Serological reactions: types, use

Laboratory diagnosis of almost allInfectious diseases are based on the detection of antibodies in the patient's blood that are produced by the pathogen antigens using serological tests. They entered medical practice from the end of the nineteenth - early twentieth century.

The development of science has helped determine antigenicmicrobial structure and chemical formulas of their toxins. This allowed us to create not only therapeutic, but also diagnostic sera. They are obtained by administering attenuated pathogens to laboratory animals. After several days of exposure, preparations used to identify microbes or their toxins using serological tests are prepared from the blood of rabbits or mice.

The appearance of such a reaction depends onconditions of its setting and the state of the antigens in the patient’s blood. If particles of microbes are insoluble, then they are precipitated, lysed, bound or immobilized in serum. If the antigens are soluble, then the phenomenon of neutralization or precipitation occurs.

Agglutination Reaction (RA)

Serological agglutination reaction ishighly specific. It is simple to perform and visually sufficient to quickly determine the presence of antigens in the patient's serum. It is used for the formulation of the Vidal reaction (diagnosis of typhoid and paratyphoid fever) and Weigl (typhus).

Она основана на специфическом взаимодействии between human antibodies (or agglutinins) and microbial cells (agglutenogens). After their interaction, particles are formed, which precipitate. This is a positive sign. For the formulation of the reaction, live or killed microbial agents, fungi, protozoa, blood cells and somatic cells can be used.

Chemically, the reaction is divided into two stages:

1. Specific combination of antibodies (AT) with antigens (AG).
2. Non-specific — precipitation of AG-AT conglomerates, that is, formation of agglutinate.

Reaction of indirect agglutination (RPGA)

This reaction has a greatersensitivity than the previous one. It is used to diagnose diseases caused by bacteria, intracellular parasites, protozoa. It is so specific that it allows you to detect even a very low concentration of antibodies.

For its production use peeled lambred blood cells and human red blood cells, pretreated with antibodies or antigens (this depends on what the laboratory technician wants to find). In some cases, human red blood cells are treated with immunoglobulins. Serological reactions of erythrocytes are considered valid if they are deposited on the bottom of the tube. One can speak of a positive reaction when the cells are arranged in the form of an inverted umbrella, occupying the whole bottom. A negative reaction is counted if the red blood cells have settled down in a column or in the form of a button in the center of the bottom.

Reaction precipitation (RP)

Serological reactions of this type are used fordetection of extremely small particles of antigens. These can be, for example, proteins (or their parts), compounds of proteins with lipids or carbohydrates, parts of bacteria, their toxins.

Serum for carrying out the reaction is obtained byartificially infect animals, usually rabbits. With this method you can get absolutely any precipitating serum. The formulation of serological precipitation reactions is similar in its mechanism of action to agglutination reactions. Antibodies contained in serum combine with antigens in a colloidal solution, forming large protein molecules that are deposited on the bottom of the tube or on the substrate (gel). This method is considered highly specific and can detect even minute amounts of a substance.

Used to diagnose plague, tularemia, anthrax, meningitis and other diseases. In addition, involved in the forensic examination.

Gel precipitation reaction

Serological reactions can be carried out not onlyin liquid medium, but also in agar gel. This is called diffuse precipitation method. With its help, study the composition of complex antigenic mixtures. This method is based on the chemotaxis of antigens to antibodies and vice versa. In the gel, they move towards each other at different speeds and, meeting, form precipitation lines. Each line is one set of AG-AT.

The reaction of neutralizing exotoxin with antitoxin (PH)

Antitoxic sera can neutralizethe action of exotoxin, which is produced by microorganisms. These serological reactions are based on this. Microbiology uses this method for the titration of sera, toxins and toxoids, as well as the determination of their therapeutic activity. The strength of the toxin neutralization is determined by conventional units - AE.

In addition, thanks to this reaction candetermine the species or type of exotoxin. This is used in the diagnosis of tetanus, diphtheria, botulism. The study can be carried out both on the glass and in the gel.

