. In this case, the reaction is sometimes called the reverse indirect hemagglutination reaction (RONHA). During the procedure, an antibody erythrocyte diagnosticum (erythrocytes with antibodies adsorbed on their surface) is added to the test material. In this reaction, red blood cells act as carriers and are passively involved in the formation of immune aggregates. With a positive reaction, passively glued red blood cells cover the bottom of the hole in an even layer with scalloped edges (“umbrella”); in the absence of agglutination, red blood cells accumulate in the central recess of the hole, forming a compact “button” with sharply defined edges.
Coagglutination reaction used to determine pathogen cells (antigens) using antibodies adsorbed on Staphylococcus aureus, containing protein A. Protein A has an affinity for the Fc fragment of immunoglobulins. Thanks to this, antibodies bind to staphylococcus indirectly through the Fc fragment, and Fab fragments are oriented outward and are able to interact with the corresponding microbes isolated from patients. In this case, flakes are formed.
Hemagglutination inhibition reaction (HAI) used in diagnostics viral infections, and only infections caused by hemagglutinating viruses. These viruses contain a protein on their surface - hemagglutinin, which is responsible for the hemagglutination reaction (HRA) when red blood cells are added to the viruses. RTGA involves blocking viral antigens with antibodies, as a result of which viruses lose their ability to agglutinate red blood cells.
Coombs reaction - RA for determination of incomplete antibodies. In some infectious diseases, such as brucellosis, incomplete antibodies to the pathogen circulate in the patient’s blood serum. Incomplete antibodies are called blocking antibodies because they have one antigen-binding site, and not two, like full-fledged antibodies. Therefore, when an antigenic diagnosticum is added, incomplete antibodies bind to antigens, but do not glue them together. To manifest the reaction, antiglobulin serum (antibodies to human immunoglobulins) is added, which will lead to agglutination of immune complexes (antigenic diagnosticum + incomplete antibodies) formed in the first stage of the reaction.
The indirect Coombs reaction is used in patients with intravascular hemolysis. In some of these patients, incomplete monovalent anti-Rhesus antibodies are detected. They specifically interact with Rh-positive erythrocytes, but do not cause their agglutination. Therefore, antiglobulin serum is added to the anti-Rh antibodies + Rh-positive erythrocytes system, which causes agglutination of erythrocytes. The Coombs test is used to diagnose pathological conditions, associated with intravascular lysis of erythrocytes of immune origin, for example, hemolytic disease of newborns caused by Rh conflict.
RA for determining blood groups is based on the agglutination of erythrocytes by immune serum antibodies to blood group antigens A(II), B(III). The control is serum that does not contain antibodies, i.e. serum AB(IV) blood group, and erythrocyte antigens of groups A(P) and B(III). Group 0(I) red blood cells are used as a negative control because they do not have antigens.
To determine the Rh factor, anti-Rh sera are used (at least two different series). If there is a Rh antigen on the membrane of the erythrocytes under study, agglutination of these cells occurs.
13.3. Precipitation reaction
RP is an immune reaction of the interaction of antibodies with antigens in the presence of electrolytes, and the antigen is in a soluble state. During precipitation, soluble antigens are precipitated by antibodies, which is manifested by cloudiness in the form of precipitation bands. The formation of a visible precipitate is observed when both reagents are mixed in equivalent ratios. An excess of one of them reduces the number of precipitated immune complexes. There are various ways to perform the precipitation reaction.
Ring precipitation reaction placed in precipitation tubes with a small diameter. The immune serum is added to the test tube and the soluble antigen is carefully layered. If the result is positive, a milky ring forms at the interface of the two solutions. The ring precipitation reaction, which is used to determine the presence of antigens in organs and tissues, the extracts of which are boiled and filtered, is called the thermoprecipitation reaction (Ascoli reaction for determining thermostable anthrax antigen).
