The indirect (passive) hemagglutination reaction (IRHA) is based on the fact that red blood cells, if a soluble antigen is adsorbed on their surface, acquire the ability to agglutinate when interacting with antibodies to the adsorbed antigen. The RNGA diagram is shown in Fig. 34. RNGA is widely used in the diagnosis of a number of infections.
Rice. 34. Scheme of the passive hemagglutination reaction (RPHA). A - obtaining an erythrocyte diagnosticum: B - RPGA: 1 - erythrocyte: 2 - antigen being studied; 3 - erythrocyte diagnosticum; 4 - antibody to the antigen being studied: 5 - agglutinate
Setting up a reaction. The test serum is heated for 30 minutes at 56° C, diluted sequentially in a ratio of 1:10 - 1:1280 and poured into 0.25 ml into test tubes or wells, where 2 drops of erythrocyte diagnosticum (erythrocytes with antigen adsorbed on them) are then added.
Controls: a suspension of erythrocyte diagnosticum with known immune serum; suspension of diagnosticum with normal serum; a suspension of normal red blood cells with test serum. In the first control, agglutination should occur, in the second and third it should not occur.
Using RIGA, you can detect an unknown antigen if known antibodies are adsorbed onto red blood cells.
The hemagglutination reaction can be performed in a volume of 0.025 ml (micromethod) using a Takachi microtitrator.
1. What does a positive X-ray analysis result indicate between red blood cells and the material being tested for the presence of the virus?
2. Will agglutination of red blood cells occur if a virus and its corresponding serum are added to them? What is the name of the reaction that reveals this phenomenon?
Exercise
Take into account and register the result RIGA.
Precipitation reaction
In the precipitation reaction, a specific immune complex is precipitated, consisting of a soluble antigen (lysate, extract, hapten) and a specific antibody in the presence of electrolytes.
The cloudy ring or precipitate formed as a result of this reaction is called precipitate. This reaction differs mainly from the agglutination reaction in the size of the antigen particles.
The precipitation reaction is usually used to determine antigen in the diagnosis of a number of infections ( anthrax, meningitis, etc.); in forensic medicine - to determine the species of blood, sperm, etc.; in sanitary and hygienic studies - when establishing falsification of products; with its help, the phylogenetic relationship of animals and plants is determined. For the reaction you need:
1. Antibodies (precipitins) - immune serum with a high titer of antibodies (not lower than 1:100,000). The titer of the precipitating serum is determined by the highest dilution of the antigen with which it reacts. The serum is usually used undiluted or in a dilution of 1:5 - 1:10.
2. Antigen - dissolved substances of protein or lipoid polysaccharide nature (full antigens and haptens).
3. Isotonic solution.
The main methods for carrying out the precipitation reaction are: ring precipitation reaction and precipitation reaction in agar (gel).
Attention! All components involved in the precipitation reaction must be completely transparent.
Ring precipitation reaction. Using a Pasteur pipette, add 0.2-0.3 ml (5-6 drops) of serum into the precipitation tube (the serum should not get on the walls of the tube). The antigen in the same volume is carefully layered onto the serum, pouring it with a thin Pasteur pipette along the wall of the test tube. The test tube is kept in an inclined position. When properly layered, there should be a clear boundary between the serum and the antigen. Carefully, so as not to mix the liquid, place the test tube in a stand. If the reaction is positive, a cloudy “ring” is formed at the interface of the antigen and antibody - a precipitate (see Fig. 48).
The reaction is accompanied by a number of controls (Table 18). The sequence of adding reaction ingredients into the test tube is very important. You cannot layer serum on the antigen (in the control - on an isotonic solution), since the relative density of the serum is greater, it will sink to the bottom of the test tube, and the boundary between the liquids will not be revealed.
Table 18. Scheme for setting up the ring precipitation reaction
Note. + presence of a “ring”; - absence of a “ring”.
