Agglutination reactions. Components, mechanism, installation methods, application. Indirect hemagglutination reaction. Coombs reaction. Coagglutination reaction. Hemagglutination inhibition reaction. Indirect hemagglutination reaction What is more accurate: rnga or rpga


The indirect or passive hemagglutination test (IRHA or RPHA) is more sensitive and specific than the agglutination test. This reaction is also used in two directions.

1) To detect antibodies in the patient’s blood serum, erythrocyte diagnostics are used, in which the antigen is adsorbed on the surface of tannin-treated erythrocytes. In relation to this reaction, the term RPHA is more often used.

The test serum is diluted in the wells of plastic plates and erythrocyte diagnosticum is added. With a positive reaction, a thin film appears along the walls of the hole in the form of a “lace umbrella”; with a negative reaction, a dense sediment of red blood cells in the form of a “button” appears.

2) To detect toxins and bacterial antigens in the test material, antibody erythrocyte diagnostics are used, obtained by adsorption of antibodies on erythrocytes. In relation to this reaction, the term RNGA is more often used. For example, with the help of antibody diagnostics, plague bacillus antigen, diphtheria exotoxin, and botulinum exotoxin are detected.

Coombs test (aptiglobulin test)

The reaction is used to detect incomplete antibodies, for example, antibodies to the Rh factor. Test serum is added to Rh + erythrocytes, in which the presence of incomplete antibodies to the Rh factor is assumed. Having attached to red blood cells, incomplete antibodies do not cause agglutination, since they have only one active center. Then antiglobulin serum containing antibodies to human globulins is added. Combining with incomplete antibodies, antiglobulin serum causes agglutination of red blood cells.

Precipitation reaction

The essence of the reaction is the sedimentation (precipitation) of the antigen under the influence of specific antibodies. To obtain a visible reaction, the presence of an electrolyte is necessary. The antigen in the precipitation reaction is molecularly dispersed substances.

Ring precipitation reaction placed in narrow precipitation tubes. Immune serum is poured into a test tube, and the test material is carefully layered onto it. If there is an antigen in it, an opaque ring of precipitate forms at the border of the two liquids.

The reaction is used in forensic medicine to determine the species of proteins in blood stains, semen, etc.; to determine the antigen in the diagnosis of anthrax (Ascoli reaction), meningitis and other infections; in sanitary and hygienic studies - to determine the falsification of food products. Immune precipitating sera are obtained by immunizing animals with the corresponding antigen. For example, human protein precipitating serum is obtained by immunizing a rabbit with human protein. The titer of the precipitating serum is the highest dilution of the antigen with which it reacts. The serum is usually used undiluted or in a dilution of 1:5.

Agar gel precipitation reaction carried out using several methods. These are the double immunodiffusion reaction, the radial immunodiffusion reaction, and the immunoelectrophoresis reaction.

Double immunodiffusion reaction(according to Ouchterlony). The melted agar gel is poured into a Petri dish and after hardening, wells are cut out in it. Antigen is placed in some wells, and immune sera are placed in others, which diffuse into the agar and form a precipitate in the form of white stripes at the meeting point.

Radial immunodiffusion reaction(according to Mancini). The immune serum is added to the melted agar gel and poured into a cup. After the agar hardens, wells are cut out and antigens are placed in them, which, diffusing into the agar, form ring-shaped precipitation zones around the wells. The higher the antigen concentration, the larger the diameter of the ring. The reaction is used, for example, to determine immunoglobulins of various classes in the blood. Immunoglobulins of the classes IgG, IgM, IgA act as antigens in this reaction, and antibodies against them are contained in specific monoreceptor sera.

Immunoelectrophoresis. Electrophoresis of protein antigens is carried out in an agar gel. Precipitating serum is introduced into the groove, which runs parallel to the direction of movement of the proteins. Antigens and antibodies diffuse into the agar and precipitation lines form where they meet.

Immune lysis reactions

An antigen (erythrocytes or bacteria), combining with specific antibodies, forms an immune complex to which complement (C1) attaches, and complement is activated along the classical pathway. Activated complement lyses red blood cells (hemolysis) or bacteria (bacteriolysis). The bacteriolysis reaction is used to identify Vibrio cholerae.