Ouchterlony double immunodiffusion reaction. This reaction is carried out in an agar gel. In a layer of gel of uniform thickness, wells are cut out at a certain distance from each other and filled with antigen and immune serum, respectively. After this, antigens and antibodies diffuse into the gel, meet each other and form immune complexes, which precipitate in the gel and become visible as precision lines.
nutrition. This reaction can be used to identify unknown antigens or antibodies, and also to test the similarity between different antigens: if the antigens are identical, the precipitation lines merge, if the antigens are not identical, the precipitation lines intersect, if the antigens are partially identical, a spur is formed.
Radial immunodiffusion reaction. Antibodies are added to the melted agar gel and the gel is applied in an even layer to the glass. Wells are cut out in the gel and a standard volume of antigen solutions of different concentrations is added to them. During incubation, antigens diffuse radially from the well and, meeting antibodies, form a precipitation ring. As long as excess antigen remains in the well, a gradual increase in the diameter of the precipitation ring occurs. This method is used to determine antigens or antibodies in the test solution (for example, to determine the concentration of immunoglobulins of different classes in blood serum).
Immunoelectrophoresis. The mixture of antigens is first electrophoretically separated, then precipitating antiserum is added to the groove running along the direction of protein movement.
Antigens and antibodies diffuse into the gel towards each other; interacting, they form arcuate precipitation lines. Flocculation reaction
(according to Ramon) - a type of precipitation reaction that is used to determine the activity of antitoxic serum or toxoid. The reaction is carried out in test tubes. In a test tube where the toxoid and antitoxin are in an equivalent ratio, turbidity is observed.
13.4. Complement fixation reaction
Antibodies, interacting with the corresponding antigen, bind added complement (1st system). An indicator of complement fixation is erythrocytes sensitized with hemolytic serum, i.e. antibodies to red blood cells (2nd system). If complement is not fixed in the 1st system, i.e. If the antigen-antibody reaction does not occur, the sensitized red blood cells are completely lysed (negative reaction). When complement is fixed by immune complexes of the 1st system after adding sensitized erythrocytes, hemolysis from
absent (positive reaction). The complement fixation reaction is used to diagnose infectious diseases (gonorrhea, syphilis, influenza, etc.).
Microbes and their toxins have a damaging effect on the organs and tissues of the human body. Antibodies are able to bind to these damaging agents and block them, i.e. neutralize. The diagnostic neutralization reaction is based on this feature of antibodies. It is carried out by introducing an antigen-antibody mixture into animals or into sensitive test objects (cell culture, embryos). For example, to detect toxins in the material of a patient, animals of the 1st group are injected with material from the patient. Animals of the 2nd group are injected with similar material, pre-treated with the appropriate antiserum. Animals of group 1 die if there is a toxin in the material. The second group of animals survives; the damaging effect of the toxin does not manifest itself, as it is neutralized.
13.6. Reactions using labeled antibodies or antigens
13.6.1. Immunofluorescence reaction (RIF, Koons method)
This method is used for express diagnostics. It can be used to detect both microbial antigens and antibodies.
Direct RIF method- an immune reaction of the interaction of antibodies with antigens, and the antibodies are labeled with a fluorochrome - a substance capable of emitting light quanta of a certain wavelength when exposed to light of a certain wavelength. The peculiarity of this method is the need to remove unreacted components in order to exclude the detection of nonspecific luminescence. To do this, wash off unreacted antibodies. The results are assessed using a fluorescence microscope. Bacteria in a smear treated with such a luminescent serum glow against a dark background along the periphery of the cell.
Indirect RIF method is used more often than the previous one. This reaction is carried out in two stages. At the first stage, antigens mutually
interact with the corresponding antibodies, forming immune complexes. All components that have not reacted (i.e., are not part of immune complexes) must be removed by washing. At the second stage, the resulting antigen-antibody complex is detected using fluorochromated antiglobulin serum. As a result, a complex of microbe + antimicrobial rabbit antibodies + antibodies to rabbit immunoglobulins, labeled with fluorochrome, is formed. The results are assessed using a fluorescence microscope.
