Indirect reef. Serological reactions using tag

Currently, serological reactions are widely used, in which labeled ag-s or at-la. These include immunofluorescence reactions, radioimmune and immunoferment methods.

They apply:

1) for serodiagnosis of infectious diseases, i.e., to identify AT using a set of known conjugate (chemically connected) with various labels (enzymes, fluorochromic dyes) of antigens;

2) to determine the microorganism or its serovara using standard labeled diagnostic antibodies (express diagnostics).

Prepare serum immunization of animals by the corresponding ag-M, then immunoglobulins are isolated and conjugate them with luminous dyes (fluorochromas), enzymes, radioisotrops.

Laughty CPs according to specificity are not inferior to another Wed, and in their sensitivity they exceed all the Wed.

No similar materials

As a label, glowing fluorochromic dyes (isothiocyonate fluoriscein, etc.) are used.

There are various modifications of reef. For rapid diagnosis of infectious diseases - to detect microbes or their antigens in the material under study, reef on Kunsu is used.

There are two methods of Reef in Kunsu: straight and indirect.

Components direct reef:

1) The material under study (the flowman separated by the nasopharynk and others);

2) labeled specific immune serum containing at-la to the desired antigen;

3) isotonic sodium chloride solution.

The smear from the material under study is treated with labeled antiserum.

AG-AT reaction occurs. With a luminescent microscopic examination in the site where the AG-AT complexes are localized, fluorescence is detected - the glow.

Components indirect reef:

1) the material under study;

2) specific anti-grades;

3) anti-globulin serum (at-la against immunoglobulin), laughing with fluorichrome;

4) sodium chloride isotonic solution.

The stroke of the material under study is first treated with immune serum to the desired antigen, and then - labeled antihyglobulin serum.

Glowing complexes AG-AT - labeled AT are detected using a luminescent microscope.

The advantage of the indirect method is that there is no need to prepare a wide set of fluorescent specific sera, and only one fluorescent anti-globulin serum is used.

Also isolated a 4-component variety of indirect reef when complement (serum guinea pig) is additionally introduced. With a positive reaction, the AG-AT complex is formed - labeled - AT-Complement.

Reef is based on the combination of antigens of bacteria, rickettsis and viruses with specific antibodies, labeled fluorescent dyes (fluorescentsiniciacianate, rhodamine, in-200, sulfochloride, etc.) having reactive groups (sulfochloride, isothiocyanite, etc.) . These groups are connected to the free amino groups of antibodies molecules that are not lost when processing with a fluorochrome of specific affinity for the appropriate antigen. Formed complexes of AG-AT become well visible, brightly luminous structures under the luminescent microscope (Fig. 7). With the help of Reef, you can detect small amounts of bacterial and viral antigens. The reef method is used in two versions: direct and indirect method.

The direct method is based on the direct connection of the antigen with a labeled antibody. Indirect method - on the phased identification of the AG-AT complex with fluorescent dyes. The first stage consists in the formation of immune complexes of a certain antigen with specific antibodies. The second stage is in identifying this complex by treating it with labeled antihamaglobulin.

The advantage of reef is simplicity, high sensitivity, the rate of obtaining the result. Reef is used as a method of early express diagnosis of influenza, dysentery, malaria, plague, tularemia, syphilis, etc. To carry out such a study, a luminescent microscope is used.

Radioimmunological analysis (RIA)

RIA is one of the most sensitive methods of immunodiagnostics. It is used to identify the antigen of the hepatitis B virus, in patients with viral hepatitis. To do this, reference serum is added to the serum under study (serum containing antibodies to hepatitis B virus). The mixture is incubated by 1-2 days at a temperature of 40 ° C, then a reference antigen is added (an antigen labeled by isotope 125 J) and continue incubation for another 24 hours. The antigen-antibody is added to the resulting antigen-antibody complex adding anti-immoglobulins against serum proteins, which leads to the formation of precipitate (Fig. 8). The result is taken into account by the presence and number of pulses in the precipitate registered by the meter. In the presence in the test serum, the antigen bound with specific antibodies, the latter do not communicate with the labeled antigen and, therefore, it is not detected in the preventation. Thus, the basis of RIA is the principle of competitive interaction of the defined antigen and the known number of labeled antigen with active antibody centers. Radioactive isotopes are used as the label.

Depending on the performance technique, two methods of RIA are distinguished.

1) Technique "Liquid Phase" (Classic RIA). The disadvantage of this technique setting is the need

special separation of free and associated labeled antigens (or antibodies).

