Methods for laboratory diagnosis of viral infections. Serological tests Serological tests used for diagnosis

Serological diagnostics based on the antigen-antibody reaction can be used to determine both, and plays a role in determining the etiology of a viral infection even with negative results of virus isolation.

The success of serological diagnosis depends on the specificity of the reaction and compliance with the temporary conditions for taking blood necessary for the synthesis of antibodies by the body.

In most cases, paired blood sera are used, taken at intervals of 2-3 weeks. A positive reaction is considered at least a 4-fold increase in antibody titer. It is known that most specific antibodies belong to the IgG and IgM classes, which are synthesized at different times of the infectious process. Wherein IgM antibodies are early, and the tests used to determine them are used for early diagnosis (it is enough to examine one serum). Antibodies of the IgG class are synthesized later and stored for a long time.

For virus typing, pH is used, for group-specific diagnostics, for example, of adenovirus infection, they use complement fixation reaction(RSK). The most used are hemagglutination inhibition reaction(RTGA), RSK, RIF, passive reactions and reverse passive hemagglutination(RPGA, ROPGA), various variants of ELISA, which almost everywhere replaced RIA, equal in sensitivity to it.

RTGA used to diagnose diseases caused by hemagglutinating viruses. It is based on the binding of antibodies to the patient's serum of the added standard virus. The indicator of the reaction is erythrocytes agglutinated by the virus (formation of a characteristic "umbrella") in the absence of specific antibodies and settling to the bottom, non-agglutinated in their presence.

RSK is one of the traditional serological tests and is used to diagnose many viral infections. Two systems take part in the reaction: antibodies of the patient's serum + standard virus and sheep erythrocytes + antibodies to them, as well as a titrated complement. If the antibodies and the virus match, this complex binds complement and lysis of sheep erythrocytes does not occur ( positive reaction). With a negative RSC, complement contributes to the lysis of erythrocytes. The disadvantage of the method is its insufficiently high sensitivity and the difficulty of standardizing the reagents.

To take into account the significance of RSK, as well as RTGA, it is necessary to titrate paired sera, that is, taken at the onset of the disease and during convalescence.

RPGA- agglutination of erythrocytes (or polystyrene beads) sensitized by viral antigens in the presence of antibodies. Any viruses can be sorbed on erythrocytes, regardless of the presence or absence of hemagglutinating activity in them. Due to the presence of non-specific reactions, sera are tested at a dilution of 1:10 or more.

RNGA- agglutination of erythrocytes sensitized by specific antibodies in the presence of viral antigens. ROPHA received the greatest distribution in the detection of the HBs antigen both in patients and in blood donors.

IF method as well ELISA used to detect antibodies in serum. ELISA for diagnostic purposes is becoming increasingly important and widespread. The viral antigen is sorbed onto the solid phase (the bottom of the wells of polystyrene plates or polystyrene beads). When the corresponding antibodies in the serum are added, they bind to the adsorbed antigens. The presence of the desired antibodies is detected using anti-antibodies (for example, human) conjugated with an enzyme (peroxidase). Substrate addition and substrate-enzyme reaction give color. ELISA can also be used to determine antigens. In this case, antibodies are adsorbed onto the solid phase.

monoclonal antibodies. Great progress in the diagnosis of viral infections has been made in the last decade, when, with the development of genetic engineering research, a system for obtaining monoclonal antibodies was developed. This dramatically increased the specificity and sensitivity diagnostic methods determination of viral antigens. The narrow specificity of monoclones, representing a small proportion of viral proteins that may not be present in clinical material, is successfully overcome by using several monoclonal antibodies to various viral determinants.

It is based on the determination of antiviral antibodies in the patient's blood in serological reactions using specific viral antigens - diagnosticums or special test systems. Serological reactions in viral infections are put in a liquid medium (RSK, RTGA, RNGA, RONGA, RTONGA, RIA), in a gel (RPG, RRG, RVIEF) or on a solid-phase carrier (for example, on the walls of a well of a polystyrene plate with fixation of one of the components of the immune response - antigen or antibody). Such solid-phase methods as ELISA, IEM, RGadsTO, RIF, RGads, RTGads are known.

Often, due to the presence in the blood of the majority healthy people natural antiviral antibodies, serological diagnosis of viral infections is based on the study paired serums, taken at the beginning and at the height of the disease or during the period of convalescence in order to determine the increase in antibody titer. An increase in antibody titer by four times or more is considered diagnostically significant.

Increasing the sensitivity of serological methods is achieved by adsorption of antigens or antibodies on erythrocytes (RNGA, RONGA, RTONGA, RGadsTO, RRG), labeled with enzymes (ELISA), radioactive isotopes (RIA, RPG) or fluorochromes (RIF), The principle of erythrocyte lysis is also used (as an indicator systems) during the interaction of antigens and antibodies in the presence of complement (RSK, RRG).