13.6.2. Enzyme immunosorbent method or assay
ELISA - the most common modern method, used for the diagnosis of viral, bacterial, protozoal infections, in particular for the diagnosis of HIV infection, viral hepatitis and etc.
There are a lot of ELISA modifications. Solid-phase non-competitive ELISA is widely used. It is carried out in 96-well polystyrene plates (solid phase). When carrying out a reaction, it is necessary to wash off unreacted components at each stage. When determining antibodies, the test blood serum is added to the wells on which antigens are sorbed, then antiglobulin serum labeled with an enzyme. The reaction is carried out by adding a substrate for the enzyme. In the presence of an enzyme, the substrate changes, and the enzyme-substrate complex is selected so that the product formed in the reaction is colored. Thus, with a positive reaction, a change in the color of the solution is observed. To determine antigens, a solid-phase carrier is sensitized with antibodies, then the test material (antigens) and enzyme-labeled serum to the antigens are sequentially added. For the reaction to occur, a substrate for the enzyme is added. A change in the color of the solution occurs with a positive reaction.
13.6.3. Immunoblotting
This method is based on a combination of electrophoresis and ELISA. When performing immunoblotting (blotting from English. blot- spot) a complex mixture of antigens is first subjected to electrophoresis in a polyacrylamide gel. The resulting fractionated anti-
gene peptides are transferred to a nitrocellulose membrane. The blots are then treated with enzyme-labeled antibodies to a specific antigen, i.e. carry out ELISA blot. Immunoblotting is used in the diagnosis of infections such as HIV.
13.6.4. Immune electron microscopy
The method consists of microscopy in electron microscope viruses (less commonly other microbes), pre-treated with appropriate immune serum labeled with electron-optically dense preparations, for example ferritin, an iron-containing protein.
13.7. Flow cytometry
Blood cells are differentiated based on laser cytofluorometry. To do this, the desired cells are stained with fluorescent monoclonal antibodies to CD antigens. The blood sample, after being treated with labeled antibodies, is passed through a thin tube and a laser beam is passed through it, which excites the fluorochrome to glow. Fluorescence intensity correlates with the density of antigens on the cell surface and can be quantitatively measured using a photomultiplier tube. The results obtained are converted into a histogram.
Flow cytometry is used to determine immune status(content of the main populations of lymphocytes, content of intracellular and extracellular cytokines, functional activity of NK cells, phagocytosis activity, etc.).
IMMUNOMICROBIOLOGICAL STUDIES
Immunological methods are used to solve many problems:
1. Condition assessment immune system person (immune status) by determining quantitative and functional characteristics cells of the immune system and their products.
2. Determination of the composition and characteristics of human tissues: blood groups, Rh factor, transplant antigens.
3. Diagnosis of infectious diseases and resistance to them by detecting and establishing antibody titers (serodiagnosis), identifying pathogen antigens in the body, and determining cellular reactions to these antigens.
4. Seroid identification of cultures of bacteria and viruses isolated from the body of humans and animals.
5. Detection in the human body and in external environment any substances with antigenic or hapten properties (hormones, enzymes, poisons, medicines, drugs, etc.).
6. Identification of immunopathological conditions, allergies, transplantation and antitumor reactions.
The process of interaction between antigen and antibody serological reactions occurs in two phases:
1) specific- the interaction phase in which a complementary combination of the active centers of antibodies (paratopes) and antigen epitopes occurs. Typically this phase lasts a few seconds or minutes;
2) nonspecific- manifestation phase, characterized external signs formation of immune complexes. This phase can develop from several minutes to several hours.
The optimal specific interaction of antibodies with antigen occurs in an isotonic solution with a pH close to neutral. The antigen-antibody reaction in an in vitro system can be accompanied by the occurrence of several phenomena
· agglutination,
· precipitation,
· lysis.
External manifestations reactions depend on the physicochemical properties of the antigen (particle size, physical state), class and type of antibodies (complete and incomplete), as well as experimental conditions (medium consistency, salt concentration, pH, temperature).