The results are recorded after 5-30 minutes, in some cases after an hour, as always starting with the controls. The “ring” in the 2nd test tube indicates the ability of the immune serum to enter into specific reaction with the corresponding antigen. There should be no “rings” in the 3-5 test tubes - there are no antibodies and antigens corresponding to each other. A “ring” in the 1st tube - a positive reaction result - indicates that the test antigen corresponds to the taken immune serum, the absence of a “ring” (a “ring” only in the 2nd tube) indicates their inconsistency - a negative reaction result.
Precipitation reaction in agar (gel). The peculiarity of the reaction is that the interaction of antigen and antibody occurs in a dense medium, i.e., in a gel. The resulting precipitate gives a turbid streak in the thickness of the medium. The absence of a band indicates a discrepancy between the reaction components. This reaction is widely used in biomedical research, in particular in the study of toxin formation in the causative agent of diphtheria.
Control questions
1. What is the main difference between agglutination and precipitation reactions?
2. Why can't cloudy ingredients be used in the precipitation reaction?
Exercise
1. Set up the ring precipitation reaction and sketch the result.
2. Study the nature of the interaction of antigen with antibody in the precipitation reaction in agar, sketch the result (get a cup from your teacher).
Lysis reaction (immune cytolysis)
Immune lysis is the dissolution of cells under the influence of antibodies when mandatory participation complement. For the reaction you need:
1. Antigen - microbes, red blood cells or other cells.
2. Antibody (lysine) - immune serum, less often patient serum. Bacteriolytic serum contains antibodies involved in the lysis of bacteria; hemolytic - hemolysins that promote the lysis of red blood cells; for the lysis of spirochetes, spirochetolysins are needed, cells - itolysins, etc.
3. Complement. Most complement in serum guinea pigs. This serum (a mixture from several animals) is usually used as a complement. Fresh (native) complement is unstable and easily destroyed by heating, shaking, or storage, so it can be used no longer than two days after receipt. To preserve complement, 2% is added to it boric acid and 3% sodium sulfate. This complement can be stored at 4°C for up to two weeks. Dry complement is most often used. Before use, it is dissolved in an isotonic solution to the original volume (indicated on the label).
4. Isotonic solution.
Hemolysis reaction(Table 19). For the reaction you need:
1. Antigen - 3% suspension of washed sheep erythrocytes at the rate of 0.3 ml of erythrocyte sediment and 9.7 ml of isotonic solution.
2. Antibody - hemolytic serum (hemolysin) against sheep erythrocytes; usually prepared in production, lyophilized and the titer indicated on the label.
Hemolysin titer is the highest dilution of serum at which complete hemolysis of a 3% suspension of red blood cells occurs in the presence of complement. For the hemolysis reaction, hemolysin is taken in triple titer, i.e. diluted 3 times less than before the titer. For example, with a serum titer of 1:1200, the serum is diluted 1:400 (0.1 ml of serum * and 39.9 ml of isotonic solution). Excess hemolysin is necessary, since some of it can be adsorbed by other reaction components.
* (You should not take less than 0.1 ml of serum - the accuracy of the measurement will suffer.)
3. Complement is diluted 1:10 (0.2 ml of complement and 1.8 ml of isotonic solution).
4. Isotonic solution.
Table 19. Hemolysis reaction scheme
Accounting for results. If the reaction is carried out correctly, hemolysis will occur in the 1st test tube - its contents will become transparent. In the controls, the liquid remains cloudy: in the 2nd tube there is not enough complement for hemolysis to occur, in the 3rd tube there is no hemolysin, in the 4th tube there is neither hemolysin nor complement, in the 5th tube the antigen does not match the antibody,
If necessary, hemolytic serum is titrated according to following diagram(Table 20).
Before titration, prepare an initial serum dilution of 1:100 (0.1 ml of serum and 9.9 ml of isotonic solution), from which necessary dilutions, For example:
From these dilutions, add 0.5 ml of serum into titration test tubes, as shown in Table. 20.
Table 20. Titration scheme for hemolytic serum (hemolysin)
In the example given in table. 20, the titer of hemolytic serum is 1:1200.
When using fresh hemolytic serum, it must be inactivated to destroy the complement present in it. To do this, it is heated for 30 minutes at 56 ° C in a water bath or in an inactivator with a thermostat. The latter method is better: it eliminates the possibility of overheating the whey, i.e., its denaturation. Denatured sera are unsuitable for testing.