Hemolysis reaction. The antigen in the reaction is erythrocytes, antibodies (hemolysins) are contained in hemolytic serum. Hemolysins attach to red blood cells, and complement is activated, which lyses red blood cells. The cloudy suspension of red blood cells turns into a transparent bright red liquid - “lacquer blood”. Since the hemolysis reaction occurs only in the presence of complement, it is used as an indicator for the detection of complement.

Local hemolysis reaction in the gel(Erne reaction) - a variant of the hemolysis reaction. Serves to determine the number of antibody-forming cells (AFC) in the spleen and lymph nodes.

The melted agar gel is mixed with a suspension of spleen cells and red blood cells, and after the agar has solidified, complement is added. A hemolysis zone forms around each hemolysin-producing cell. The number of hemolysin-producing cells is determined by the number of such zones.

Direct microbial agglutination reaction (RA). In this reaction, antibodies (agglutinins) directly agglutinate corpuscular antigens (agglutanogens). They are usually represented by a suspension of inactivated microorganisms (microbial agglutination reaction). Based on the nature of the agglutinate formed, granular and flocculent agglutination are distinguished. Granular agglutination occurs when microbes containing O-antigen stick together. Bacteria with flagella (H-antigen) agglutinate to form large flakes.

To determine the type of microorganisms, standard diagnostic agglutinating sera are used. They are obtained by hyperimmunizing laboratory animals with a suspension of bacteria. The titer of such a serum is its highest dilution at which clear agglutination of the corresponding antigen is observed. However, due to the complexity of the antigenic structure of bacteria, agglutinating sera contain antibodies not only to species-specific, but also to group antigens and can give group agglutination with related species of bacteria. Antibody titers to species-specific antigens in serum are always higher than to group antigens. To remove group-specific antibodies, microorganisms containing group antigens are sequentially added to the serum (Castellani method). This method produces adsorbed sera that contain antibodies to a specific type of microbe.

Agglutination reaction methods. The most common are lamellar (approximate) and expanded RA.

The plate RA is placed on the glass. In this reaction, slightly diluted or undiluted sera are used. It is used as an accelerated method for detecting antibodies or identifying microorganisms. A drop of serum is applied to the glass, into which an unknown bacterial culture is introduced with a loop, mixed, and after 2-3 minutes the appearance of fine-grained or flaky agglutination is observed. For control, a drop of saline solution is used, in which turbidity is observed after adding bacteria. When using non-adsorbed serums, the reaction on glass is only indicative.

Expanded RA is carried out in test tubes or plate wells. In this case, the diagnostic serum is diluted to titer and equal amounts of antigen are added. If the result is positive, a loose sediment in the form of an “umbrella” is formed at the bottom of the test tube; if the result is negative, a sediment in the form of a “button” is formed. Since titers of group-specific antibodies in serum are much lower than titers of species-specific antibodies, group reactions are observed only in small dilutions of serum. If agglutination occurs before the titer or up to half the titer of the serum, it is species specific.

To determine antibodies in the patient's serum (serological diagnosis), a standard microbial diagnosticum is used, containing a suspension of known microbes or their antigens. In this case, it is also possible to install plate and deployed RA.

Direct cell agglutination reaction. To determine blood groups, standard donor blood sera containing known anti-A or anti-B antibodies are used. Reactions are performed on glass or plates. If the erythrocytes have A (2nd blood group), B (3rd blood group) or both antigens (4th blood group), the corresponding sera agglutinate the erythrocytes. A blood compatibility test is also used, where drops of blood from the donor and recipient are mixed and agglutination is assessed.

Clinics use the agglutination reaction of leukocytes, platelets and other cells to identify autoantibodies, as well as to determine antigens on these cells.

The hemagglutination reaction is based on the phenomenon of red blood cell gluing, which occurs under the influence of various factors. There are direct and indirect hemagglutination.
In the direct hemagglutination reaction, red blood cells stick together when certain antigens, such as viruses, are adsorbed onto them.