The polyvalency of antigens and antibodies ensures the formation of aggregates visible to the naked eye. This occurs in accordance with the theory of network formation, according to which other antibody and antigen molecules are sequentially attached to the resulting antigen-antibody complex. As a result, network structures are formed, which turn into aggregates that precipitate. The nature and severity of the reaction depend on the quantitative ratio of antigens and antibodies. Reactions are most intense when the reactants are in equivalent proportions.
Prerequisite formation of a lattice (network) - the presence of more than three antigenic determinants for each antigen molecule and two active centers for each antibody molecule. Antigen molecules are lattice nodes, and antibody molecules are connecting links. The region of optimal ratios (equivalence zone) of antigen and antibody concentrations, when neither free antigens nor free antibodies are detected in the supernatant after sediment formation.
Aggregates that can precipitate are formed when antigens combine with full antibodies. Incomplete antibodies (monovalent) do not cause the formation of network structures and large aggregates. To detect such antibodies, use special methods based on the use of antiglobulins (Coombs reaction).
Serological tests, due to their high specificity and sensitivity, are used to detect and quantification antigens and antibodies. The amount of immunoreagents in reactions is expressed by titer - the maximum dilution of serum or antigen at which a reaction is still observed.
Serological reactions in microbiological and immunological laboratories are used for two purposes:
1) for seroidentification of microorganisms, toxins, antigens in general using a known antibody (immune diagnostic serum),
2) for serodiagnosis - determining the nature of the antibody in the patient’s blood serum for bacterial, viral, and less often other infectious diseases using a known antigen (diagnosticum).
To determine the generic, species and type of antigen, known immune diagnostic sera are required. They are obtained by repeated administration to animals (usually rabbits) in increasing doses of killed or live microorganisms, their decay products, neutralized or native toxins. After a certain cycle of immunization of animals, massive bloodletting or total bleeding of the animal is performed. Blood collected in a sterile container is first placed in a thermostat at a temperature of 37°C for 4 - 6 hours to accelerate clotting, then in an icebox for a day. The resulting transparent serum is sucked into a sterile container, preservatives are added, the antibody titer is determined, checked for sterility and poured into ampoules.
Are used non-adsorbed And adsorbed diagnostic sera. Unadsorbed serums have high titers antibodies, but are capable of giving group (cross) reactions.
Adsorbed sera are characterized by strict specificity of action (they react only with a homologous antigen). Sera containing antibodies to only one specific antigen are called monoreceptor.
They also produce serums labeled with fluorochromes, enzymes, and radioisotopes, which allow even traces of the antigen to be detected with a high degree of accuracy.
Suspensions of living or killed bacteria, their breakdown products, toxins, and viruses are used as antigens (diagnosticums) in serological reactions. In some cases, extracts or chemically isolated antigens from microorganisms and animal tissues are used.
All immunomicrobiological methods can be divided into 3 groups:
1) based on direct interaction of antigen with antibody(phenomena of agglutination, precipitation, hemagglutination, immobilization, etc.);
2) based on mediated interaction of antigen with antibody(reactions indirect hemagglutination, coagglutination, latex agglutination, carbon agglomeration, bentonite agglutination, complement fixation, etc.);
3) using labeled antibodies or antigens(fluorescent antibody method, enzyme-linked immunosorbent and radioimmunoassays and other methods).
AGGLUTINATION REACTIONS
These reactions involve antigens in the form of particles (microbial cells, red blood cells and other corpuscular antigens), which are glued together by antibodies and precipitate.
To perform an agglutination reaction(RA) three components are needed: 1) antigen (agglutinogen);
2) antibody (agglutinin)
3) electrolyte (isotonic sodium chloride solution).
Approximate agglutination reaction (RA)
An indicative, or plate, RA is placed on a glass slide at room temperature. To do this, use a Pasteur pipette to apply a drop of serum at a dilution of 1:10 to 1:20 and a control drop of isotonic sodium chloride solution separately onto the glass. Colonies or a daily culture of bacteria (a drop of diagnosticum) are introduced into both bacteriological loops and mixed thoroughly. Reactions are taken into account visually after a few minutes, sometimes using a magnifying glass (x5). With positive RA, the appearance of large and small flakes in a drop of serum is noted; with negative RA, the serum remains uniformly cloudy.
