SEROLOGICAL RESEARCHES (Latin serum serum + Greek logos the doctrine) — the methods of immunology studying specific properties of blood of the person or animals for the purpose of identification of antigens or antibodies by means of serological tests.
Page and. are widely used in medicine for a lab. diagnoses of infectious and parasitic diseases, blood typing and fabric antigens, specific accessory of protein, recognition of autoimmune diseases, nek-ry types of pathology of pregnant, hormonal disturbances, reactivity of an organism to antibiotics and studying of many other questions. Establishment of the diagnosis by means of S. and. call a serodiagnosis (serological diagnosis).
S.'s beginning and. it is necessary at the end of the last century after it was established that compound of antigen with an antibody (see. Antigen — an antibody reaction ) is followed by a number of phenomena available to sighting — agglutination (see), precipitation (see) or lysis. There was a possibility of specific recognition antigens (see) or antibodies (see) if one of these components is known.
In 1897 F. Vidal reported that blood serum of patients with a typhoid selectively agglutinates typroid bacteria and therefore this reaction (see. Vidalya reaction ) it can be applied for a lab. diagnoses of a typhoid. It was the same year shown that filtrates of cultures of plague, typroid and cholera bacteria at connection with the corresponding immune serums form flakes, or precipitated calcium superphosphate.
The precipitation test was suitable for detection of any proteinaceous antigens. In 1900 — 1901 K. Landshteyner found that in erythrocytes of people there are two various antigens (And yes In), and in blood sera two agglutinins (and and P) that promoted use of hemagglutination reaction for definition blood groups (see).
Zh. Borde's opening in 1898. complement (see), at presence to-rogo immune antieritrotsitar-ny serums gained ability to lyse erythrocytes, allowed to develop reaction of binding complement (see), widely used in infectious and noninfectious immunology.
Since 40th 20 century in serological researches began to apply immunokhikhmichesky methods, namely a tag of antibodies of a flyuorokhromama (see. Immunofluorescence ), isotopes (see. Radio immunological method ) or enzymes (see. Enzimimmunologichesky method ). It promoted substantial increase of sensitivity serol. reactions, i.e. possibility of identification of the minimum quantities of antigens or antibodies. Serol. reactions differ on ability to reveal separate types of antibodies. The agglutination test, e.g., well reveals IgM-antibodies, but is less sensitive for detection of IgG-antibodies. Reactions of binding complement and hemolysis (see), to-rye demand participation of a complement, do not reveal the antibodies which are not attaching a complement, e.g. IgA-antibodies and IgE-antibodies. Only the antibodies directed against the antigenic determinants of a surface of virion connected with pathogenicity participate in a neutralization test of viruses (e.g., protein of gp 56 of a virus of the Venezuelan encephalomyelitis of horses).
- 1 The main types of serological tests
- 2 The comparative characteristic and use of methods of serological researches in medical practice
The main types of serological tests
the Reactions based on a phenomenon of agglutination
the Agglutination test of bacteria with use of the corresponding antibacterial serum — the simplest serol. reaction. The suspension of bacteria is added to various cultivations of ispytuyemy blood serum and through certain time of contact at t ° 37 ° registered at what highest cultivation of blood serum there is an agglutination. The agglutination test of bacteria is used for diagnosis of many inf. diseases; a brucellosis, a tularemia, a typhoid and paratyphus, bacillar dysentery (see. Wright reaction , Haddlsona reaction ), sapropyra (cm. Veylya — Felix reaction ).
The agglutination test is applied to blood typing and a Rhesus factor in obstetric practice, at hemotransfusions and transplantation of fabrics. Antibodies against Rhesus factor (see) represent monovalent antibodies, they are not capable to forward reaction with Rh-positive erythrocytes therefore they for their detection use Koombs's reaction (see. Koombs reaction ), based on identification of monovalent antibodies by means of antiglobulinovy serums. Add the studied blood serum and after this antiglobulinovy serum against IgG (indirect reaction of Koombs) to erythrocytes of the known specificity. Fab-fragments of monovalent antibodies of the studied blood serum join erythrocytes, and antibodies against IgG join free Fc-fragments of these antibodies, and there is an agglutination of erythrocytes. For diagnosis of hemolitic anemia use forward reaction of Koombs, In an organism of such patients erythrocytes connect to the antibodies circulating in blood against a Rhesus factor. That to reveal them, add an antibody against IgG to the erythrocytes taken from the patient. Emergence of agglutination of erythrocytes confirms the diagnosis of a disease.