Indirect (passive) hemagglutination reaction (IRHA, RPHA) is based on the use of erythrocytes (or latex) with antigens or antibodies adsorbed on their surface, the interaction of which with the corresponding antibodies or antigens of the patients' blood serum causes the erythrocytes to stick together and fall out to the bottom of the test tube or cell in the form of a scalloped sediment.

Components. To perform RNGA, erythrocytes from sheep, horses, rabbits, chickens, mice, humans, and others can be used, which are stored for future use by treating them with formaldehyde or glutaraldehyde. The adsorption capacity of erythrocytes increases when they are treated with solutions of tannin or chromium chloride.

Antigens in RNGA can be polysaccharide antigens of microorganisms, extracts of bacterial vaccines, antigens of viruses and rickettsiae, as well as other substances.

Red blood cells sensitized by hypertension are called erythrocyte diagnosticums. For the preparation of erythrocyte diagnosticum, sheep erythrocytes, which have high adsorbing activity, are most often used.

Application. RNGA is used to diagnose infectious diseases, determine gonadotropic hormone in urine when establishing pregnancy, to identify hypersensitivity to drugs, hormones, and in some other cases.

In serological studies, a direct hemagglutination inhibition reaction is used, when the virus isolated from a patient is neutralized with specific immune serum and then combined with red blood cells. The absence of hemagglutination indicates the consistency of the virus and the immune serum used.

The indirect hemagglutination reaction (passive hemagglutination) is observed in cases where immune serum or patient serum containing appropriate antibodies is added to red blood cells that have been previously treated (sensitized) with various antigens. Specific gluing of red blood cells occurs, their passive hemagglutination.

The indirect or passive hemagglutination reaction is superior in sensitivity and specificity to other serological methods, and is used in the diagnosis of infections caused by bacteria, rickettsia, and protozoa.

The method for performing an indirect hemagglutination reaction consists of several stages.

· First, red blood cells are washed with an isotonic sodium chloride solution, then, if necessary (when using protein antigens), they are treated with a tannin solution of 1: 20,000 and sensitized with soluble antigens.

· After washing with a buffered isotonic sodium chloride solution, the erythrocyte antigen is ready for use.

· The test sera are diluted with an isotonic solution of sodium chloride in test tubes or special plastic plates with wells, then an erythrocyte diagnosticum is added to each dilution of the serum.

· The results of the indirect hemagglutination reaction are taken into account by the nature of the red blood cell sediment formed at the bottom of the test tube.

· A reaction result in which red blood cells evenly cover the entire bottom of the test tube is considered positive. In a negative reaction, red blood cells in the form of a small disk or “button” are located in the center of the bottom of the test tube.

· After 2 hours of incubation at 37 °C, the results are taken into account, assessing the appearance of the red blood cell sediment (without shaking): with a negative reaction, a sediment appears in the form of a compact disk or ring at the bottom of the well; with a positive reaction, a characteristic lacy sediment of red blood cells appears, a thin film with uneven edges

Coagglutination reaction.

This reaction is based on the unique property of Staphylococcus aureus, which contains protein A in its cell wall, to bind to the Fc fragments of IgG and IgM.

In this case, the active centers of antibodies remain free and can interact with specific determinants of antigens. A drop of a 2% suspension of staphylococci, sensitized with appropriate antibodies, is applied to a glass slide, and a drop of a suspension of the bacteria being studied is added. If the antigen matches the antibodies, a clear agglutination of the staphylococci loaded with antibodies occurs within 30-60 s.

Requirements for the immune serum used to sensitize staphylococcal cells and the conduct of the sensitization process. To obtain a coagglutinating reagent, a suspension of staphylococci should be treated with immune serum against the desired antigen. The serum must be taken from an animal whose IgG has an affinity for protein A. The immunoglobulins of humans, pigs, dogs and guinea pigs have the greatest affinity for it, the least - donkey and rabbit, and the IgG of sheep, horses, rats and mice interacts with it very weakly .