2) Technique "solid phase".

AG or AT known specificity binds to sorbents (solid phase) - walls of a polystyrene well or plastic tube. For immobilized ag (at), the rest of the IR components are sorbed by the rest of the IR.

Depending on the nature of the reaction, the following methods distinguish:

1) Competitive method - a method based on competition AG.

Reaction components:

a) the determined AG (the material under study - blood, sputum, etc.);

b) identical to the studied AG-in antigen, labeled by radioisotope;

c) Specific AT-LA known concentrations associated with sorbent;

d) standard ag (control);

e) buffer solution.

First, the reaction is introduced by the studied AG. The formation of the AG-AT complex on the sorbent surface occurs. The sorbent is washed, the labeled HA is administered, the greater the content of the AG under study, the less labeled AG binds to the atm on the surface of the sorbent. The concentration of labeled HA is determined by measuring the radioactivity of the reaction using counters. The magnitude of the radioactivity of the reaction will be inversely proportional to the number of AG in the test sample.

2) Non-competitive method.

Reaction components:

a) defined ag;

b) specific at-and known concentrations associated

on sorbent;

c) identical to the knitted antibody antibody labeled

radioisotope;

d) standard ag;

e) buffer solution.

A hypertension is added to the associated AT. In the process of incubation on the sorbent, AG-AT complexes are formed. The sorbent is washed from free components and labeled ATs, which bind to free valences of AG-on as part of the complex. The magnitude of radioactivity is proportional to the concentration of the AG studied.

3) "Sandwich method" (indirect method) is the most common method.

Components:

a) the test serum (or the AG studied);

b) AG-s, associated with sorbent (or at-la, associated with sorbent when determining AG-A);

c) diagnostic AT against immunoglobulins labeled with radioisotrops;

d) control serums (or ag-s);

e) buffer solutions.

The at-a (or ag-s) studies are reacting with solid-phase AG-MI (AT-MI), after which the incubate is removed and labeled anti-globulin ATs are introduced into the reaction, which are associated with the specific complexes of AG-AT on the surface of the sorbent. The magnitude of the radioactivity of the reaction is directly proportional to the number of studied AT (or AG).

The advantages of RIA:

1) high specificity and sensitivity;

2) simplicity of the technique of production;

3) accuracy of quantitative evaluation of results;

4) Easily succumb to automation.

Disadvantage: The use of radioactive isotopes.

No similar materials (

Immunoenimen method (ELISA)

The method is used to detect antigens using the appropriate antibodies conjugated with the enzyme-label (chronic peroxidase, B - galactose or alkaline phosphatase). After connecting the antigen with the enzyme-labeled immune serum, a substrate and chromogen are added to the mixture. The substrate is split by the enzyme, and its degradation products cause chromogen chemical modification. At the same time, chromogen changes its color - the intensity of the color is directly proportional to the number of connected antigen molecules and antibodies (Fig. 9).

The most common solid-phase IFA, in which one of the components of the immune reaction (antigen or antibody) is sorbed on a solid carrier. Polystyrene micropaneels are used as a solid carrier. In determining the antibodies to the wells with a sorbed antigen, the blood serum of patients, antihyglobulin serum, labeled by an enzyme and a mixture of substrate solutions for enzyme and chromogenic, is sequentially added. Each time after the addition of the next component from the holes remove non-tied reagents by thorough washing. With a positive result, the color of the chromogen solution changes. The solid-phase carrier can be sensitized not only by an antigen, but also an antibody. Then the desired antigen is introduced into the wells with sorbed antibodies, the immune serum is added against the antigen, labeled by the enzyme, and the mixture of substrate solutions for the enzyme and chromogen.

ELISA is used to diagnose diseases caused by viral and bacterial pathogens. In the quality of the label, enzymes are used: peroxidase, alkaline phosphatase, etc.

The reaction indicator is the ability of enzymes to cause color reactions under action to the corresponding substrate. For example, the substrate for peroxidase is an orthophenyldiamine solution.

The most widely applied solid-phase ELISA. The essence of IFA is similar to Ria.

The results of the IFA can be assessed visually and measuring the optical density on the spectrophotometer.

The benefits of the ELISA include:

Lack of contact with radioactive substances;

Simplicity of reaction assessment methods;

Stability of conjugates;

Easily succumb to automation.

However, compared to RIA, the lower sensitivity of the method is noted, but in some cases the sensitivity exceeds Reef and Rome.

The following types of IFA are given as examples:

Competitive type.

Purpose.