Bacteriolysis reaction. In this reaction, complement lyses bacteria in the presence of appropriate (homologous) serum. The reaction scheme is fundamentally similar to the hemolysis reaction scheme. The difference is that after a two-hour incubation, all test tubes are seeded onto Petri dishes with a medium favorable for the microorganism taken into the experiment to find out whether it is lysed. If the experiment is carried out correctly, cultures from 2-5 test tubes (controls) should show abundant growth. Lack of growth or weak growth in inoculation from the 1st test tube (experiment) indicates the death of microbes, i.e., that they are homologous to the antibody.
Attention! The bacteriolysis reaction must be carried out under aseptic conditions.
Control questions
1. What will happen to red blood cells if distilled water is used instead of isotonic sodium chloride solution? What is the basis of this phenomenon?
2. What reaction will occur when erythrocytes interact with homologous immune serum in the absence of complement?
Exercise
Set up the hemolysis reaction. Record and sketch the result.
Related information.
It uses red blood cells or neutral synthetic materials(for example, latex particles), on the surface of which antigens (bacterial, viral, tissue) or antibodies are sorbed. Their agglutination occurs when appropriate sera or antigens are added. Red blood cells sensitized with antigens are called antigenic erythrocyte diagnosticum and are used to detect and titrate antibodies. Erythrocytes sensitized with antibodies. are called immunoglobulin erythrocyte diagnosticums and are used to detect antigens.
The passive hemagglutination reaction is used to diagnose diseases caused by bacteria (typhoid and paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.), protozoa (malaria) and viruses (influenza, adenoviral infections, viral hepatitis B, measles, tick-borne encephalitis, Crimean hemorrhagic fever, etc.), as well as for determining certain hormones, identifying hypersensitivity patient to medicines and hormones such as penicillin and insulin.
Passive hemagglutination reaction (RPHA). The passive hemagglutination test is a sensitive method serological diagnostics and is used for both early and retrospective diagnosis, as well as to determine the immunopogic state of vaccinated people. In patients with tularemia, antibodies are usually detected at the end of the 1st or 2nd week of the disease; after 1-1.5 months, RPHA titers reach maximum performance(1:100000-1:20000, rarely higher), after which they decrease and remain at the level of 1:100-1:200 for a long time.
In vaccinated people, antibodies are also constantly detected, however, in lower titers, not exceeding 1:2000-1:5000 1-1.5 months after vaccination, and remain for several years at a low level of 1:20-1:80.
The antigen for staging RPHA is tularemia erythrocyte diagnosticum (antigenic). The drug is formalinized sheep red blood cells, sensitized with tularemia antigen, available in liquid and dry form. Liquid preparation - a 10% suspension of red blood cells in a formaldehyde solution of 10% concentration. Dry lyophilized preparation is a vacuum-dried 10% suspension of red blood cells without a preservative. Before use, it is diluted according to the directions on the label. To set up the reaction in polystyrene plates, both drugs are used in a 2.5% concentration, and when setting up the reaction in microvolumes - at a 0.5% concentration.
Technique for setting up RPGA. The test sera are diluted with physiological solution 1:5 (1:10) and heated at 56 degrees C for 30 minutes. After this, in order to remove heterogeneous antibodies to sheep erythrocytes, the sera are treated with a 50% suspension of formalinized sheep erythrocytes. To do this, add red blood cells at the rate of 2 drops (0.05 ml) per 1 ml of serum and mix thoroughly by shaking. The serum is left until the erythrocytes have completely settled, or is centrifuged after one hour at room temperature, after which it is ready for examination.