Complement fixation reaction (CFR) in the form of complement fixation in the cold (during the night at a temperature of +4 0 C) is often used in virology for the retrospective diagnosis of a number of viral infections and for the determination of virus-specific antigens in materials from patients.

Radial hemolysis reaction (RRH) in agarose gel is based on the phenomenon of hemolysis of erythrocytes sensitized with an antigen under the influence of virus-specific antibodies in the presence of complement and is used for serological diagnosis of influenza, ARVI, rubella, mumps, and togavirus infections.

To set up the reaction, 0.1 ml of undiluted viral antigen is added to sheep erythrocytes (0.3 ml of a 10% suspension), and the mixture is incubated for 10 minutes at room temperature. 0.3 ml of sensitized erythrocytes and 0.1 ml of complement are added to 1.2% agarose at a temperature of 42 0 C, the mixture is poured onto glass slides or into the wells of polystyrene plates, holes are cut out in the frozen agarose gel with a punch and filled with the studied and control sera. Glasses or panels are closed with a lid and placed in a humid chamber for 16-18 hours in a thermostat. Accounting for the reaction is carried out according to the diameter of the hemolysis zone around the holes filled with serum. There is no hemolysis in the control.

With the majority viral infections develop immune reactions applied to diagnostics. Cellular responses are usually assessed in tests of lymphocyte cytotoxicity against infectious agents or target cells infected by them, or the ability of lymphocytes to respond to various antigens and mitogens. In the work of practical laboratories, the severity of cellular reactions is rarely determined. Methods for identifying antiviral ATs have become more widespread.

RN is based on suppression of the cytopathogenic effect after mixing the virus with specific AT. The unknown virus is mixed with known commercial antisera and, after appropriate incubation, introduced into the cell monolayer. The absence of cell death indicates a mismatch between the infectious agent and known antibodies.

Inhibition of hemagglutination

RTGA is used to identify viruses capable of agglutinating various erythrocytes. To do this, the culture medium containing the pathogen is mixed with a known commercial antiserum and introduced into the cell culture. After incubation, the ability of the culture to hemagglutination is determined and, in its absence, a conclusion is made about the mismatch of the virus with antiserum.

Inhibition of the cytopathic effect by virus interference

The reaction of inhibition of the cytopathic effect due to the interference of viruses used to identify a pathogen that interferes with a known cytopathogenic virus in a culture of susceptible cells. To do this, a commercial serum (for example, to the rubella virus if it is suspected) is introduced into the culture medium containing the virus under study, incubated and infect the second culture; after 1-2 days, a known cytopathogenic virus (for example, any ECHO virus) is introduced into it. In the presence of a cytopathogenic effect, it is concluded that the first culture was infected with a virus corresponding to the applied AT.

Direct immunofluorescence

Among other tests, the most widely used direct immunofluorescence reaction(the fastest, most sensitive and reproducible). For example, the identification of CMV by cytopathogenic effect requires at least 2-3 weeks, and when using labeled monoclonal antibodies, identification is possible after 24 hours. examine using fluorescent microscopy (allows you to detect the presence of fluorescence of infected cells).

Immunoelectron microscopy

Immunoelectron microscopy(similar to the previous method) allows you to identify different kinds viruses detected by electron microscopy (for example, various types of herpesviruses), which cannot be done based on morphological features. Instead of antisera, AT labeled in various ways are used for identification, but the complexity and high cost of the method limit its application.

Most viral infections develop immune responses that are used for diagnosis. Cellular responses are usually assessed in tests of lymphocyte cytotoxicity against infectious agents or infected target cells, or the ability of lymphocytes to respond to various antigens and mitogens.

In the work of practical laboratories, the severity of cellular reactions is rarely determined. Methods for identifying antiviral ATs have become more widespread.

RN based on the suppression of the cytopathogenic effect after mixing the virus with specific antibodies. The unknown virus is mixed with known commercial antisera and, after appropriate incubation, introduced into the cell monolayer. The absence of cell death indicates a mismatch between the infectious agent and known antibodies.

Inhibition of hemagglutination RTHA used to identify viruses capable of agglutinating various erythrocytes. To do this, the culture medium containing the pathogen is mixed with a known commercial antiserum and introduced into the cell culture. After incubation, the ability of the culture to hemagglutination is determined and, in its absence, a conclusion is made about the mismatch of the virus with antiserum. Inhibition of the cytopathic effect by viral interference The reaction of inhibition of the cytopathic effect due to the interference of viruses is used to identify a pathogen that interferes with a known cytopathogenic virus in a culture of susceptible cells. To do this, a commercial serum (for example, to the rubella virus if it is suspected) is introduced into the culture medium containing the studied virus, incubated and infect the second culture; after 1-2 days, a known cytopathogenic virus (for example, any ECHO virus) is introduced into it. If there is a cytopathogenic effect, it is concluded that the first culture was infected with the virus corresponding to the applied AT.