Designed to detect the surface antigen of the hepatitis B virus (HP) in serum and blood plasma in the diagnosis of viral hepatitis B and determining the NV5 Hepatic carrier.

Components:

1) studied material serum or blood plasma;

2) antibodies to nvz blood pressure adsorbed on the surface of the polystyrene microplating well;

3) conjugate - mouse monoclonional antibodies to NVZ Hell labeled with peroxidase,

4) Ortophenylenediamine (OFD) -Substrat;

5) phosphate-salt buffer;

6) Control serums:

Positive (serum with HVE hell);

Negative (serum without NVZ AD). Progress

1) The introduction of control and test serums.

2) incubation 1 hour at 37 "C.

3) Loading holes.

4) Making a conjugate.

5) incubation 1 hour at 37 "S.

6) Laundering holes.

7) Making an OFD. In the presence of the NVZ, the heap is a solution and the wells shine.

IFA accounting is carried out by optical density with a photometer. The degree of optical density will be inversely proportional to the concentration of the pressure under investigated NVZ.

Mechanism

The reaction proceeds into three phases:

1) NVZ Hell under study serum (plasma) is associated with homologous AT adsorbed on the surface of the well. IK AG-AT is formed. (NVZ AGL \\ NVZ AT).

2) NVZ HD antibodies labeled with peroxidase, are associated with the remaining free determinants of NVZ AG-AT complex. AT-AG-labeled AT (AP! 1 NVZ AT-NVZ AD-AP (1 NVZ AT, labeled peroxidase).

3) OFD interact (with peroxidase) of the AT-AG-AT complex and yellow painting occurs.

Indirect type

It is the main test reaction to the diagnosis of HIV infection.

Objective: Serological diagnosis of HIV infection - detection of antibodies to HIV antigens, components:

1) the material under study - blood serum;

2) synthetic

With this method, the phenomenon of luminescence is used.

The essence of the luminescence phenomenon is that when absorbing various types of energy (light, electric, etc.), the molecules of some substances are transmitted to the excited state, and then returning to the initial state, the absorbed energy in the form of light radiation is distinguished.

The reef luminescence is manifested in the form of fluorescence - this is a glow that occurs at the time of irradiation with the exciting light and stopped immediately after its end.

Many substances and live microorganisms have their own fluorescence (the so-called primary), but its intensity is very small. Substances with intense primary fluorescence and used to impart fluorescent properties with petroleumusing substances, obtained the name of fluorochromas. Such an induced fluorescence is called secondary.

For the excitation of fluorescence with luminescent microscopy, the ultraviolet or blue-purple part of the spectrum is most often used (wavelength is 300-460 nm). For these purposes, laboratories have luminescent microscopes of various models - ML-1-Ml-4, Lumam.

In virological practice, two basic methods of luminescent microscopy are used: fluorochromic and fluorescent antibodies (or reef).

Fluorochromization - This processing of drugs with fluorochromes in order to increase the strength and contrast of their glow. The greatest interest is an acredigne orange fluorochrome, which causes polychromatic fluorescence of nucleic acids. Thus, when processing drugs with these fluorochromes, deoxyribonucleic acid is bright fluorescents with green, and ribonucleic - ruby-red.

The reef method is that antibodies connected to fluorochroma retain the ability to enter into a specific bond with a homologous antigen. The forms of the antigen + antibody complex due to the presence of fluorochrome in it is found under a luminous microscope according to a characteristic luminescence.

To obtain antibodies, high-active hyperimmune serums are used, from which antibodies are isolated and their fluorochromes. Fitz fluorescene isotocyonda (green glow) and RSX-rhodamine sulfochloride (red glow) are most often used as fluorochrome. Antibodies labeled with fluorochroma are called conjugate.

The method of preparation and painting of drugs is as follows:

• prepare on the subject glasses of strokes, prints from organs or on coating glasses - infected cell culture; You can use histopreach;
• preparations are dried in air and fixed in a cooled acetone at room temperature or at minus 15 ° C (from 15 minutes to 4-16 hours);
• painted on a direct or indirect method; Borrowed under a luminescent microscope on the intensity of the glow, estimated in the crosses.

In parallel, prepare and color drugs from a healthy animal - control.

There are two basic methods of use of fluorescent antibodies: straight and indirect.

Direct method (single-stage). A fixed drug is applied conjugate (fluorescent serum to an alleged virus), withstand 30 minutes at a temperature of 37 ° C in a wet chamber. The preparation is then washed from an unbound conjugate with a physiological solution (pH 7.2 - 7.5), dried in air, they are applied to an oil-based oil and is examined under the microscope.