Hemagglutination-inhibition reaction — RTGA (see. Hemagglutination ) — it is based on a phenomenon of prevention (braking) by immune serum of hemagglutination of erythrocytes viruses. The phenomenon of virus hemagglutination is not serol. reaction also results from connection of a virus with receptors of erythrocytes, however RTGA represents a serological test, is-polzuyekhmy for identification and titration of antiviral antibodies. RTGA — the main method of serodiagnosis of flu, measles, rubella, epidemic parotitis, a tick-borne encephalitis and other viral infections, activators to-rykh have the hemagglutinating properties.
Reaction passive, or indirect, hemagglutinations, In it use erythrocytes or neutral synthetic hchateriala (e.g., particles of latex), on a surface to-rykh antigens (bacterial, virus, fabric) or antibodies are occluded (see. Boydena reaction ). Their agglutination happens at addition of the corresponding serums or antigens. Erythrocytes, sensibilized antigens, call an antigenic erythrocyte diagnosticum and use for identification and titration of antibodies. Erythrocytes, sensibilized anti-that-lakhmi, call immunoglobin erythrocyte diagnosticums (see) also use for identification of antigens:
Reaction of passive hemagglutination is applied to diagnosis of the diseases caused by bacteria (a typhoid and paratyphus, dysentery, a brucellosis, plague, cholera, etc.), protozoa (malaria) and viruses (flu, adenoviral infections, tick-borne encephalitis, the Crimean hemorrhagic fever, etc.). Reaction of passive hemagglutination on sensitivity does not concede to a method of allocation of a virus at arenovirusny diseases (see), in particular at a lymphocytic choriomeningitis. The viral antigen of a lymphocytic choriomeningitis comes to light at virus carriers (house mice) in reaction of passive hemagglutination with the suspensions of the taken bodies divorced in tens of thousands of times. At a salmonellosis in reaction of passive hemagglutination bacteria at concentration to several hundred microbic bodies in 1 g of excrements are defined, dysenteric bacteria in foodstuff come to light at contents in 1 g of material not less than 500 microbic bodies.
Reaction of passive hemagglutination is used in diagnosis and prevention of a viral hepatitis of V. V the Soviet Union for identification of HBs-antigen (see. Australian antigen ) in blood of patients with an acute hepatitis In the diagnosticum representing erythrocytes of hens, sensibilized goat immunoglobulin against HBs-antigen is made. The drop of a diagnosticum is connected to the equal volume of blood serum of the inspected people, and if at it there is a HBs-antigen, there is an agglutination. Reaction is capable to catch to 1,5 ng/ml of HBs-antigen. For detection of HBs-antibodies use erythrocytes with the HBs-antigen occluded on them emitted from blood of patients. Reaction of passive hemagglutination is applied also to detection of hypersensitivity of the patient to medicines and hormones, napr, to penicillin or insulin. In this case erythrocytes sensibilize 0 blood groups of the person medicinal substance and then use for identification of agglutinins to it in blood serum of the patient.
Reaction of passive hemagglutination is used for identification of gonadotropic hormone in urine for the purpose of establishment of pregnancy (see. Chorionic gonadotrophin ). For this purpose standard serum to this hormone is incubated with the studied urine. At the subsequent addition of erythrocytes with the hormone occluded on them agglutination does not occur (affirmative answer) since the hormone which is contained in urine neutralized the agglutinating antibodies.
