Laboratory diagnosis of almost all infectious diseases is based on the detection of antibodies in the patient’s blood, which are produced against pathogen antigens, by the methods of serological reactions. They entered the practice of medicine from the late nineteenth to early twentieth centuries.
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 introducing attenuated pathogens to laboratory animals. After several days of exposure from the blood of rabbits or mice, preparations are prepared that are used to identify microbes or their toxins 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 the 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 is manifested.
Agglutination Reaction (RA)
Serological agglutination reaction is highly specific. It is simple to execute and visual enough to quickly determine the presence of antigens in the patient's blood serum. It is used to formulate the Vidal reaction (diagnosis of typhoid fever and paratyphoid fever) and Weigl (typhus 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. 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:
- A specific compound of antibodies (AT) with antigens (AH).
- Nonspecific - the deposition of AG-AT conglomerates, that is, the formation of agglutinate.
Indirect Agglutination Reaction (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 even a very low concentration of antibodies can be detected.
For its production, purified lamb erythrocytes and human red blood cells, pre-treated with antibodies or antigens, are used (this depends on what the laboratory assistant wants to find). In some cases, human red blood cells are treated with immunoglobulins. Serological reactions of red blood cells are considered completed if they precipitated to the bottom of the tube. You can talk about a positive reaction 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 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. This may be, for example, proteins (or parts thereof), compounds of proteins with lipids or carbohydrates, parts of bacteria, their toxins.
Reaction sera are obtained by artificially infecting animals, usually rabbits. In this way, 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 precipitate on the bottom of the tube or on the substrate (gel). This method is considered highly specific and can detect even negligible amounts of substance.
It is 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 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 the gel, they move towards each other at different speeds and, meeting, form lines of precipitation. Each line - one set of AG-AT.
Antitoxin (PH) neutralization reaction of exotoxin
Antitoxic sera can neutralize the action of exotoxin, which is produced by microorganisms. This is the basis for these serological reactions. Microbiology uses this method to titrate sera, toxins and toxoids, as well as determine their therapeutic activity. The strength of the neutralization of the toxin is determined by conventional units - AE.
In addition, due to this reaction, it is possible to determine the species or type of exotoxin. This is used in the diagnosis of tetanus, diphtheria, botulism. Research can be carried out both "on glass", and in gel.
Lysis reaction (RL)
The immune serum that 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 entering the patient's body. Depending on the specificity of the antibodies that make up the serum, bacteriolysins, cytolysins, spirochetolizins, hemolysins, and others are isolated.
These specific antibodies are called complement. It is found in almost all body fluids, has a complex protein structure and is extremely sensitive to fever, shaking, the action of acids and direct sunlight. But in the dried state it is able to maintain its lysing properties for up to six months.
There are such types of serological reactions of this type:
- bacteriolysis;
- hemolysis.
Bacteriolysis is carried out 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, then the researcher will see the lysis of bacteria, and the reaction will be considered positive.
The second serological reaction of the blood is that a suspension of red blood cells of the patient 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 titer of complement (i.e., its smallest amount that provokes the lysis of red blood cells) in blood serum and to perform a complement binding assay. It is in this way that a serological reaction to syphilis is carried out - the Wasserman reaction.
Complement Binding Reaction (CSC)
This reaction is used to detect antibodies to an infectious agent in the blood serum of a patient, as well as to identify the pathogen by its antigenic structure.
Up to this point, we have described simple serological reactions. CSC 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 antigen occurs only in the presence of compliment proteins, which are adsorbed on the surface of the formed AG-AT complex.
Antigens themselves, after the addition of complement, undergo 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:
- The formation of the antigen-antibody complex, which is not visually noticeable to the researcher.
- Change in antigen by complement. This phase can most often be traced with the naked eye. If the reaction is not visible visually, then use an additional indicator system to detect changes.
Indicator system
This reaction is based on complement binding. Purified sheep erythrocytes and a complement-free hemolytic serum are added to the test tube one hour after the CSC. If an unbound complement remains in the test tube, it will attach to the AG-AT complex formed between sheep blood cells and hemolysin and cause their dissolution. This will mean that the CSC is negative. If the red blood cells remained intact, then, accordingly, the reaction is positive.
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, red blood cells interact with antibodies.
And the second reaction does not belong to serological ones, since red blood cells react with hemagglutinins produced by viruses. Since each pathogen acts only on specific red blood cells (chicken, lamb, monkeys), this reaction can be considered highly specific.
To understand whether a positive reaction or a negative one is possible by the location of blood cells at the bottom of the tube. If their pattern resembles an inverted umbrella, then the desired virus is present in the patient’s blood. And if all the red blood cells have formed like a coin column, then the desired pathogens are not.
Hemagglutination Inhibition Reaction (RTHA)
This is a highly specific reaction that allows you to establish the type, type of virus or the presence of specific antibodies in the patient's blood serum.
Its essence lies in the fact that antibodies added to the test tube with the test material interfere with the deposition of antigens on red blood cells, thereby stopping hemagglutination. This is a qualitative sign of the presence in the blood of specific antigens to a particular virus of interest.
Immunofluorescence Reaction (RIF)
The reaction is based on the ability to detect AG-AT complexes under
fluorescence microscopy after their treatment with fluorochrome dyes. This method is easy to use, does not require a clean 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 that is pretreated with fluorescent serum. And indirectly, the drug is first treated with a conventional diagnosticum containing antigens for the desired antibodies, and then luminescent serum, which is specific for the proteins of the AG-AT complex, is reapplied, and microbial cells become noticeable under microscopy.