The dilution liquid is poured in a volume of 0.5 ml into a row of wells on a polystyrene plate. During the preliminary study of sera, it is advisable to test them by setting up the reaction in a short row of the plate (6-wells). If antibodies are detected in a short series, the sera are retested in a long series of dilutions (12 wells). After spilling the dilution liquid, add 0.5 ml of test sera in a 1:5 dilution to the first well of each row (short or long). Then the same volumes of serum are titrated with twofold dilutions. Thus, serum dilutions are obtained in the short series from 1:10 to 1:320, and in the long series from 1:10 to 1:20480. After titration of the sera, one drop (0.05 ml) of a working 2.5% suspension of sensitized erythrocytes is added to each well. The contents of the plates are thoroughly shaken until a homogeneous suspension is obtained. The plates are left at room temperature on a stationary table surface. Preliminary recording of the reaction is carried out after 2-3 hours, the final determination of the titer is made after complete sedimentation of red blood cells in the wells. The following controls are provided for the reaction: 1) test serum diluted 1:10 in a volume of 0.5 ml + 1 drop of a 2.5% suspension of unsensitized erythrocytes; 2) dilution liquid in a volume of 0.5 ml + 1 drop of a 2.5% suspension of unsensitized erythrocytes; 3) dilution liquid in a volume of 0.5 ml + 1 drop of a 2.5% suspension of sensitized erythrocytes. All controls should give a clearly negative reaction.
Accounting and assessment of RPGA. The reaction is assessed according to the following scheme:
1) sharply positive reaction(++++) - red blood cells fall to the bottom of the hole in an even layer in the form of an “umbrella”, which often has a scalloped melting of the edges;
2) positive reaction (+++) - red blood cells cover at least 2/3 of the bottom of the well;
3) weakly positive reaction (++) - the agglutinate is small and located in the very center of the well;
4) questionable reaction (+) - around the sediment of erythrocytes in the very center of the well there are individual grains of agglutinate;
5) negative (-) - at the bottom of the hole, red blood cells settle in the form of a “button” or a small ring with smooth, sharply defined edges.
The serum titer is taken into account based on the last dilution of the serum, which gave a very clear reaction (at least three pluses). A dilution of 1:100 or higher is considered a diagnostic titer; however, just as in the case of RA, it is necessary to monitor its increase.
RPGA for tularemia is quite specific and detects some cross reactions only with brucellosis serums. Differential diagnosis possible by the height of titers in RPGA, which are significantly higher with the homologous antigen.
Technique for setting up RPHA in microvolumes. RPGA can be performed in microvolumes using a Takachi-type microtitrator (or round-bottomed microplates with micropipettes), which allows titration of material in volumes of 25 μl and 50 μl. The reaction technique and the sequence of all operations are the same as when studying in polystyrene plates. It should, however, be borne in mind that the sensitivity of the micromethod is usually one dilution (i.e., 2 times) lower than that of the macromethod.
To set up the reaction in a microtitrator, a dilution liquid in a volume of 50 μl is added to each well using a dropper pipette. Then, using titrators with a 50 μl head, the test serum is collected by immersing the head in it. Make sure that the liquid has filled the titrator head. The titrator with serum is transferred to the first well and, holding it in vertical position, do several rotational movements round trip. Then the titrator is transferred to the next well and the manipulation is repeated. Titration can be carried out simultaneously in several rows. After titrating the entire series, the titrator is washed with distilled water (changing 2 portions) by rotating movements, the water is removed from the head using a swab and burned on a burner flame.
After titration, add 25 μl of erythrocyte diagnostic fluid to the wells. The concentration of diagnosticum for RPHA in microvolumes should be 0.5% (i.e., a 2.5% suspension of red blood cells is additionally diluted 5 times). After adding red blood cells, the plates should be shaken slightly until a homogeneous suspension is obtained. The results can be recorded within 1-1.5 hours, which is a significant advantage of RPGA in a microtiter. In addition, this technique requires small quantities of all reaction ingredients and test sera.
The reaction is taken into account according to the following scheme:
1) “+” – complete hemagglutination, in which red blood cells fall to the bottom of the well in an even layer in the form of an “umbrella”, occupying at least 2/3 of the bottom;
2) “+-“ - partial hemagglutination, in which red blood cells fall to the bottom in the form of a loose ring of small size;
3) “-“ – absence of hemagglutination, when red blood cells fall to the bottom in the form of a small button or ring with a smooth edge.
Specificity positive result, obtained in RPHA, can be tested using a three-component reaction - the reaction of inhibition of passive hemagglutination (RPHA).