Detailed agglutination reaction in order to identify the titer of specific antibodies in a patient.
Full-blown RA for serodiagnosis is made in the serum of patients. It is also diluted in isotonic sodium chloride solution from 1:50 - 1:100 to 1:800 or 1: 1600. Since lower serum titers may contain normal agglutinins found in healthy people or patients with another diagnosis (diagnostic titer). As an antigen in this reaction, diagnosticums are used - known suspensions, usually of killed bacteria.
1 ml of isotonic sodium chloride solution is first poured into agglutination tubes. 1 ml of serum diluted 1:100 is added to the first of them, and after mixing it, 1 ml is transferred to the second, from the second to the third, etc. 1-2 drops of a bacterial suspension containing 3 billion microbial bodies in 1 ml are added to the resulting two-fold dilutions of sera (from 1:100 to 1:1600 or more). The tubes are shaken and placed in a thermostat at 37°C for 2 hours, then kept at room temperature for 24 hours.
The detailed agglutination reaction is taken into account by evaluating each test tube sequentially, starting with the control ones, with gentle shaking. There should be no agglutination in control tubes. The intensity of the agglutination reaction is marked with the following signs: ++++ - complete agglutination (agglutinate flakes in an absolute transparent liquid); +++ - incomplete agglutination (flakes in a slightly opalescent liquid); ++ - partial agglutination (flakes are clearly visible, the liquid is slightly cloudy); + - weak, questionable agglutination - the liquid is very cloudy, the flakes in it are difficult to distinguish; - - absence of agglutination (the liquid is uniformly cloudy).
The serum titer is taken to be its last dilution, in which the intensity of agglutination is assessed as no less than two pluses (++)
Agglutination reaction Agglutination reaction
(RA) is a method for identifying and quantifying Ag and Ab, based on their ability to form agglomerates visible to the naked eye. In the department of infectious diseases. diseases or for other purposes is used to identify unknown microbes and cells, to determine the presence and amount of Ab in blood and other liquids. The determination principle is based on the specificity of the interaction between Ag and Ab and consists in finding the known from the unknown. There are many options for RA: quantitative and qualitative, test tube and glass, volumetric and droplet, conventional, accelerated and express methods. To stage RA you need: 1) s-ka blood. In the variant with determining the type (var) of bacteria, industrial agglutinating tests are used, produced by immunizing rabbits. In the variant with determination of the type of Ab, a blood sample is taken from the test. people or animals. The solution must be sterile and free of suspended particles. Prepare the basic dilution in saline solution. It should be 2-4 times lower than the diagnostic titer for this disease; 2) Ag. In the version of the reaction with determination of the Ab type, industrial diagnostic kits are used; in the variant with the determination of Ag, diagnosticums are prepared themselves in the form of a 1-3 billion suspension in a saline solution of 18-20-hour agar (less often broth) test. microbe inactivated by heating in a water bath at 70°C for 1 hour or by 24-hour incubation at 37°C with formaldehyde (final concentration 0.2%); 3) electrolyte in the form of saline solution. Staging technique volumetric serial tube RA to determine the Ab titer in s-ki: several rows of working dilutions are prepared from the main dilution of s-ki. The number of rows depends on the number of diagnosticums taken into the experiment; the number and dilution factors are determined by the diagnostic titer of the suspected disease. The series must at least contain a dilution corresponding to the diagnostic Ab titer, two dilutions below and two dilutions above it. for example, if the diagnostic titer is 1:100, then with the volumetric method of staging RA the following dilutions should be prepared: 1:25, 1:50, 1:100, 1:200, 1"400; with the drip method, the first dilution (1:25) is not necessary, but another higher dilution is required - 1:800 B. scientific research s-ku is titrated to. It is diluted as follows: 0.25 ml of saline solution is poured into all test tubes, except the 1st one, when the reaction is carried out in a volume of 0.5 ml, and 0.5 ml when the reaction is carried out in a volume of 1 