Laboratory diagnosis of almost all infectious diseases is based on the detection of antibodies in the patient's blood, which are produced against the pathogen's antigens, by the methods of serological reactions. They entered medical practice from the late nineteenth to early twentieth century.
The development of science has helped determine the antigenic structure of microbes and the chemical formulas of their toxins. This made it possible to create not only therapeutic, but also diagnostic sera. They are obtained by administering attenuated pathogens to laboratory animals. After several days of exposure, the blood of rabbits or mice is used to prepare preparations used to identify microbes or their toxins using serological tests.
The external manifestation of such a reaction depends on the conditions of its setting and on the state of antigens in the patient's blood. If microbial particles are insoluble, they precipitate, lyse, bind or immobilize in the serum. If the antigens are soluble, then the phenomenon of neutralization or precipitation appears.
Agglutination reaction (RA)
The serological agglutination test is highly specific. It is easy to perform and quitevisual, to quickly determine the presence of antigens in the patient's blood serum. It is used to test the Vidal reaction (diagnosis of typhoid and paratyphoid fever) and Weigl (typhoid fever).
It is based on a specific interaction between human antibodies (or agglutinins) and microbial cells (agglutenogens). After their interaction, particles are formed that precipitate. This is a positive sign. Live or killed microbial agents, fungi, protozoa, blood cells and somatic cells can be used to set up the reaction.
Chemically, the reaction is divided into two steps:
- Specific connection of antibodies (AT) with antigens (AG).
- Non-specific - precipitation of AG-AT conglomerates, that is, the formation of agglutinate.
Indirect Agglutination Reaction (IPHA)
This reaction is more sensitive than the previous one. It is used to diagnose diseases caused by bacteria, intracellular parasites, and protozoa. It is so specific that it can detect even very low concentrations of antibodies.
Purified sheep erythrocytes and human red blood cells pre-treated with antibodies or antigens are used for its production (depending 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 to have taken place if they have settled on the bottom of the tube. About a positive reactionsay when the cells are arranged in the form of an inverted umbrella, occupying the entire bottom. A negative reaction is counted if the erythrocytes settled in a column or in the form of a button in the center of the bottom.
Precipitation reaction (RP)
Serological reactions of this type are used to detect extremely small particles of antigens. These can be, for example, proteins (or parts thereof), compounds of proteins with lipids or carbohydrates, parts of bacteria, their toxins.
Sera for the reaction are obtained by artificially infecting animals, usually rabbits. By this method, you can get absolutely any precipitating serum. The setting of serological precipitation reactions is similar in mechanism of action to agglutination reactions. 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 on the substrate (gel). This method is considered highly specific and can detect even negligible amounts of a substance.
Used to diagnose plague, tularemia, anthrax, meningitis and other diseases. In addition, he is involved in a forensic medical examination.
Gel precipitation reaction
Serological reactions can be carried out not only in a liquid medium, but also in agar gel. This is called the diffuse precipitation method. 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 a gel they movetowards each other at different speeds and, meeting, form precipitation lines. Each line is one set of AG-AT.
Exotoxin neutralization reaction with antitoxin (PH)
Antitoxic serums are able to neutralize the action of exotoxin produced by microorganisms. These serological reactions are based on this. Microbiology uses this method to titrate sera, toxins and toxoids and determine their therapeutic activity. The power of toxin neutralization is determined by conventional units - AE.
In addition, thanks to this reaction, it is possible to determine the species or type of exotoxin. This is used in the diagnosis of tetanus, diphtheria, botulism. The study can be carried out both “on glass” and in gel.
Reaction of lysis (RL)
Immune serum, which enters the patient's body, has, in addition to its main function of passive immunity, also lysing properties. It is able to dissolve microbial agents, cellular foreign elements and viruses that enter the patient's body. Depending on the specificity of the antibodies included in the serum, bacteriolysins, cytolysins, spirochetolizins, hemolysins and others are isolated.
These specific antibodies are called "complement". It is found in almost all human body fluids, has a complex protein structure and is extremely sensitive to temperature rise, shaking, acids and direct sunlight. But in the dried state it is able to retainits lysing properties up to six months.
There are these types of serological reactions of this type:
- bacteriolysis;
- hemolysis.
Bacteriolysis is carried out using the patient's blood serum and specific immune serum with live microbes. If enough complement is present in the blood, then the researcher will see the bacteria lyse, and the reaction will be considered positive.
The second serological reaction of the blood is that a suspension of the patient's red blood cells is treated with serum containing hemolysins, which are activated only in the presence of a certain compliment. If there is one, then the laboratory assistant observes the dissolution of red blood cells. This reaction is widely used in modern medicine to determine the complement titer (that is, its smallest amount that provokes erythrocyte lysis) in blood serum and to perform an analysis for complement fixation. It is in this way that a serological test for syphilis is carried out - the Wasserman reaction.
Complement fixation reaction (CFR)
This reaction is used to detect antibodies to an infectious agent in the patient's blood serum, as well as to identify the pathogen by its antigenic structure.
Up to this point, we have described simple serological reactions. RSK is considered a complex reaction, since not two, but three elements interact in it: antibody, antigen and complement. Its essence lies in the fact that the interaction between the antibody and antigenoccurs only in the presence of complement proteins, which are adsorbed on the surface of the formed AG-AT complex.
The antigens themselves, after complement addition, undergo significant changes, which show the quality of the reaction. It can be lysis, hemolysis, immobilization, bactericidal or bacteriostatic action.
The reaction itself occurs in two phases:
- Formation of an antigen-antibody complex that is not visually visible to the examiner.
- Change in the antigen under the action of complement. This phase can most often be traced with the naked eye. If the reaction is not visually visible, then an additional indicator system is used to identify changes.
Indicator system
This reaction is based on complement fixation. Purified ram erythrocytes and complement-free hemolytic serum are added to the test tube an hour after the RSC is set. If an unbound complement remains in the test tube, then it will join the AG-AT complex formed between sheep blood cells and hemolysin, and cause them to dissolve. This will mean that RSK is negative. If the erythrocytes remained intact, then, accordingly, the reaction is positive.
Hemagglutination test (RGA)
There are two fundamentally different hemagglutination reactions. One of them is serological, it is used to determine blood groups. In this case, red blood cells interact with antibodies.
And the secondthe reaction does not apply to serological, since red blood cells react with hemagglutinins produced by viruses. Since each pathogen acts only on specific erythrocytes (chicken, lamb, monkey), this reaction can be considered highly specific.
You can tell if a reaction is positive or negative by the location of the blood cells at the bottom of the test tube. If their pattern resembles an inverted umbrella, then the desired virus is present in the patient's blood. And if all the erythrocytes have formed like a coin column, then there are no desired pathogens.
Hemagglutination inhibition test (HITA)
This is a highly specific reaction that allows you to determine the type, type of viruses 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 erythrocytes, thereby stopping hemagglutination. This is a qualitative sign of the presence in the blood of specific antigens for the specific virus being sought.
Immunofluorescence reaction (RIF)
The reaction is based on the ability to detect AG-AT complexes with fluorescent microscopy after their treatment with fluorochrome dyes. This method is easy to handle, does not require isolation 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 fromantigen, which is pre-treated with fluorescent serum. And the indirect one is that first the drug is treated with a conventional diagnosticum containing antigens for the antibodies of interest, and then the luminescent serum, which is specific for the proteins of the AG-AT complex, is re-applied, and microbial cells become visible under microscopy.