The direct method allows you to detect and identify the antigen. For this you need to have on each virus fluorescent serum.

Indirect method (two-stage). An unknown serum containing antibodies to an intended virus is applied to the fixed drug at 37 ° C, unbound antibodies are laundered. Fluorescent anti-love serum is applied to the drug, corresponding to the type of animal - the producer of homologous antiviral antibodies, is kept for 30 minutes at 37 ° C. Then, the drug is washed from unrelated labeled antibodies, dried in air, they are applied to the oil oil and is examined under a luminescent microscope.

Anti-love serums are obtained by immunizing globulins of animals of those species that serve as produced antivirus antibodies. So, if antiviral antibodies were obtained on rabbits, then used fluorescent serum anticrolly.

The indirect method allows not only to detect and identify the antigen, but also to identify and determine the titer of antibodies. In addition, this method can be detected by one labeled serum antigen of various viruses, as it is based on the use of anti-love serums. More often used anti-cycling, antibysy, antisold serums and serums against governors of the guinea pig.

Several modifications of the indirect method have been developed. The most attention is the method using complement. The method is that an inactivated petrolevascular specific serum is applied to a fixed drug and complement of the guinea pigs, withstand 30 minutes at 37 ° C, washed, and to detect the antigen + anti-antibody complex + complement, the fluorescent anti-frequency serum is applied to 30 minutes at 37 ° C , washed, dried in air and are investigated under a luminescent microscope.

Advantages reef: high specificity and sensitivity; simplicity of performance; The minimum number of components is required. This is an express diagnostic method, since within a few hours you can get an answer. The disadvantages include subjectivism in assessing the intensity of the glow and, unfortunately, sometimes fluorescent serums are poor quality. Currently, the reef is widely used in the diagnosis of viral animal diseases.

If you have found a mistake, please select the text fragment and click Ctrl + Enter..

Immunofluorescent method (reef, immunofluorescence reaction, kuns reaction) - method of detecting specific ags using AT conjugated with fluorochroma. It has high sensitivity and specificity.

It is used for rapid diagnosis of infectious diseases (identification of the pathogen in the material under study), as well as to determine the AT and surface receptors and leukocyte markers (immunophenotyping) and other cells.

Detection of bacterial and viral antigens in infectious materials, tissues of animal and cultures of cells with the help of fluorescent antibodies (serums) gained widespread use in diagnostic practice. The preparation of fluorescent serums is based on the ability of some fluorochromes (for example, an isothiocyanate of fluorescein) to join chemical bond with serum proteins without violating their immunological specificity.

There are three varieties of the method: straight, indirect, with complement. The direct reef method is based on the fact that the antigens of tissues or microbes treated with immune serums with antibodies marked with fluorochromas are capable of glowing in the UV ray of the fluorescent microscope. Bacteria in a smear treated with such luminescent serum are glowing along the periphery of the cells in the form of a border of green.

The indirect reef method is to identify the antigen complex - antibody using anti-globulin (anti-antibodies) with a whey labeled with fluorochroma. For this, the strokes from the suspension of microbes are treated with antimicrobial rabbit diagnostic serum antibodies. Then antibodies that are not contrary to microbial antigens are washed, and the antibodies remaining on the microbes reveal, treating the smear of anti-globulin (anti-cycling) serum with fluorochromas. As a result, the microbe complex is formed + antimicrobial rabbit antibodies + anti-cycling antibodies labeled with fluorochroma. This complex is observed in a luminescent microscope, as well as with a direct method.

Mechanism. A stroke from the test material is prepared on the subject glass, fixed on the flame and is treated with immune rabbit serum containing antibodies against causative agent antigens. To form an antigen complex - antibody, the drug is placed in a wet chamber and incubated at 37 ° C for 15 minutes, after which it is thoroughly washed with an isotonic solution of sodium chloride to remove the antibodies that have not connected to the antigen. Then, the drug is applied with a fluorescent anti-globulin serum against rabbit globulins, kept for 15 minutes at 37 ° C, and then the drug is thoroughly washed with a sodium chloride isotonic solution. As a result of binding fluorescent anti-globulin serum with antigen-fixed antigen, antigen - antibody, which are found in fluorescent microscopy are formed.

4. In the air of the children's bedroom, 75 Mt / m3 of Streptococcus, 12 Mt / m3 of staphylococcal and 1 Mt / m3 tuberculosis bacteria was found. To give a sanitary and bacteriological assessment of the air and outline its sanitation plan.