ml. Pour 0.25 (0.5) ml of the main dilution into the 1st and 2nd test tubes, from the 2nd test tube, into the cut volume and breeding s-k and increased 2 times, 0.25 (0.5) ml is transferred to the 3rd, from the 3rd to the 4th, etc. to the last, from the cut 0.25 (0.5) ml is poured into everything to balance the volumes. Each dilution is carried out using a separate pipette. If several diagnosticums are taken into experiment, then for each of them its own series of dilutions is prepared in the same way. Diagnosticum is added to each dilution of the test tube in a volume equal to the volume of the test tube, as a result of which the dilution in each test tube is doubled. The experiment corresponds to the s-ki control (0.25 - 0.5 ml of the main dilution of s-ki and the same amount of saline solution) and the Ag control (0.25 - 0.5 ml of diagnosticum and the same amount of saline solution). If several diagnosticums are used in the experiment, then each has its own antigen control. The rack with test tubes is shaken well and placed in a thermostat at 37°C for 4 hours, and then left at room temperature until the next day, after which the PA is recorded based on the amount of sediment and the degree of clearing of the liquid. Determination of these indicators, depending on the nature of the agglutinates, is carried out with the naked eye against a dark background, in an agglutinoscope or over the concave surface of a microscope mirror. Accounting begins with controls: control C should be transparent, Ag should be uniformly cloudy (after shaking the tube). If the controls are good, establish the presence and degree of agglutination in all test tubes, which are designated by pluses: large sediment and complete clearing of the liquid - 4 pluses; large sediment and incomplete clearing of the liquid - 3 pluses; noticeable sediment and noticeable clearing of the liquid are 2 pluses. After this, the titer is determined: the highest dilution with an agglutination intensity of at least 2 pluses. Titre research s-ki are compared with the diagnostic titer for this disease. If the titer is examined. s-ki is 2 times lower than the diagnostic value, the reaction is assessed as doubtful; if the titer is equal diagnostic - how weakly positive; if it is 2-4 times higher, it is considered positive; if it is 8 or more times higher, it is considered sharply positive. When Ab is widespread in healthy people, an increase in Ab titer is used to assess RA. To determine the type of Ar in serial RA, the number of rows must correspond to the number taken for identification diagnostic tests. From the main dilution of the diagnostic test, a series of successive two-fold dilutions are prepared in the same way as in RA to determine the Ab titer. Dilution factors depend on the titer of the agglutinating test. In the experiment, it is necessary to have a dilution equal to the titer of the test, as well as 2, 4, 6, 8 times lower than it. For example, if the titer of the diagnostic test is 1 3200, then you should use dilutions 1 3200, 1 1600, 1 800, 1 400, 1 200 The same volume of tested Ag is added to the dilutions of the test, as a result, the dilution of the test increases by 2 times. 2 controls of test and Ag are added to the experiment. If in the experiment several s-k are involved, then each of them needs its own control. Upon completion of the reaction, the stand is shaken vigorously and placed in a thermostat at 37 ° C. The results are taken into account as described above. Evaluation of the reaction has features. In order to draw a conclusion about the compliance of the study. Ag taken into the experiment, the titer of the reaction must correspond to at least half the titer of the standard diagnostic test. Titers of 1 4 and below are considered as a group reaction Drip md staging of RA differs from volumetric in that s-ku is diluted in a volume of 1 ml, Ag is used in a higher concentration (10 billion/ml) and it is added 1 - 2 drops into a test tube Dilution of the drug after adding Ag is considered unchanged. Otherwise, the method of setting, recording and evaluation is similar to the volumetric method
(Source: Dictionary of Microbiology Terms)
Agglutination reaction.
An agglutination reaction is the gluing and precipitation of microbial or other cells (erythrocytes) under the influence of antibodies in the presence of an electrolyte. The visible effect of the reaction (agglutination phenomenon) is the formation of a precipitate called agglutinate.