Direct immunofluorescence.

Among other tests, the direct immunofluorescence reaction (the fastest, most sensitive and reproducible) has found the greatest distribution. For example, the identification of CMV by cytopathogenic effect requires at least 2-3 weeks, and when using labeled monoclonal antibodies, identification is possible after 24 hours. using fluorescent microscopy (allows you to detect the presence of fluorescence of infected cells).

Immunoelectron microscopy (similar to the previous method) allows you to identify different types of viruses detected by electron microscopy (for example, different types of herpesviruses), which cannot be done based on morphological features. Instead of antisera, AT labeled in various ways are used for identification, but the complexity and high cost of the method limit its application.

Detection of antiviral antibodies (AT) in blood serum. RTGA. RSK. REEF.

Immunosorptive methods for the detection of antiviral antibodies.

A simpler and more accessible approach is the detection of antiviral antibodies (AT) in serum. Blood samples should be taken twice: immediately after the onset of clinical signs and after 2~3 weeks. It is extremely important to examine exactly two serum samples. The results of a single study cannot be considered conclusive due to the inability to link the appearance of AT with the present case. It is possible that these antibodies circulate after a previous infection. In such a situation, the role of the study of serum obtained during the period of convalescence can hardly be overestimated. The presence of the disease during the period of taking the first sample is indicated by at least a fourfold increase in the AT titer, which was detected during the study of the second sample.

The methods listed below do not allow differentiation of antibodies (AT) formed during illness and circulating after recovery (the duration of this period is variable for different infections). Since for adequate diagnosis it is necessary to confirm a significant increase in AT titers in two samples, the first sample is examined in the acute phase, and the second - during the recovery period (after 2-3 weeks). The results obtained are retrospective and more suitable for epidemiological surveys. RTGA detects antibodies synthesized against the hemagglutinins of viruses (for example, the influenza virus).

The method makes it easy to detect such antibodies (AT) in the patient's serum. RSK is the main method of serodiagnosis of viral infections (among the available ones). The reaction detects complement-fixing IgM and IgG, but does not differentiate them; to optimize the results obtained, the formulation of the reaction requires certain skills of the personnel.

REEF. If a biopsy of infected tissue is available and commercial fluorescein-labeled AT kits are available, direct immunofluorescence can confirm the diagnosis.

The formulation of the reaction includes incubation of the studied tissue with AT, their subsequent removal and fluorescent microscopy of the sample. Immunosorptive methods for detecting antiviral antibodies Immunosorptive methods (for example, ELISA and RIA) are more informative, since they detect IgM and IgG separately, which makes it possible to draw certain conclusions about the dynamics of the infectious process or the state of convalescence. To detect AT, a known antigen is adsorbed on a solid substrate (for example, on the walls of test tubes, plastic microplates, Petri dishes) and various dilutions of the patient's serum are added. After appropriate incubation, unbound ATs are removed, enzyme-labeled antiserum against human Ig is added, the procedure for incubation and washing of unbound ATs is repeated, and any chromogenic substrate (sensitive to the action of the enzyme) is added. Since the color change is proportional to the content of specific antibodies, it is quite possible to determine their titer by spectrophotometric method. In the diagnosis of HIV infection, the method of immunoblotting has found the greatest distribution.

Detection of viral antigens (AH). ELISA. Currently, commercial kits have already appeared for the detection of AH of some pathogens, allowing them to be identified within 5-10 minutes. To detect AG on the solid phase, known AT are adsorbed and serum containing AG is added; after incubation, unbound AG is decanted, the system is washed, and labeled antibodies specific to adsorbed antibodies are added. Repeat the procedure of incubation and washing, make a chromogenic substrate, positive result fixed when the color of the system changes. DNA hybridization is a highly specific method that allows the identification of the virus genome after its hybridization with complementary DNA molecules. Enzymes and isotopes are used as markers.

The method determines the ability of viral DNA to hybridize with labeled complementary DNA; the specificity of the method is directly proportional to the length of the complementary chain. A promising method for in situ hybridization of nucleic acids. To set up the reaction, labeled DNA is applied to tissue biopsies (including those fixed with formalin or enclosed in paraffin blocks) and the interaction with complementary DNA is recorded. The method is used to detect herpes simplex viruses, human papilloma, Epstein-Barr, etc.

PCR. The method significantly increases the sensitivity of the hybridization method, increasing the content of viral DNA in the material obtained from the patient, and also speeds up the time to obtain the result.