Exam ticket number _54

Retroviruses. HIV infection (AIDS), and its pathogens.

The human immunodeficiency virus causes HIV infection by developing the development of the acquired immune deficit syndrome.

The causative agent of HIV infection is a lymphotropic virus belonging to the Retroviridae family by the genus Lentivirus.

Morphological properties: RNA-containing virus. The viral particle of the spherical shape of the shell consists of a double layer of lipids penetrated by glycoproteins. The lipid shell comes from the plasma membrane of the host cell, in which the virus is reproduced. The glycoprotein molecule consists of 2 subunits located on the surface of the virion and penetrating his lipid shell.

The core of the cone virus, consists of capsid proteins, a number of matrix proteins and protease proteins. The gene forms two RNA threads, for the implementation of the process of reproduction, HIV has a reverse transcriptase, or reverse.

The virus gene consists of 3 main structural genes and 7 regulatory and functional genes. Functional genes perform regulatory functions and ensure the implementation of reproduction processes and the participation of the virus in the infectious process.

The virus affects mainly T- and B-lymphocytes, some monocytic cells (macrophages, leukocytes), nervous cell cells.

Cultural properties: on the culture of cells of T-lymphocytes and human monocytes (in the presence of IL-2).

Antigenic structure: 2 types of virus - HIV-1 and HIV-2 HIV-1, has more than 10 genotypes (subtypes): A, B, C, D, E, F ..., differing among themselves on the amino acid composition of proteins.

HIV-1 is divided into 3 groups: M, N, O. Most of the isolates belong to the group M, in which 10 subtypes are distinguished: A, B, C, D, FL, F-2, G, N, I, K. Stability : It is sensitive to physical and chemical factors, dies in when heated. The virus can continue to be kept in the dried state, in dried blood.

Factors of pathogenicity, pathogenesis: The virus is attached to the lymphocyte, penetrates the cell and reproduces in the lymphocyte. As a result of the breeding of HIV in the lymphocyte, the latter are destroyed or lose their functional properties. As a result of the breeding of the virus in various cells, it is accumulated in organs and tissues, and it is found in the blood, lymph, saliva, urine, sweat, hiding masses.

In HIV infection, the number of T-4-lymphocytes is reduced, the function of the B-lymphocytes is disturbed, the function of natural killers is suppressed and the response to antigens decreases and the products of complement, lymphokinov and other factors regulating immune functions (Il) are reduced, resulting in the immune dysfunction Systems.

Clinic: The respiratory system is affected (pneumonia, bronchitis); CNS (abscesses, meningitis); The gastrointestinal tract (diarrhea), malignant neoplasms arise (internal organs tumors).

HIV infection takes place in several stages: 1) the incubation period, which is an average of 2-4 weeks; 2) the stage of primary manifestations, characterized in initially acute fever, diarrhea; The stage of the asymptomatic phase and the persistence of the virus, the restoration of well-being, is determined, but HIV antibodies, 3) are determined in the blood, the stage of secondary diseases that manifest itself by the respiratory lesion, the nervous system. HIV infection is worse, the 4th terminal stage of AIDS.

Microbiological diagnostics.

Virological and serological studies include methods for determining antigens and HIV antibodies. For this use ELISA, IB and PCR. The serum of HIV-1 patients and HIV-2 contain antibodies to all virus proteins. However, antibodies to proteins GP41, GPL20, GPL60, P24 in HIV-1 and antibodies are determined to confirm the diagnosis, GP36, GPL05, GPL40 in HIV-2 proteins. HIV antibodies appear in 2-4 weeks after infection and are determined at all HIV stages.

The method of identifying the virus in the blood, lymphocytes. However, with any positive sample, the IB reaction is set to confirm the results. PCR is also used capable of identifying HIV infection in the incubation and early clinical period, but its sensitivity is somewhat lower than the ELISA.

Clinical and serological diagnoses are confirmed by immunological studies, if they indicate the presence of immunodeficiency in the patient's surveyed.

The diagnostic immunoferment test system for determining the antibodies to HIV includes a viral aga, adsorbed on a carrier, ATI against IG. Used to serodiagnostics AIDS.

Treatment: The use of reverse transcriptase inhibitors operating in activated cells. Preparations are tremidine derivatives - azidothimidine and phosphazide.

Prevention. Specific - no.

The influence of physical and chemical factors on the microbes. Mutation and its value for practical medicine. Examples. Ecology value.

The effect of chemical and biological factors.