In addition to strict specificity for the desired antigen, the serum used in RCOA should not contain antibodies to staphylococcus in order to avoid agglutination of the staphylococcal reagent due to the specific effect of the antigen and antibodies, which should be excluded in the IgG - protein A system. Control is carried out by mixing one drop of serum and a 10% suspension of staphylococcal reagent on a glass. If after 3...5 minutes no agglutinate flakes are formed, then the serum is considered suitable for the reaction.

If the available serum samples to this antigen agglutinate staphylococcus, then they can be adsorbed by a suspension of staphylococcal cells that do not have protein A (for example, Wood-46 strains). In this way, antibodies that react with staphylococcus due to Fab fragments are removed.

Thus, the serum used to prepare the coagglutinating reagent must meet the following requirements:

  • obtained from a producing animal whose IgG has affinity for protein A;
  • must have specificity for the desired antigen;
  • be free from anti-staphylococcal antibodies.

· Preparation of diagnosticum. The prepared 10% staphylococcal reagent is combined with an equal volume of immune serum in the previously determined optimal working dilution. The mixture is shaken for 60 minutes at 40...42 °C in a shutgel apparatus at 90 vibrations per minute. Then, after 15 minutes, they are washed twice with PBS, resuspended to a 2% suspension and preserved with sodium merthiolate (1: 10,000).

Lesson 14

Topic: Indirect serological reactions. Indirect hemagglutination reactions (IRHA), complement fixation (IFR).

Antibodies

Antibodies are protein molecules capable of specifically binding to antigens. Antibodies belong to gamma globulins. Another name for antibodies is immunoglobulins. In mammals, there are 5 classes of immunoglobulins, differing in their structure and some properties: IgG, IgM, IgA, IgE, IgD.

The structure of immunoglobulins. IgG has the most “typical” structure. The molecule consists of 4 protein chains: two light (L) and two heavy (H), which are connected by disulfide bonds. The region of the antibody that binds to the antigen is called the active site of the antibody. There are 2 active centers in the IgG molecule. It is formed by the N-terminal regions of the heavy and light chains. The region of the heavy chains located near the disulfide bonds is called the hinge region. With the help of the papain enzyme, the IgG molecule above the hinge region is split into 3 fragments: 2 of them contain a light chain and part of the heavy chain (Fab fragments); and the third fragment consists of only part of the heavy chains (Fc fragment). Thanks to the movable hinge region, Fab fragments can change their relative position in space.

The amino acid sequences of light and heavy chains are divided into constant and variable regions. Variable regions are located at the N-termini of the light and heavy chains (VL and VH). Constant regions are located at the C-termini of the chains (CL and CH). In light and heavy chains, amino acid sequences form several globular structures called domains.


The active center of an antibody is formed by the variable domains of the light and heavy chains and is a cavity ( paratope), having a certain configuration and distribution of electrical charges on its surface. The size, shape and charge distribution of the active site determines its specificity, that is, the ability to bind to a specific antigenic determinant ( epitope), having a complementary structure.

Antigenic determinants are areas protruding on the surface of antigen molecules. Therefore, the epitope-paratope interaction occurs according to the “key-lock” principle.

The strength of the connection between the active center of antibodies and the antigenic determinant is characterized by the concept of affinity. Affinity is a measure of the affinity of the active site and the antigenic determinant.

IgG class immunoglobulins account for 75% of the total amount of serum immunoglobulins. An important property of IgG is its ability to pass through the placenta. Thus, maternal antibodies enter the child’s body and protect him in the first months of life from infection (natural passive immunity).

About 10% of the total pool of immunoglobulins belongs to the IgM class. The IgM molecule is a pentamer, that is, it consists of 5 identical molecules, similar in structure to the IgG molecule, and has 10 active centers. The subunits are connected to each other by disulfide bonds. The IgM molecule has an additional J chain that links the subunits. IgM antibodies do not cross the placental barrier.