The reactions based on a phenomenon of precipitation
them use for definition of the most various antigens and antibodies. The elementary example of qualitative test is formation of an opaque strip of precipitation on border of stratification of antigen on an antibody in a test tube. Various kinds of a precipitation test in semi-fluid gels of an agar or agarose are widely applied (a method of double immunodiffusion on Oukhterlonya, a method of radial immunodiffusion, an immunoelectrophoresis), to-rye have at the same time qualitative and quantitative character (see. Immunodiffusion , Immunoelectrophoresis ).
For statement of double immunodiffusion pour a layer of the kindled gel on a glass plate and after hardening cut out holes with a diameter of 1,5 — 3 mm. In the holes located around place the studied antigens, and in the central hole — immune serum of the known specificity. Diffusing towards each other, homologous serums and antigens form precipitated calcium superphosphate. At radial immunodiffusion (by a method Mang-repair) immune serum is brought in an agar. The antigen placed in holes diffuses through an agar, and as a result of precipitation with immune serum around holes opaque rings, external diameter are formed to-rykh it is proportional to concentration of antigen. Modification of this reaction is used in diagnosis of flu for recognition of IgM-and IgG-antibodies (see. Immunoglobulins ). In an agar bring influenzal antigen, and in holes of blood serum. Then plates process immune serums against IgM-or IgG-antibodies that promotes identification of reaction of the corresponding antibodies with antigens. The method allows to define at the same time antiserum capacities and their belonging to a certain class of immunoglobulins.
A kind of an immunoelectrophoresis is radioimmunoforez. In this case after electrophoretic division of antigens in the flute which is cut out parallel to the movement of antigens in gel pour at first marked a radioiodine immune serum against the defined antigens, and then immune serum against IgG-antibodies, edges pretsipitirut the formed complexes of an antibody with antigen. All not communicated reagents wash away, and antigen — an antibody find a complex by method autoradiography (see).
Reactions with participation of a complement. Reactions with participation complement (see) are based on ability of a subcomponent of a complement of Cl (Clq) and then other components of a complement to join cell-bound immune complexes.
Reaction of binding complement allows to titrate antigens or antibodies on extent of complement deflection by a complex antigen — an antibody. This reaction consists of two phases: interactions of antigen with ispytuyemy blood serum (the studied system) and interactions of hemolitic serum with mutton erythrocytes (indicator system). At positive reaction in the studied system there is fixation of the complement, and then at addition sensibilized antibodies of erythrocytes do not observe hemolysis (see. Reaction of binding complement ). Reaction is widely applied to serodiagnosis of visceral syphilis (see. Wasserman reaction ) and viral infections (see. Virologic researches ).
Cytolysis. Antibodies against cellular structures can dissolve the cells bearing these structures with the participation of a complement. It is easy to estimate lysis of erythrocytes on degree and intensity of release of hemoglobin. Lysis of nuclear cells is estimated by calculation of percent of dead cells, to-rye are not painted by methylene blue. Often also use ^радиоак-тивный chrome, to-ry previously chemically connect with cells. The number of the destroyed cells determine by amount of the untied chrome which is released at killing.
Reaction of radial hemolysis of erythrocytes can proceed in gel. The suspension of erythrocytes of a ram is shsheshchat in agarose gel, having added a complement there; in the layer which stiffened on glass do holes and bring in them hemolitic serum. Around holes as a result of radial diffusion of antibodies the active area will be formed. Radius of an active area is directly proportional to a caption of serum. If to occlude on erythrocytes any antigen, napr, glycoprotein hemagglutinin of an influenza virus, a rubella or tick-borne encephalitis, then it is possible to reproduce a phenomenon of hemolysis by immune serums to these viruses. Reaction of radial hemolysis in gel found application in diagnosis of viral infections thanks to simplicity of statement, nonsensitivity to serumal inhibitors, an opportunity to titrate blood sera on diameter of an active area, without resorting to serial dilutions.