Action of chemicals

Chemicals can slow down or completely suppress the growth of microorganisms. If the chemical substance suppresses the growth of bacteria, but after removal, their growth is renewed again.

Antimicrobial substances, taking into account the chemical structure and the mechanism of their bactericidal action on bacteria, can be divided into the following groups: oxidizing agents, halogens, compounds of metals, acids, and alkali, surfactants, alcohols, dyes, phenol derivatives and formaldehyde.

Oxidifiers. This group includes hydrogen peroxide and potassium permanganate.

Halogens. Chlorine, iodine and their drugs: chlorine lime, chlorine b, panticide, iodine alcohol solution 5%, iodinol, iodoform.

Compounds of heavy metals (salt of lead, copper, zinc, silver, mercury; silver metal connections: Potargol, collargol). These compounds are able to provide both antimicrobial and dirty local action on macro-organism fabric.

Acids and alkalis. The bactericidal action of acids and alkalis are the dehydration of microorganisms, a change in the pH of the nutrient medium, the hydrolysis of colloidal systems and the formation of acidic or alkaline albums.

Dyes have properties to delay the growth of bacteria. They act slowly, but more selectively.

Formaldehyde-colorless gas. In practice, a 40% aqueous solution of formaldehyde (formalin) is used. Gaseous and dissolved in water formaldehyde destructively affects the vegetative and dispute shapes of bacteria.

The effect of biological factors

The effect of biological factors is primarily in the antagonism of the microbes, when the products of the vital activity of some microbes determine the death of others.

Antibiotics (from Greek. Anti - Anti-BIOS - Life) - Biologically active substances formed during the life of mushrooms, bacteria, animals, plants and created by synthetic, capable of selectively suppress and kill microorganisms, mushrooms, rickettsia, large viruses, simplest and Separate helminths.

3. The reaction of the biological activity of bacterial enzymes during rheumatism, diagnostic and practical importance, the protective role of antibodies against enzymes in the acquired immunite (definition of antihialuromanonidase and anti-streptolisin).

Rheumatism is the total disease of an infectious-allergic nature, in which the connecting tissue is affected, mainly the cardiovascular system, as well as joints, internal organs, central nervous system. It is believed that the cause of the development of rheumatism is the activation of pathogenic microorganisms, mainly beta-hemolytic streptococcus Group A. It is his main role in the etiology and pathogenesis of rheumatic disease. First, the disease develops against the background of streptococcal infection. Secondly, in the blood of patients there is a large number of antibodies to the microorganisms of this group. Thirdly, the prevention of the disease is successfully carried out by antibacterial drugs.

With rheumatoid arthritis, synovial membranes, by unclear reasons, secrete a large amount of enzyme glucose-6-phosphate dehydrogenase which also destroys disulfide bonds in the cell membrane. In this case, there is a "leakage" of proteolytic enzymes from cell lysosomes, which cause damage to nearby bones and cartilage. The body responds to this by producing cytokines, among which there is also a tumor necrosis factor α TNF-α. Cascades of reactions in cells that are launched by cytokines, further aggravate the symptoms of the disease. Chronic rheumatoid inflammation associated with TNF-α, very often causes damage to the cartilage and joints leading to physical disability.

61. Immunofluorescence reaction. Mechanism, components, application. Direct and indirect methods of production.

There are three main varieties of the method: straight, indirect (Fig. 13.10), with complement. The Kuns reaction is an express diagnostic method for detecting microbes or antibody definitions.

Direct Reef method It is based on the fact that the antigens of tissues or microbes treated with immune serums with antibodies labeled fluorochromas are capable of glowing in the UV-ray of the fluorescent microscope. Packteries in a smear treated with such lumine serum are glowing along the periphery of the cells in the form of a green comb.

Indirect Reef method It is to identify the antigen antigen complex with the help of anti-globulin (against the antibody) of the serum labeled with fluorochroma. For this, strokes from the suspension of microbes are treated with antimicrobial rabbit rabbit antibodies of diagnostic serum. Then antibodies that are not contrary to microbial antigens are washed, and the antibodies remaining on the microbes reveal, treating the smear of anti-globulin (anti-cycling) serum with fluorochromas. As a result, the microbe complex is formed + antimicrobial rabbit antibodies + anti-cycling antibodies labeled with fluorochroma. This complex is observed in a luminescent microscope, as well as with a direct method.

As a label, glowing fluorochroma dyes (isothiocyonate fluoriscein, etc.) are used.

There are various modifications of reef. For rapid diagnosis of infectious diseases - to detect microbes or their antigens in the material under study, reef on Kunsu is used.