Antibodies of the IgA class make up 15-20% of the total content of immunoglobulins. The IgA molecule consists of 2 light and 2 heavy chains and has 2 active centers. In blood serum, IgA is present in monomeric form, while in the secretions of mucous membranes, IgA is presented in the form of dimers and is called secretory or sIgA, and has 4 active centers. The C-termini of the heavy chains in the sIgA molecule are connected to each other by the J-chain and a protein molecule called the secretory component. The secretory component protects sIgA from breakdown by proteolytic enzymes, which are contained in large quantities in the secretions of the mucous membranes. The main function of sIgA is to protect mucous membranes from infection. IgA does not penetrate the placental barrier. High concentrations of sIgA are found in human breast milk, especially in the first days of lactation. They protect the newborn's gastrointestinal tract from infection.

IgD is mainly found on the membrane of B lymphocytes. They have a structure similar to IgG, 2 active centers. The biological role is not fully known.

IgE - the concentration of this class of immunoglobulins in blood serum is extremely low. IgE molecules are mainly fixed on the surface of mast cells and basophils. IgE is similar in structure to IgG and has 2 active centers. It is assumed that IgE is essential in the development of anthelmintic immunity. IgE plays a major role in the pathogenesis of some allergic diseases (bronchial asthma, hay fever) and anaphylactic shock.


Indirect hemagglutination reaction

In indirect serological reactions, the complex of antigens with antibodies is not visible to the naked eye. In such cases, antigens are adsorbed on larger carrier particles (erythrocytes, latex particles), obtaining an antigenic erythrocyte diagnosticum. Subsequent agglutination of such particles with specific antibodies allows the agglutinate (precipitate) to be seen with the naked eye. The indirect (passive) hemagglutination reaction (IRHA) detects blood serum antibodies using the antigenic erythrocyte diagnosticum, which is erythrocytes with antigens adsorbed on them.

Red blood cells with antigens adsorbed on them interact with the corresponding antibodies in the blood serum, which causes the red blood cells to stick together and fall out to the bottom of the test tube or cell in the form of a scalloped sediment. In a negative reaction, red blood cells settle in the form of a button.

RNGA is placed in plastic tablets or in test tubes with dilutions of blood serum, to which an erythrocyte diagnosticum is added.

Sometimes an antibody erythrocyte diagnosticum is used - red blood cells on which antibodies are adsorbed. This reaction is called RONGA - reverse indirect hemagglutination reaction.

RNGA components:

Patient's blood serum (diluted 1:25);

Erythrocyte diagnosticum (erythrocytes loaded with the antigen of the pathogen under study);

Wash solution.

Staged by RNGA. Two drops of phosphate buffer solution are added to seven wells of the plate for immunological studies. Two drops of the patient’s blood serum are added to the first well, after which 2 drops are transferred from the first well to the second well, from the second to the third, etc. 2 drops are removed from the sixth well. Add 2 drops of erythrocyte diagnosticum to all seven wells (6 experimental and 1 control). After each operation, it is necessary to rinse the pipette in a washing solution. The plates are left at room temperature for 45 minutes, after which the results are recorded.

Complement fixation reaction

The complement fixation reaction is that when an antigen combines with an antibody, an immune complex is formed, to which complement is attached through the Fc fragment of antibodies. If the antigen-antibody complex is not formed, then complement remains free. Free complement is detected by adding to the mixture a hemolytic system consisting of sheep red blood cells and antibodies against them. A positive reaction is the absence of hemolysis as a result of complement binding to the antigen + antibody complex. A negative reaction is the presence of hemolysis as a result of complement binding to the erythrocyte + anti-erythrocyte antibody complex.

RSK components:

Healthy blood serum;

Patient's blood serum (diluted 1:5);

The antigenic component of the reaction is an inactivated pathogen;

Complement in a dilution corresponding to the working dose. Complement is obtained from the blood serum of guinea pigs. The complement titer is its minimum dose, which in the presence of hemolytic serum causes complete hemolysis of red blood cells. The working dose of complement used in the production of RSC is 30% greater than its titer;

The hemolytic system is a suspension of sheep erythrocytes treated with rabbit antibodies to sheep erythrocytes.

Wash solution.