Technique for setting up RTPGA. This reaction is used to confirm the specificity of a positive RPGA result when it is in doubt or is of particular epidemiological interest. The mechanism of the reaction is the specific inhibition of hemagglutination when a suspension of killed tularemia bacteria is added to the test serum. Three components interact in the reaction: test serum, specific tularemia antigen and antigenic erythrocyte diagnosticum RTPHA is usually placed in a row of 7-8 wells. It is advisable to install a repeated RPGA in parallel with the RTPGA. 0.25 ml of dilution liquid is poured into two rows of wells, then the test serum in a volume of 0.25 ml is added to the first wells of both rows and titration is performed. Two identical rows of serum dilutions are obtained. Add 0.25 ml of dilution liquid to all wells of the second row, and 0.25 ml of a suspension of tularemia bacteria to the wells of the first row. Tularemia diagnosticum is used (containing 25 billion tularemia bacteria in 1 ml), previously diluted 50 times. This suspension contains 500 million bacteria in 1 ml or 125 million in a volume of 0.25 ml. After adding the antigen, the plate is left for 1 hour at room temperature, after which one drop (0.05 ml) of erythrocyte diagnosticum is added to all wells of both rows, the plate is shaken and left on a flat table surface. Accounting is carried out after 2-3 hours.
Accounting and assessment of RTPGA. If the test serum contains specific tularemia antibodies, they are neutralized by the added antigen and hemagglutination will not occur in the first row of wells, or, if high titer serum, hemagglutination will be observed in a smaller (2-4) number of wells than in the row with RPGA. In this case, the specificity of the results was confirmed. If hemagglutination is noted in both rows, i.e. If the results of RTPGA and RPGA coincide, this indicates the absence of tularemia antibodies in the test serum. In this case, the primary result of RPGA is considered nonspecific.
Technique for staging RTHG in microvolumes. RTPGA, like RPGA, can be performed in microvolumes using a Takachi-type microtiter. To do this, add 0.25 μl of diluting liquid into the wells of microplates in two rows of 7-8 wells each. Then, using a titrator, 0.25 μl of the test serum is added and titrated in both rows. After this, 25 μl of tularemia antigen (the concentration of which is 500 million tularemia bacteria in 1 ml) is added to each well in the first row, and 25 μl of dilution liquid is added to the second row. The plates are left for 1 hour at room temperature, after which 25 μl of antigenic zritrocytic diagnosticum (0.5% concentration) is added to all wells of both rows. Accounting and evaluation of the results are carried out similarly to reactions in macrovolumes.
Table of contents of the topic "Immunomodulators. Immunodiagnosis of infectious diseases.":Passive agglutination reactions. Indirect agglutination reactions. Indirect or passive hemagglutination reaction (IRHA, RPHA). Reverse RNGA. Passive hemagglutination inhibition reaction (PHA).
These reactions are called indirect (passive), since they use Ag (or AT) artificially sorbed on the surface of various corpuscular particles.
Indirect or passive hemagglutination reaction (RNGA, RPGA) is one of the most sensitive serological reactions. It is based on the ability of AT to interact with Ag fixed on various red blood cells, which then agglutinate. For greater stability of diagnosticums, red blood cells are formalinized.
Reverse RNGA used to detect Ag in blood serum; For this purpose, not Ag, but AT are fixed on erythrocytes. Reactions of this type are widely used to diagnose infectious diseases, establish pregnancy, detect hypersensitivity to drugs, etc.
Passive hemagglutination inhibition reaction (RTPGA) - further development RNGA; in a sense controls its specificity. Unlike RNGA, includes three components; Ag, AT and Ag (AT) adsorbed on erythrocytes. Initially, Ag reacts with AT (standard antiserum), then erythrocytes sensitized with the same Ag (or AT) are added to the mixture. If, during the interaction of Ag with AT, no free AT (or Ag) remains in the system, then agglutination of the erythrocyte diagnosticum is not observed.
11621 0
Serological reactions are designated in accordance with the phenomena accompanying the formation of the antigen-antibody complex during the interaction of components with different properties. There are reactions of agglutination, precipitation and lysis.