There are two methods of Reef in Kunsu: straight and indirect.

Components direct reef:
1) The material under study (the flowman separated by the nasopharynk and others);
2) labeled specific immune serum containing at-la to the desired antigen;
3) isotonic sodium chloride solution.
The smear from the material under study is treated with labeled antiserum.
AG-AT reaction occurs. With a luminescent microscopic examination in the site where the AG-AT complexes are localized, fluorescence is detected - the glow.

Components indirect reef:
1) the material under study;
2) specific anti-graders;
3) anti-globulin serum (at-la against immunoglobulin), laughing with fluorichrome;
4) sodium chloride isotonic solution.

The stroke of the material under study is first treated with immune serum to the desired antigen, and then - labeled antihyglobulin serum.

Glowing complexes AG-AT - labeled AT are detected using a luminescent microscope.
The advantage of the indirect method is that there is no need to prepare a wide set of fluorescent specific sera, and only one fluorescent anti-globulin serum is used.

Also isolated a 4-component variety of indirect reef when complement (serum guinea pig) is additionally introduced. With a positive reaction, the AG-AT complex is formed - labeled - AT-Complement.

Table of contents:

Direct methods

Microscopy in a dark field

Pale treponami cannot grow on nutrient media and are not visualized under a light microscope. Since the pathogen detection is impossible using conventional microscopy, a special microscope with a dark field is used, where the causative agent is visible in the form of a spiral on a dark background.

For microscopy, biomaterial is closed with suspicious focus. Microscopy in the dark field is a possible way to evaluate skin lesions, such as the Shankre of primary syphilis or the secondary syphilis. If the material of the maculopapulous focus is dry, investigate aspirate of the lymphatic node.

The negative result does not exclude the pathological process, statistically detection of the pathogen is possible only in 80%.

PCR diagnostics

The reaction aimed at the repeated increase in the DNA of the Pale Treponém allows us to conclude about the infection with syphilis or its absence.

The biomaterial for analysis can be any: blood, the contents of syphilide, the spinal fluid, etc. The test is suitable for the incubation period.

PCR is fully specific.

Indirect serological analyzes on syphilis: treponem and non-deplete tests

Serological analyzes (CER or Serological Reaction Complex) are considered to be the most common way to diagnose all stages of syphilis. Allocate the following reactions:

• agglutination;
• precipitations;
• immunofluorescence;
• immuno enzyme analysis, etc.

Surgeological analyzes on syphilis are subdivided into treponem and unplaced.

Frameless

In suspected of the acquired syphilis, screening testing is performed, for which they use frameless tests defining antibodies to lipoid tissue antigens or pathogen in a variety of modifications. In the Russian Federation, the reaction of microprecipitation (RMP), which allows you to identify antibodies to damaged by pathogen cells in the blood. The authenticity of screening is high, but the specificity is small, so testing is suitable for primary mass examination in preventive purposes.

The sensitivity of express tests is estimated at 78-86% to detect primary syphilis, 100% for secondary syphilis and 95-98% for the detection of tertiary syphilis.

Specificity - from 85-99%, sometimes less, which is found in the following states:

• gestration;
• menstruation;
• oncology;
• connective tissue diseases;
• viral diseases;
• liver disease;
• vaccination;
• "Fresh" them;
• repatic title, etc.

In addition, excess fat in the diet, the use of alcohol-containing beverages and the reception of some drugs can lead to false positive conclusions.

Screening test results become positive after 1-2 weeks after the formation of Shankra. Frameless tests give a negative response after some time after treatment. In the HIV status, non-renewable antibodies can be detected for a long time, sometimes throughout life (which is confirmed by the results of the corresponding randomized study).

Other varieties of unnecessary tests: VDRL, plasma-magic test (RPR), sample with toloidin red, complement binding response with cardiolipin antigen (RSCK).

Vasserman reaction (RW)

Combination of complement - the response of the immune system for infection, the result varies from the negative (set "-"), to a sharply positive "++++" or 4 plus.

In the initial stage of primary syphilis RW is negative.

Tremponen

Due to the possibility of false positive results, to confirm any positive or dubious result of non-renewable analysis, use trempone tests:

• immunofluorescence reaction (reef);
• hemagglutination (RPGA),
• immunoassimensional analysis (ELISA) for immunoglobulins of class G (IgG) and immunoglobulin M (Igm);
• immunoblotting;
• Ribt / Rit (reaction of the immobilization of pale treponam).

Trempone tests are not used to assess the effectiveness of therapy.