Staged by RSK. RSC is placed in two test tubes - experimental and control. 0.5 ml of the patient’s blood serum is added to the test tube, 0.5 ml of the blood serum of a healthy donor is added to the control tube, 0.5 ml of the pathogen lysate is added to both test tubes, as well as 0.5 ml of complement. After each operation, it is necessary to rinse the pipette in a washing solution. The tubes are placed in a thermostat at 37°C for 30 minutes. After incubation, 1.0 ml of hemolytic system is added to both tubes. The tubes are shaken and placed in a thermostat at 37°C for 30 minutes. If the reaction is positive, there is a delay in hemolysis in the test tube (colorless liquid and sediment of red blood cells), and in the control tube there is hemolysis of red blood cells.

Literature to prepare for the lesson:

1. Borisov microbiology, virology, immunology. M., 2002.

2. Medical microbiology, virology and immunology. Ed. . M., 2004.

3. Pozdeev microbiology. M., GEOTAR-MEDIA, 2005.

It is based on the fact that red blood cells, on which antigens are previously adsorbed, acquire the ability to agglutinate in the presence of homologous sera (antibodies).

In this case, red blood cells act as carriers with specific determinants, the agglutination of which occurs as a result of the antigen + antibody reaction.

Red blood cells to the surface of which antigens are firmly attached are called erythrocyte antigen diagnosticum, or erythrocytes sensitized with antigen.

Another type of RNGA - antibodies are adsorbed on the surface of erythrocytes and their subsequent agglutination occurs in the presence of a homologous antigen. In this case, such erythrocytes are called erythrocyte antibody diagnosticum, or erythrocytes sensitized by antibodies.

Based on these two fundamental methodological approaches, many modifications of the RNGA have been developed and used. Thus, small standard latex particles are used as carriers. In this case, the reaction is called latex agglutination reaction (RLA) or Staphylococcus aureus is used - coagglutination reaction, etc. Usually, erythrocyte diagnostics are prepared at biological industry enterprises, and the main experience of RNGA is carried out in diagnostic laboratories.

Preparation of erythrocyte diagnosticums includes the following steps:

  • fixation of red blood cells with formaldehyde or glutaric or acrylic aldehydes. Such treated red blood cells are stored for a long time. More often, erythrocytes from sheep, humans, chickens, etc. are used for this purpose;
  • treatment of fixed erythrocytes with tannin solution. As a result, red blood cells acquire the ability to irreversibly adsorb proteins (viruses and antibodies) on their surface;
  • sensitization of tanized erythrocytes by viruses or antibodies.

It should be noted that the methods for preparing erythrocyte diagnostics for viral infections are different.

The procedure for performing RNGA to detect and determine antibody titer is as follows:

  • equal doses of erythrocytes sensitized with antigen are added to successive 2-fold dilutions of serum;
  • the mixture is left for 2-3 hours at room temperature or for 16-18 hours at 4 °C;
  • take into account the results. If the serum contains antibodies to the virus with which the red blood cells were sensitized, hemagglutination is observed, which is assessed in crosses.

The antibody titer in the serum is taken to be the highest serum dilution that still provides hemagglutination by at least two crosses.

RNGA is accompanied by all relevant controls. Usually the reaction is carried out using the micromethod.

RNGA allows you to solve the following diagnostic problems:

  • detect antibodies and determine their titer in blood serum using a known erythrocyte antigen diagnosticum;
  • detect and identify an unknown virus using a known erythrocyte antibody diagnostic.

Advantages of RNGA: high sensitivity, simplicity of placement technique and speed of response. However, it is important to note that great difficulties arise in the preparation of stable erythrocyte diagnostics (great dependence on the purity of the components used, the need to select a mode of fixation, tanization and sensitization of erythrocytes for each type of virus).

The site provides reference information for informational purposes only. Diagnosis and treatment of diseases must be carried out under the supervision of a specialist. All drugs have contraindications. Consultation with a specialist is required!

Serological blood analysis is a method for laboratory testing of certain antigens or antibodies ( specific proteins) in the patient's blood serum, based on the body's immune responses. This method is used when diagnostics infectious diseases to detect the presence of antibodies in the patient’s blood to a certain type of virus or bacteria, as well as to determine the blood group.