Agglutination reaction (RA)
The agglutination reaction (RA) is based on the use of a corpuscular antigen (a suspension of bacteria, sensitized erythrocytes, latex particles, etc.) interacting with specific antibodies, as a result of which the resulting antigen-antibody complex precipitates. This reaction is widely used in laboratory practice for serological diagnosis. bacterial infections and for identification of isolated microorganisms.
RA is used to diagnose many infectious diseases: brucellosis (Wright, Heddleson reaction), tularemia, leptospirosis (RAL - Leptospira agglutination and lysis reaction), listeriosis, typhus (RAR - Rickettsia agglutination reaction), shigellosis, yersiniosis, pseudotuberculosis, etc.
Indirect or passive agglutination reaction (RIGA or RPGA).
To stage this reaction, red blood cells of animals (sheep, monkey, guinea pigs, some birds) sensitized with antibodies or antigen are used, which is achieved by incubating a suspension of red blood cells and a solution of antigen or immune serum.
Diagnosticums obtained on the basis of erythrocytes sensitized with antigens are called antigen erythrocyte diagnosticums. They are intended for the determination of antibodies in serial dilutions of blood sera, for example, erythrocyte shigella diagnosticums, erythrocyte salmonella O-diagnosticums.
Accordingly, diagnostics based on erythrocytes sensitized with specific immunoglobulins are called antibodies(immunoglobulin) diagnosticums and they serve to detect antigens in various materials, for example, erythrocyte immunoglobulin diphtheria diagnosticum for RIGA, used to detect diphtheria exotoxin of corynebacteria in a liquid nutrient medium when material from the nose and oropharynx is inoculated into it.
The hemagglutination reaction is used to diagnose both bacterial (typhoid fever, paratyphoid fever, dysentery, brucellosis, plague, cholera, etc.) and viral (influenza, adenoviral infections, measles, etc.) infections. In terms of sensitivity and specificity, RIGA is superior to RA.
Hemagglutination inhibition reaction (HAI)
The hemagglutination inhibition reaction (HAI) is used to titrate antiviral antibodies in blood serum, as well as to establish the type of isolated viral cultures. RTGA can be used to diagnose those viral infections, the pathogens of which have hemagglutinating properties.
The principle of the method is that serum containing antibodies to a specific type of virus suppresses its hemagglutinating activity and the red blood cells remain non-agglutinated.
Passive hemagglutination inhibition (delay) reaction (RPHA).
Three components are involved in RTPGA: immune serum, antigen (test material) and sensitized erythrocytes.
If the test material contains an antigen that specifically reacts with immune antibodies standard serum, then it binds them, and with the subsequent addition of erythrocytes sensitized with an antigen homologous to serum, hemagglutination does not occur.
RTPHA is used to detect microbial antigens, for their quantitative determination, and also to control the specificity of RTPGA.
Latex agglutination reaction (RLA)
Latex particles are used as a carrier of antibodies (immunoglobulins). RLA is an express method for diagnosing infectious diseases, taking into account the time required (up to 10 minutes) and the ability to detect antigen in a small volume of test material.
RLA is used to indicate antigens of Streptococcus pneumoniae, Haemophilus influenzae type b, Neisseria meningitidis in cerebrospinal fluid, to detect group A streptococci in throat swabs, to diagnose salmonellosis, yersiniosis and other diseases. The sensitivity of the method is 1-10 ng/ml, or 10³ -10⁶ bacterial cells in 1 µl.
Coagglutination reaction (CoA)
The coagglutination reaction (CoA) is based on the ability of protein A of staphylococci to attach specific immunoglobulins. RCA - a method of express diagnostics - serves to identify soluble thermostable antigens in human secretions and in the composition of circulating immune complexes (CIC). Detection of specific antigens in the composition of the CEC requires their preliminary precipitation from blood serum.