Reef on the definition of treponine at class IgG is used after a positive result of express tests (84% sensitivity for primary syphilis and 100% at other stages, specificity 96%). For diagnosis, newborns are not applicable.

Some laboratories use "reverse" screening research.

CDC (CENTERS FOR DISEASE CONTROL AND PREVENTION, USA) recommends traditional studies, with verification of quantitative non-renewable tests, treatment is carried out at a positive result.

Immunofluorescence reaction (reef)

A serum is applied to the assembled material with labeled fluorochroma antibodies specific to the Pale Treponam Antigen, the pathogen attracts immune complexes to itself, which is why it begins to glow in a luminescent microscope.

Passive hemoagglutination or RPGA reaction

Prior to the appearance of hemagglutination (gluing), erythrocytes from the moment of the introduction of pale treponema should pass at least 4 weeks.

Prepared erythrocytes with fixed protein pathogen fractions interact with plasma, if there are antibodies to syphilis, the reaction occurs.

Suitable for confirming any stage of the disease.

Linked immunosorbent assay

The reaction is based on the antigen antibody. Antibodies of different classes are detected, which can be quantified.

The results obtained allow us to judge the duration of the pathological process, the success of the treatment, immunological status, the activity of pathogens.

Immunoblotting is a variety of ELISA, used for in-depth diagnostics with all dubious results.

Sensitivity and specificity is close to 100%, today the ultra-sensitive method of identifying proteins.

Ribt.

The method is based on the antigen antibody reaction. Antigen serve pale treponams cultivated in rabbit tests. When interacting with antibodies of an infected person, pathogens lose mobility. The reaction is estimated by microscopy in a dark field.

note

Ribt is currently applied due to labor intensiveness less often, but the analysis may be useful for solving controversial issues (false-positive reactions to syphilis).

Differential diagnosis

The greatest complexity is the diagnosis of tertiary syphilis, which is caused by symptoms from the cardiovascular and nervous system, as well as manifestations of the skin.

Patients must be examined on, and.

We list the disease with which a differential diagnosis in syphilis are carried out:

• dermatological manifestations;
• genital warts ();
• donovanosis;
• venusic lymphogranul;
• virus;
• yaws.

Where does the diagnosis of syphilis begins

It is initially a conversation with a patient, during which the details are specified: when there was a suspicious sexual contact and what complaints are.

After collecting anamnesis, they move to physicial inspection, special attention is paid to the region of genitals and anus, mucous membranes and lymph nodes. A preliminary diagnosis can already be installed. Final verification occurs with laboratory tests.

If you just say about the complex, then some analyzes detect the causative agent of syphilis, while others reflect the body's reaction to the introduction of pale treponama.

To install the final diagnosis of RPGs, 1 should be complemented by 1 treponem and 1 non-empty analysis.

Diagnosis of syphilis in pregnant women

A mandatory study on syphilis is carried out several times during pregnancy.

The direction for the analysis of the CCR is issued during the first visit of the woman in the consultation, and during pregnancy three times the survey is performed. Patients from a high risk group with an imaginary history:, asocial, dependent, etc. require particular attention.

If the analysis results are positive, carry out deeper diagnostics, and according to the testimony, treatment is prescribed, which depends on the stage and clinical manifestations.

Diagnosis of congenital syphilisa

Most children are born in untreated mothers, or gaining therapy too late.

Tremponium tests using neonatal serum are not recommended due to the passive transfer of IgG antibodies. All children born from mothers with syphilis should be examined using a quantitative unrepressive serological test (RPR or VDRL), performed using a newborn serum.

How to interpret the results of serological studies

The reaction of microprecipitation, reef and RPGA are negative - the norm is positive - confirmation of syphilis.

The reaction of microprecipitation is negative, the remaining positive - syphilis in history after conducting specific therapy, or late stage.

Negative reef with positive RPGA and microprocypitation reaction - the result causes doubts, repeated integrated assessment.

A negative result of reef and micropreitation, but positive RPGA is a state after successful antibiotic therapy or a false positive result.

Positive reef with negative RPGAs and microprocipatition reactions - early stage, treatment or unreliability.

The positive reaction of microprocipatition, not confirmed by nor RPGA, nor Reef - the absence of syphilis.

Syphilis Tool Study

Instrumental diagnostics is carried out depending on the involvement of the organs. For example, granulomatous liver damage can be seen at the abdominal cavity.

Patients with tertiary syphilis can show the dilatation of aorta. Linear calcification along the aorta testifies in favor of syphilitic aortics.