Material under study

First of all, for serological analysis, biological material collected from the patient is used:
  • blood serum
  • saliva
  • fecal matter
In some cases, material isolated from certain environmental objects is examined:
  • the soil

Method of analysis or blood sampling

This analysis does not require special preparation of the patient. Blood sampling is carried out in the morning on an empty stomach and is performed in treatment rooms of medical institutions, according to generally accepted hematological methods. For serological testing, blood is collected using two methods: venous blood is taken from the patient’s ulnar vein, and capillary blood is taken from the ring finger. The blood is placed in sterile sealed tubes.

Features of blood transportation and serum storage

Immediately after blood collection, it is transported to a special laboratory, where serum is prepared on the same day. Whey can be stored for no more than 4–6 days in a refrigerator at a temperature of 2–4 degrees. If longer storage is required, the whey is frozen. To avoid compromising the quality of the whey, it is allowed to freeze it once and, accordingly, defrost it. During long-term storage, antibodies lose their properties and become partially inactive; immunoglobulins of the class are most sensitive to freezing M. After thawing the serum, it is necessary to thoroughly mix it until smooth, which helps restore the concentration of antibodies contained in this serum.


What are serological tests used for?

Serological laboratory diagnostic methods are used to identify diseases such as echinococcosis, trichinosis, toxocarosis, opisthorchiasis, cysticercosis, toxoplasmosis, amebiasis, giardiasis, to determine the effectiveness of the course of treatment and, finally, to detect recurrent disease after the patient has fully recovered.

Basic methods of serological diagnosis

Immunofluorescence reaction (RIF)

This reaction is an indirect version of a serological test, that is, it is carried out in two stages. At the first stage, the required antibody is identified in the circulating antigen-antibody complex using antiglobulin, which is similar in structure to immune serum proteins. Identification of the desired antibody is also possible by studying a pre-prepared antigen preparation under a fluorescent microscope, without the use of antiglobulins.

The immunofluorescence reaction is a very labor-intensive process that requires a lot of time and responsibility from a specialist. The reaction begins with the preparation of solutions, then the sera and their control samples are titrated ( a process that allows you to determine the content of a certain substance by gradually mixing the test solution with a controlled amount of the reagent). Previously prepared dilutions and their control samples are carefully applied to glass slides with the antigen. Then the preparations are incubated, followed by washing and air drying. A thin layer of antiserum is applied to the slides with the antigen, after which a secondary incubation of the preparations is performed and the entire previous chain of actions is repeated, ending with the drying of the preparation. As a result, the preparation on a glass slide is treated with glycerol and examined under a fluorescence microscope.

The results of the reaction are assessed on a four-point scale, which is characterized by the intensity of the surface yellow-green glow of antigen cells:
+ very faint glow of the cell, noticeable only at its periphery
++ faint glow of the cell periphery, but with a clearly noticeable green tint
+++/++++ bright green glow of the cell periphery
The reaction titer is considered to be a serum dilution where at least 50% of the antigen cells exhibit a clear surface glow, that is, the result of the reaction +++ or ++++ . The reaction range value is from 1/80 to 1/100.

The intensity of the reaction depends on the number of precipitated red blood cells at the bottom of the formed holes:
– a negative reaction, which is characterized by the deposition of red blood cells to the bottom of the wells in the form of a small ring with smooth edges or a “button”
+ low intensity, this reaction is characterized by small single deposits at the bottom of the hole. Non-agglutinated red blood cells form a “small ring” in the center of the well
++ medium intensity, it is characterized by the formation at the bottom of the hole of a “wide dense ring” of non-agglutinated erythrocytes
+++ intense reaction, agglutinated erythrocytes form so-called “umbrellas”, in the center of which rings formed by settled non-agglutinated erythrocytes are clearly visible
++++ a sharply intense reaction in which all red blood cells agglutinate and, forming “umbrellas,” line the bottom of the wells.
The titer of this reaction is considered to be the last serum dilution at which obvious agglutination of erythrocytes is detected at least +++ , that is, with an intense or sharply intense reaction.

The reliability and quality of serological diagnostic methods depend on the organization of internal and external laboratory control, consisting of several independent procedures designed to assess the quality of the results of the analysis.

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