Immune sticking. Erythrocytes, thrombocytes and other blood cells have on a surface receptors to the third component of a complement (SZ). If (bacteria, viruses, etc.) to add the corresponding immune serum and a complement to antigen, then the complex antigen — an antibody, covered with the SZ-component of a complement is formed. During the mixing with thrombocytes thanks to the SZ-component of a complement a complex antigen — the antibody will settle on cells and will cause their agglutination (see. Immune sticking ). This reaction is applied to definition of antigens of the HLA system (see. Immunity transplant ) and during the studying of a number of virus - infections (a tick-borne encephalitis, fever of a dengue), to-rye are followed immu-nopatol. processes and circulation in blood of viral antigens in a complex with antibodies.
The neutralization test is based on ability of antibodies to neutralize nek-ry specific functions of macromolecular or soluble antigens, napr, activity of enzymes, toxins of bacteria, pathogenesity of viruses. In bacteriology this reaction is used for detection of antistreptolysins, anti-Streptokinasa and anti-staphylolysins. The neutralization test of toxins can be estimated on biol. to effect, so, e.g., titrate anti-tetanic and anti-botulinic serums (see. Toxin — antitoxin reaction ). The mix of toxin with an antiserum entered by an animal prevents their death. Various options of a neutralization test apply in virology. During the mixing of viruses with the corresponding antiserum and administration of this mix an animal or in cellular cultures pathogenicity of viruses is neutralized.
the Immunofluorescence developed by Koons (A. N. of Coons) in 1942, consists in use for serol. reactions marked flyuorokhromy serums (see. Immunofluorescence ). Marked flyuorokhromy serum forms with antigen a complex antigen — an antibody, to-ry becomes available to observation under a microscope in the ultraviolet rays exciting a luminescence of a flyuorokhrom. Reaction of a direct immunofluorescence is used for studying of cellular antigens, by detection of a virus in the infected cells and detection of bacteria and rickettsiae in smears. So, for diagnosis of rage prints of pieces of a brain of the animals suspected on a carriage of virus process the luminescing antirabic serum. At a positive take in protoplasm of nervous cells glybk of bright green color are observed. Express diagnosis of flu, a parainfluenza and an adenoviral infection is based on detection of antigens of viruses in cells of prints from a mucous membrane of a nose.
The method of an indirect immunofluorescence based on identification of a complex antigen — an antibody by means of the luminescing immune serum against IgG-antibodies and used for detection not only antigens, but also titration of antibodies is more widely applied. The method found application in a serodiagnosis of herpes, cytomegalindens, Lass's fevers. In laboratory it has to be stored at ° — 20 ° a reserve of drugs of antigensoderzhashchy cells, napr, the cells of VERO or chicken fibroblasts recorded by acetone which are grown up on pieces of thin glass and infected with a virus. On drugs layer the studied blood serum, place drug in the thermostat at f 37 ° for formation of cell-bound immune complexes, and then after washing of not communicated reagents reveal these complexes the marked luminescing serum against globulins of the person. Applying marked immune serums against IgM-or IgG-antibodies, it is possible to differentiate type of antibodies and to find an early immune response on existence of IgM-antited.
The immunofluorescence is widely used not only in bacteriology, virology, parasitology, but also in immunopathologies (see) for detection of humoral antibodys to fabric antigens of the person: to mitochondrions, smooth muscles, cells of a stomach.
In enzyme - an immunological method apply the antibodies conjugated with enzymes, hl. obr. peroxidase of horse-radish or alkaline phosphatase. To find compound of marked serum with antigen, add the substrate decaying the enzyme attached to serum with the advent of coloring in yellowy-brown (peroxidase) or flavovirent (phosphatase) color. Use also the enzymes decomposing not only chromogenic, but also lyumogenny substrate. In this case at positive reaction the luminescence appears. Like an immunofluorescence, the enzimimmunologichesky method is applied to detection of antigens in cells or titration of antibodies on antigensoderzhashchy cells.