Precipitation reaction
In the precipitation reaction (RP), as a result of the interaction of antibodies with highly dispersed soluble antigens (proteins, polysaccharides), complexes are formed with the participation of complement - precipitates. It is a sensitive test used to detect and characterize a variety of antigens and antibodies. The simplest example of high-quality RP is the formation of an opaque precipitation band in a test tube at the boundary of the antigen layering on the immune serum - the ring precipitation reaction. Various types of RP in semi-liquid agar or agarose gels are widely used (double immunodiffusion method, radial immunodiffusion method, immunoelectrophoresis).
Complement fixation reaction (CFR)
The complement fixation reaction (CFR) is based on the phenomenon of hemolysis with the participation of complement, i.e. capable of detecting only complement-fixing antibodies.
RSC is widely used for the diagnosis of many bacterial and viral infections, rickettsial infections, chlamydia, infectious mononucleosis, protozoal infections, helminthiases. RSK is complex serological reaction, in which two systems are involved: the test system (blood serum), represented by the antigen-antibody and complement system, and the hemolytic system (sheep red blood cells + hemolytic serum). Hemolytic serum is heat-inactivated rabbit blood serum immunized with sheep erythrocytes. It contains antibodies against sheep red blood cells.
A positive RSC result - the absence of hemolysis - is observed if the test serum contains antibodies homologous to the antigen. In this case, the resulting antigen-antibody complex binds complement, and in the absence of free complement, the addition of the hemolytic system is not accompanied by hemolysis. If there are no antibodies corresponding to the antigen in the serum, the formation of an antigen-antibody complex does not occur, complement remains free and the serum causes hemolysis of red blood cells, i.e. the presence of hemolysis is a negative result of the reaction.
Yushchuk N.D., Vengerov Yu.Ya.
Agglutination reaction - RA(from lat. agglutinatio- adhesion) is a simple reaction in which antibodies bind corpuscular antigens (bacteria, erythrocytes or other cells, insoluble particles with antigens adsorbed on them, as well as macromolecular aggregates). It occurs in the presence of electrolytes, for example, when an isotonic sodium chloride solution is added.
Apply various options agglutination reactions: extensive, indicative, indirect, etc. The agglutination reaction is manifested by the formation of flakes or sediment
RA is used for:
determination of antibodies in the blood serum of patients, for example, with brucellosis (Wright, Heddelson reactions), typhoid fever and paratyphoid fever (Vidal reaction) and others infectious diseases;
determination of the pathogen isolated from the patient;
determination of blood groups using monoclonal antibodies against erythrocyte alloantigens.
To determine antibodies in a patient putexpanded agglutination reaction: add to dilutions of the patient's blood serum diagnosticum(suspension of killed microbes) and after several hours of incubation at 37 °C, the highest serum dilution (serum titer) is noted, at which agglutination occurred, i.e., a precipitate formed.
The nature and speed of agglutination depend on the type of antigen and antibodies. An example is the peculiarities of interaction of diagnosticums (O- and R-antigens) with specific antibodies. Agglutination reaction with O-diagnosticum(bacteria killed by heat, retaining heat-stable 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.
If it is necessary to determine the pathogen isolated from the patient, put indicative agglutination reaction, using diagnostic antibodies (agglutinating serum), i.e., serotyping of the pathogen is carried out. An indicative reaction is carried out on a glass slide. A pure culture of the pathogen isolated from the patient is added to a drop of diagnostic agglutinating serum at a dilution of 1:10 or 1:20. A control is placed nearby: instead of serum, a drop of sodium chloride solution is applied. When a flocculent sediment appears in a drop containing serum and microbes, a extensive agglutination reaction in test tubes with increasing dilutions of agglutinating serum, to which 2-3 drops of the pathogen suspension are added. Agglutination is taken into account by the amount of sediment and the degree of clearness of the liquid. The reaction is considered positive if agglutination is observed in a dilution close to the titer of the diagnostic serum. At the same time, controls are taken into account: serum diluted with isotonic sodium chloride solution should be transparent, the suspension of microbes in the same solution should be uniformly cloudy, without sediment.
Different related bacteria can be agglutinated by the same diagnostic agglutinating serum, which makes their identification difficult. Therefore they use adsorbed agglutinating sera, from which cross-reacting antibodies have been removed by adsorption to related bacteria. Such sera retain antibodies that are specific only to a given bacterium. The production of special monoreceptor diagnostic agglutinating sera was proposed by A. Castellani (1902).