Reaction lysis (RL)

Immune serum that enters the bodythe patient has, in addition to its main function of passive immunity, also lysing properties. It is capable of dissolving microbial agents, cellular alien elements and viruses entering the patient’s body. Depending on the specificity of the antibodies that make up the serum, bacteriolysins, cytolysins, spirochetolysins, hemolysins and others are isolated.

These specific antibodies are called"complement". It is contained in almost all body fluids of a person, has a complex protein structure and is extremely sensitive to temperature increase, shaking, the action of acids and direct sunlight. But in the dried state is able to maintain its lysing properties up to six months.

There are these types of serological reactions of this type:

- bacteriolysis;

- hemolysis.

Bacteriolysis is performed using blood serum.patient and specific immune serum with live microbes. If there is a sufficient amount of complement in the blood, the researcher will see the lysis of bacteria, and the reaction will be considered positive.

The second serological blood reaction isthat a suspension of erythrocytes of the patient is treated with serum containing hemolysins, which are activated only in the presence of a certain compliment. If there is one, the lab technician observes the dissolution of the red blood cells. This reaction is widely used in modern medicine to determine the titer of complement (that is, its smallest amount, provoking the lysis of red blood cells) in the serum and for setting the assay for complement binding. This method is carried out serological reaction for syphilis - Wasserman reaction.

Complement Binding Reaction (RSC)

This reaction is used to detect antibodies to the infectious agent in the patient’s serum, as well as to identify the pathogen by its antigenic structure.

Up to this point, we have described simpleserological reactions. CSC is considered to be a complex reaction, since not two, but three elements interact in it: an antibody, an antigen and complement. Its essence lies in the fact that the interaction between the antibody and the antigen occurs only in the presence of complement proteins, which are adsorbed on the surface of the formed complex AG-AT.

Themselves antigens, after the addition of complement,subjected to significant changes, which show the quality of the reaction. This may be lysis, hemolysis, immobilization, bactericidal or bacteriostatic action.

The reaction itself occurs in two phases:

1. The formation of a complex of antigen-antibody, which is not visible to the researcher.
2. Изменение антигена под действием комплемента.This phase can most often be traced to the naked eye. If visually the reaction is not visible, then use an additional indicator system that allows you to identify changes.

Indicator system

This reaction is based on the binding of complement.An hour after the RSC was added, purified sheep red blood cells and hemolytic serum that does not contain complement are added to the test tube. If there is an unrelated complement in the test tube, it will join the complex AG-AT, formed between the ram blood cells and hemolysin, and cause their dissolution. This will mean that the RAC is negative. If the red blood cells remained intact, then, accordingly, the reaction is positive.

Hemagglutination Reaction (DSA)

There are two fundamentally different hemagglutination reactions. One of them is serological, it is used to determine blood groups. In this case, the red blood cells interact with antibodies.

And the second reaction does not apply to serological,as red blood cells react with hemagglutinins produced by viruses. Since each pathogen acts only on specific red blood cells (chicken, mutton, monkey), this reaction can be considered narrowly specific.

Understand, positive reaction or negative,possible by the location of blood cells at the bottom of the tube. If their drawing resembles an inverted umbrella, then the virus in question is present in the patient’s blood. And if all the red blood cells are like a coin column, then the pathogens are not.

Hemagglutination inhibition reaction (rtga)

This is a highly specific reaction, allowing to establish the type, type of viruses or the presence of specific antibodies in the patient's serum.

Its essence is that antibodies,added to the tube with the test material, preventing the deposition of antigens on erythrocytes, thereby stopping the hemagglutination. This is a qualitative sign of the presence in the blood of specific antigens to the specific desired virus.

Immunofluorescence Reaction (RIF)

The basis of the reaction is the ability to detectAG-AT complexes under fluorescent microscopy after their treatment with fluorochromic dyes. This method is easy to use, does not require the allocation of pure culture and takes little time. It is indispensable for the rapid diagnosis of infectious diseases.

In practice, these serological reactions are divided into two types: direct and indirect.

Direct RIF is produced with an antigen thatpretreated with fluorescent serum. And indirect is that first the drug is treated with conventional diagnosticum containing antigens to the desired antibodies, and then re-applied fluorescent serum, which is specific for proteins of the complex AG-AT, and microbial cells become visible during microscopy.