Enzyme - an immunological method is the most popular version immunosorption. On the firm carrier, the Crimea there can be a cellulose, polyacrylamide, a dextran and various plastic, occlude antigen. The surface of holes of micropanels serves more often the carrier. With sorbed antigen bring the studied blood serum in holes, then marked enzyme an antiserum and substrate. Positive takes consider on discoloration of fluid medium. For detection of antigens on the carrier occlude antibodies, then bring the studied material in holes and show reaction marked enzyme antimicrobic serum.
Radioimmunologicheski y a method is based on use of a radio isotope tag of antigens or antibodies. Originally it was developed as a specific method of measurement of level of the hormones circulating in blood. Test system was marked isotope hormone (antigen) and an antiserum to it. If to add the material containing required hormone to such antiserum, then it will connect a part of antibodies, at the subsequent introduction of the marked titrated hormone its quantity reduced in comparison with control will contact antibodies. The result is estimated on comparison of curves of the connected and untied radioactive label. This kind of a method carries the name of competitive reaction. There are also other modifications of a radio immunological method. Radio immunological method — the most sensitive method of definition of antigens and antibodies used for definition of hormones, medicinal substances and antibiotics, for diagnosis of bacterial, viral, rickettsial, protozoan diseases, a research of blood proteins, fabric antigens.
The comparative characteristic and use of methods of serological researches in medical practice
Methods C. and. are continuously improved in the direction of sensitization and universality of use. Initially serol. diagnosis was based on identification of antibodies. With emergence in the middle of 20 century of reactions of an immunofluorescence and the passive hemagglutination having bigger sensitivity there was an opportunity to find not only antibodies, but also antigen directly in material from patients. En-zim-immunologichesky and radio immunological methods, on sensitivity on 2 — 3 orders the exceeding immunofluorescence and a passive-nuyugemagglyutinatsiyu, approach methods biol. detection of bacteria and viruses. The area of their use for detection both antigens, and antibodies is theoretically not limited.
Serodiagnosis inf. diseases is based on emergence of antibodies to the allocated or estimated activator irrespective of whether the activator in an acute stage of a disease was found. Investigate vapors of the blood sera taken in an onset of the illness and 2 — 3 weeks later. The gain of antibodies in the second blood serum not less than by 4 times in comparison with the first is diagnostically significant. Matters also what class of immunoglobulins presented antibodies. IgM-antibodies find at the end of the acute period of a disease and in an early stage of reconvalescence. IgG-antibodies appear in later terms of reconvalescence and circulate long. If in the first trimester of pregnancy find IgM-antibodies to a virus of a rubella in the woman, then it forms the basis for abortion since during this period the fruit is especially sensitive to a virus. At different inf. diseases selectively use the most specific and convenient performed by methods.
Page and. widely apply in epidemiology. Systematic gathering of and a research of samples of blood of various groups of the population allow to understand contacts of the population with a source of activators inf. diseases. Studying of level of collective immunity allows to reveal groups of the increased risk and to plan inoculative actions, to study geographical spread of infections. Page and. various age groups of the population allowed to reveal, e.g., retrospectively circulation of different options of an influenza virus in certain spans.
Page and. are of great importance in studying hereditary diseases (see) and the autoimmune diseases which are followed by emergence tkane-and the organospetsifichesky antibodies destroying the corresponding target cells and also in oncology for detection of tumoral antigens. So, the immunodiagnosis of cancer of liver is based on definition in blood serum of patients of alpha-fetoprotein and other embryonic antigens by method of immunodiffusion and a radio immunological method.
Significant progress of science in studying of a fine antigenic structure of cellular antigens, antigens of bacteria and viruses is reached thanks to use in serol. reactions of monoclones, to-rye it is possible to receive to separate determinants of antigen.
See as well mmunodiagnostika .
Bibliography: Methods of researches in immunology, under the editorship of I. Lefkovits and B. Per-nis, lane with English, M., 1981; The Guide to immunology, under the editorship of O. E. Vyazov and Sh. of X. Hodzhayeva, M., 1973; The Guide to clinical laboratory diagnosis, under the editorship of V. V. Menshikov, M., 1982; Immunology, ed. by J. - F. Bach, N. Y., 1978.
S. Ya. Gaydamovich.