Indirect (passive) hemagglutination reaction(RNGA, RPGA) is based on the use of red blood cells with antigens or antibodies adsorbed on their surface, the interaction of which with the corresponding antibodies or antigens of the blood serum causes sticking and precipitation of red blood cells to the bottom of the test tube or cell V in the form of scalloped sediment (Fig. 13.2). At negative reaction red blood cells settle ■ in the form of a “button”. Typically, antibodies are detected in the RNGA using an antigenic erythrocyte diagnosticum, which is erythrocytes with adsorbed on them with antigens. Sometimes anti-A erythrocyte diagnostics are used, on which antibodies are adsorbed. For example, botulinum toxin can be detected by adding erythrocyte antibody botulinum toxin to it (this reaction is called reverse indirect hemagglutination reaction- RONG). RNGA is used to diagnose infectious diseases and determine gonadotropic hormone V urine when establishing pregnancy, to identify hypersensitivity to medications, hormones and in some other cases.
COMBES REACTION
(Coombs test) - a method for determining Rh antibodies on the surface of red blood cells, which cause the precipitation of globulins in the blood serum. This test is used for diagnosis hemolytic anemia in infants with Rh incompatibility who experience destruction of red blood cells.
Coagglutination reaction. The pathogen cells are determined using staphylococci pre-treated with immune diagnostic serum. Staphylococci containing protein A, having an affinity for the Fc fragment of immunoglobulins, nonspecifically adsorb antimicrobial antibodies, which then interact with active centers with the corresponding microbes isolated from patients. As a result of coagglutination, flakes are formed consisting of staphylococci, diagnostic serum antibodies and the detected microbe.
Hemagglutination inhibition reaction(RTGA) is based on blockade, suppression of viral antigens by immune serum antibodies, as a result of which viruses lose their ability to agglutinate red blood cells (Fig. 13.3). RTGA is used to diagnose many viral diseases, the causative agents of which (influenza viruses, measles, rubella, tick-borne encephalitis, etc.) can agglutinate the red blood cells of various animals.
- A group of doctors from WHO arrived in India to identify patients with polio and assist in carrying out universal vaccination against polio. In one of the villages surveyed, a 6-year-old boy was brought to the doctors from a large family who fell ill 5 days ago. The temperature suddenly rose, there was a severe headache, repeated vomiting, pain in the arms and legs. Upon inspection: heat, severe weakness, meningeal symptoms, on right leg reduced muscle tone, tendon reflexes are sharply weakened, the foot hangs down. During puncture of the spinal canal, cerebrospinal fluid flowed out under high blood pressure, the number of lymphocytes is increased, bacteria are not isolated. Delivered preliminary diagnosis"paralytic form of polio." How could the boy become infected? How is specific active prevention of polio carried out? Is there a danger of infecting other children in this family, what should be done?
Answer: The boy could have become infected through the fecal-oral route through food, water, and also through airborne droplets. The main measure for the prevention of polio is immunization with a live culture vaccine from 3 serotypes of Sabin strains. In Russia it is used polio vaccination for oral administration produced by the Institute of Poliomyelitis and Viral Encephalitis named after. M.P. Chumakova. The vaccine is a trivalent preparation of attenuated Sabin strains of polio virus types 1, 2, 3, obtained from a primary culture of African green monkey kidney cells. . Children aged 3 months to 6 years are subject to routine polio vaccinations. Vaccinations with oral polio vaccine are carried out with 2 or 4 drops of vaccine orally three times at 3, 4, 5 and 6 months with three revaccinations at 18, 20 months and 14 years. The sick boy must be hospitalized, and all other children of this family must be vaccinated with live polio vaccine.
- Composition and use of trivalent polymer-subunit liquid influenza vaccine (influenza)
This is a molecular vaccine. Composition: surface Ag of influenza virus of three subtypes (A/H1N1, A/H3N2, B). Application: for the prevention of influenza
Examination card No. 23