This reaction is used for serodiagnosis And seroidentification. RA is used for serodiagnosis (detection of antibodies in the blood serum of patients) typhoid fever and paratyphoid(Vidal reaction), brucellosis(Wright's reaction) tularemia and leptospirosis. RA is used for seroidentification (determining the type of pathogen isolated from a patient) when intestinal infections, whooping cough, cholera and etc.
Componentsreactions:
1. A ntigen (agglutinogen) – these are whole (not destroyed) microbial or other cells ( corpuscular, insoluble antigen). Agglutinogens- this is a suspension alive or killed microbial cells or any other cells. Antigens can be either unknown or known. An unknown agglutinogen is a microbial culture isolated from the patient’s body that needs to be determined. Known antigen – diagnosticum– diagnostic drug - suspension of the dead microbes known species in saline solution. This suspension cloudy (opaque), because microbial cells do not dissolve, but remain intact. A known agglutinogen will be used to detect unknown antibodies in the blood serum of patients.
2. Antibody (agglutinin)- found in blood serum. Antibodies can also be either unknown or known. Unknown antibodies to be determined are in the blood serum sick person. Known antibodies are found in immune diagnostic sera which are called agglutinating sera. They are used for sero-identification, i.e. to determine an unknown antigen - a type of microbial culture.
3. Electrolyte– 0.9% sodium chloride solution.
Methods for staging RA.
1. Approximate (lamellar) RA– carried out on glass. Apply 2 drops of serum and 1 drop of isotonic solution to a glass slide. A microbial culture is added in a loop into one of the drops of serum and into a drop of isotonic solution and mixed. A drop of isotonic solution with germs – antigen control, a drop germ-free serums – antibody control, a drop serums with microbes – experience. If the serum contains antibodies corresponding to microbial antigens that mix with it, then the antibodies and antigens will specifically bind to each other and after 1–3 minutes agglutinate flakes will appear in the test drop. The antigen control should be cloudy and the antibody control should be clear. The results of the reaction are recorded based on the appearance of agglutinate flakes . If flakes fall out, the reaction is positive, i.e. An antigen corresponds to an antibody and the antigen can be used to determine the antibody or vice versa. If cloudiness remains, the reaction is negative.
2. Detailed agglutination reaction - carried out in test tubes. First, prepare 2-fold dilutions of the blood serum of a sick person from 1:50 to 1:1600. 1 ml of isotonic sodium chloride solution is poured into 6 test tubes. 1 ml of the patient’s blood serum at a dilution of 1:50 is added to the first test tube, mixed and a dilution of 1:100 is obtained, then 1 ml of a dilution of 1:100 is transferred to the second test tube and a dilution of 1:200 is obtained, etc. Two tubes are kept for antigen and serum control. To the serum control add only serum at a dilution of 1:50, to the antigen control - only the antigen. Add 0.1 ml of antigen - diagnosticum (O- or H-) to all other test tubes and place all test tubes in a thermostat at 37°C for 18-20 hours. The results of the reaction are recorded based on the nature, amount of precipitate (agglutinate) formed and the degree of turbidity. Accounting is carried out only with the following results in the controls: serum control - transparent, antigen control - cloudy. O-antibodies give a fine-grained precipitate. H-antibodies – coarse-grained. Based on the last test tube in which the agglutination reaction is still visible, it is established diagnostic titer.
When serodiagnosis of diseases, it is important not only to detect specific antibodies to a particular pathogen, but also to identify their quantity, i.e. establish such an antibody titer when we can talk about the presence of a disease caused by this pathogen. This titer is called the diagnostic titer. For example, to diagnose typhoid fever, you need to identify an antibody titer of 1:400, but not less. Even more accurate results are obtained by detecting the increase in antibodies in paired sera. The patient’s serum is collected at the onset of the disease and after 3–5 or more days. If the antibody titer increases at least 4 times, therefore, we can talk about the current disease.
