Serological reactions: species, use

Laboratory diagnostics of almost all infectious diseases is based on the detection of antibodies in the blood of the patient, which are developed for the antigens of the pathogen, by methods of serological reactions. They entered the medical practice from the late nineteenth - early twentieth century.

The development of science helped to determine the antigenic structure of microbes and the chemical formulas of their toxins. This allowed to create not only therapeutic, but also diagnostic serums. They are obtained by introducing weakened pathogens into laboratory animals. After several days of exposure from the blood of rabbits or mice, preparations used to identify microbes or their toxins are prepared using serological reactions.

The external manifestation of such a reaction depends on the conditions of its formulation and on the state of antigens in the patient's blood. If particles of microbes are insoluble, then they precipitate, lyse, bind or immobilize in serum. If antigens are soluble, then the phenomenon of neutralization or precipitation occurs.

The agglutination reaction (PA)

Serological reaction agglutination is highly specific. It is simple in execution and is sufficiently clear to quickly determine the presence of antigens in the serum of the patient. It is used for the formulation of the Vidal's reaction (diagnosis of typhoid and paratyphoid) and Weigl (typhus).

It is based on the specific interaction between human antibodies (or agglutinins) and microbial cells (agglutinogens). After their interaction, particles are formed which precipitate. This is a positive sign. Live or killed microbial agents, fungi, protozoa, blood cells and somatic cells can be used to formulate the reaction .

Chemically, the reaction is divided into two stages:

1. Specific compound of antibodies (AT) with antigens (AH).
2. Nonspecific - precipitation of conglomerates of AG-AT, that is, the formation of agglutinate.

The reaction of indirect agglutination (RPHA)

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

To produce it, we use purified lamb erythrocytes and red blood cells of a person, pretreated with antibodies or antigens (this depends on what the lab technician wants to find). In some cases, human erythrocytes are treated with immunoglobulins. Serological reactions of erythrocytes are considered to have taken place if they have deposited on the bottom of the test tube. A positive reaction can be said when the cells are arranged in the form of an inverted umbrella, occupying the entire bottom. A negative reaction is counted if the red blood cells have settled in a column or in the form of a button in the center of the bottom.

The precipitation reaction (RP)

Serological reactions of this type serve to detect extremely small antigen particles. These can be, for example, proteins (or parts thereof), protein compounds with lipids or carbohydrates, parts of bacteria, and their toxins.

Sera for carrying out the reaction are obtained by artificially infecting animals, usually rabbits. This method can receive absolutely any precipitating serum. The staging of serological precipitation reactions is similar to the mechanism of action on the agglutination reaction. Antibodies contained in the serum combine with antigens in a colloidal solution, forming large protein molecules that are deposited on the bottom of the tube or onto a substrate (gel). This method is considered highly specific and can detect even negligible amounts of matter.

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

Gel precipitation reaction

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

The reaction of neutralization of exotoxin with antitoxin (PH)

Antitoxic serums are able to neutralize the effect of exotoxin, which produces microorganisms. This is the basis for these serological reactions. Microbiology uses this method for titrating serums, toxins and toxoids, as well as determining their therapeutic activity. The neutralization of toxin is determined by conventional units - AE.

In addition, due to this reaction, it is possible to determine the specific or typical accessory 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.

The lysis reaction (RL)

Immune serum, which enters the body of the patient, has, in addition to its main function of passive immunity, also lysing properties. It is capable of dissolving microbial agents, cellular foreign elements and viruses entering the patient's body. Depending on the specificity of the antibodies in the serum, the bacteriolysins, cytolysins, spirohetholysins, hemolysins, and others are isolated.

These specific antibodies are called "complement". It is contained in virtually all body fluids, has a complex protein structure and is extremely sensitive to fever, shaking, acid and direct sunlight. But in the dried state it is able to maintain its lysing properties up to six months.

There are types of serological reactions of this type:

- bacteriolysis;

- hemolysis.

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

The second serological response of the blood is that the patient's erythrocyte suspension is treated with serum containing hemolysins, which are activated only in the presence of a certain compliment. If there is one, the laboratory assistant observes dissolution of red blood cells. This reaction is widely used in modern medicine to determine the complement titer (i.e., its smallest amount provoking the lysis of red blood cells) in the serum and for the analysis of complement binding. This is the way the serological response to syphilis is carried out - the Wasserman reaction.

The complement fixation reaction (RCC)

This reaction is used to detect in the blood serum of a patient an antibody to an infectious agent, and also to identify the pathogen from its antigenic structure.

Up to this point, we have described simple serological reactions. RSK is considered a complex reaction, because it interacts not with two, but with three elements: antibody, 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 AG-AT complex.

The antigens themselves, after the addition of complement, undergo significant changes, which indicate the quality of the reaction. It can be lysis, hemolysis, immobilization, bactericidal or bacteriostatic action.

The reaction itself occurs in two phases:

1. The formation of an antigen-antibody complex, which is visually invisible to the researcher.
2. Antigen change under the action of complement. This phase can often be traced to the naked eye. If the visual response is not visible, then an additional indicator system is used to detect changes.

Indicator system

This reaction is based on binding complement. In the test tube one hour after the DSC formulation, purified erythrocytes of the ram and non-complemented hemolytic serum are added. If there was an unbound complement in the tube, it will join the AG-AT complex formed between mutton cells of the blood and hemolysin and cause their dissolution. This will mean that the RCC is negative. If the red blood cells remained intact, then, respectively, the reaction is positive.

The hemagglutination reaction (RGA)

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

And the second reaction does not refer to serological, since the red blood cells react with the hemagglutinins produced by the viruses. Since each agent acts only on specific erythrocytes (chicken, lamb, monkey), then this reaction can be considered narrowly specific.

Understand, a positive reaction or negative, you can by the location of blood cells on the bottom of the test tube. If their picture resembles an inverted umbrella, then the desired virus is present in the patient's blood. And if all the red blood cells are formed like a coin column, then there are no unknown pathogens.

The reaction of inhibition of hemagglutination (RTGA)

This is a highly specific reaction that allows you to determine the type, type of virus, or the presence of specific antibodies in the patient's blood serum.

Its essence lies in the fact that the antibodies added to the test tube with the test material prevent the deposition of antigens on the red blood cells, thereby stopping hemagglutination. This is a qualitative indication of the presence of specific antigens in the blood to a particular sought-for virus.

The reaction of immunofluorescence (RIF)

The reaction is based on the ability to detect AG-AT complexes in luminescent microscopy after their treatment with fluorochromic dyes. This method is easy to handle, 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, which is pre-treated with fluorescent serum. And indirectly, the drug is first treated with a conventional diagnosticum containing antigens to the desired antibodies, and then luminescent serum, which is specific for the proteins of the AG-AT complex, is re-applied and the microbial cells become noticeable by microscopy.