IMMUNOLOGY TEXTBOOK - Mercury Podillia 2013

IMMUNOLOGICAL RESEARCH METHODS

Immunological (Serological) Methods for Investigating Infectious Diseases

Serological reactions are used in two main directions:

- Detection of Antibodies in the patient's Blood serum for diagnostic purposes based on the presence of a panel of known Antigens. Suspensions of microorganisms inactivated by chemical or physical Methods, or diagnostic agents representing microorganism fractions, are used as antigens. As a rule, serological Diagnosis results are obtained by examining paired blood sera of patients taken in the first days of the illness and at specific intervals thereafter.

- Determination of the genus, species, and type affiliation of a microorganism or its antigens using known immune sera. Immune sera must contain antibodies at a high titer and be strictly specific. Laboratory practice employs serological reactions based on the direct interaction of antigen and antibody (agglutination, precipitation) and indirect reactions (indirect hemagglutination assay, Complement fixation test), as well as reactions using labeled antibodies or antigens (enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent antibody technique).

Agglutination reaction is used in laboratory practice to identify isolated microorganisms or to detect specific antibodies in blood serum. The Mechanism of the reaction is based on the interaction of the antigenic determinant groups with the active centers of the immunoglobulin in an electrolyte medium.

Precipitation reaction. The precipitation phenomenon consists of the interaction of finely dispersed antigens (precipitinogens) with corresponding antibodies (precipitins), resulting in The formation of a precipitate. Precipitation assays are performed using two methods: in a liquid medium—in the form of a flocculation reaction or ring precipitation, or in a solid medium in Agar (gel). The precipitation reaction is used for two purposes: detecting antigens using a known immune serum, or detecting antibodies using known antigens. There are many variants of this reaction, but the most frequently used methods include the Ouchterlony double immunodiffusion in gel, Mancini radial immunodiffusion, Immunoelectrophoresis, flocculation, and ring precipitation tests.

Complement fixation test (CFT) is used for the Laboratory Diagnosis of Sexually Transmitted Diseases, rickettsioses, viral infections (Influenza, measles, tick-borne encephalitis, etc.) and is based on the ability of complement to bind to the antigen-antibody complex. Complement is adsorbed onto the Fc fragment of IMMUNOGLOBULINS G and M. The reaction proceeds in two phases. The first phase is the interaction of antigen and antibody. The material containing antibodies is the test serum to which a known antigen is added. Standard complement is added to this system and incubated at 37 °C for one hour.

The second phase is the detection of the reaction results using an indicator hemolytic system (sheep red Blood Cells and rabbit hemolytic serum containing hemolysins against sheep erythrocytes). The indicator system is added to the antigen + antibody + complement mixture (1st phase) and incubated again at 37 °C for 30–60 min, after which the reaction results are evaluated. Erythrocyte destruction occurs if complement binds to the hemolytic system.

Indirect hemagglutination assay (IHA). IHA is used in two variants: with a known antigen to detect antibodies, or with known antibodies to detect antigen. This reaction is specific and is used for the diagnosis of diseases caused by Bacteria and rickettsiae. To perform IHA, erythrocyte Diagnostics prepared by adsorbing antigens or antibodies onto erythrocytes depending on the purpose of the study are used. In positive cases, the degree of erythrocyte agglutination is graded with plus signs.

Hemagglutination assay (HA) and hemagglutination inhibition assay (HIA). HA is based on the ability of erythrocytes to clump together when certain antigens are adsorbed onto them. Allantoic and Amniotic Fluid, suspensions of the chorioallantoic membranes of chicken embryos, suspensions and extracts from cultures or Organs of animals infected with Viruses, and native infectious material are used as test Materials in hemagglutination. HA is not a serological test, as it occurs without the participation of immune serum; it is used to select the working dilution of an antigen for performing HIA or to determine the presence of an antigen (virus) in the test material (e.g., in influenza). The assay utilizes erythrocytes from animals, birds, and humans with blood group I (0). If the HA result is positive, the study continues by determining the type of the isolated virus using hemagglutination inhibition with type-specific sera.

HIA is based on the property of antiserum to suppress viral hemagglutination, since a virus neutralized by specific antibodies loses its ability to agglutinate erythrocytes.

Immunofluorescence assay (IFA). IFA is based on the combination of bacterial, rickettsial, and viral antigens with specific antibodies labeled with fluorescent Dyes (fluorescein isothiocyanate, rhodamine, B-isothiocyanate, lissamine rhodamine B-200, sulfochloride, etc.) containing reactive groups (sulfochloride, isothiocyanate, etc.). These groups bind to the free amino groups of antibody molecules, which do not lose their specific affinity for the corresponding antigen during fluorochrome Treatment. The resulting antigen-antibody complexes become clearly visible, brightly glowing structures under a fluorescence Microscope. IFA can be used to detect small amounts of bacterial and viral antigens.

Enzyme-linked immunosorbent assay (ELISA) is used to detect antigens using their corresponding antibodies conjugated to an enzyme label. After the antigen binds to the enzyme-labeled immune serum, a substrate and a chromogen are added to the mixture. The substrate is cleaved by the enzyme, and its degradation products cause a chemical Modification of the chromogen. In this process, the chromogen changes color—the color intensity is directly proportional to The amount of bound antigen and antibody molecules. ELISA is used to diagnose diseases caused by viral and bacterial pathogens.