REEF

The immunofluorescence reaction is based on the interaction of antihahlamicidium AT with a trop-specific chlamydial antigen. There are two types of immunofluorescence reaction - direct and indirect. In the first case, the specific antibody is directly labeled with fluorochroma and the reaction passes into one stage, which significantly reduces the period of research. In the second case, the specific antibody does not have a tag, and to identify the AG-AT complex formed at the first stage, the second labeled antibodies, specific to antihamdial antibodies, use. The result of the reaction is estimated visually using a luminescent microscope.

In addition to imported domestic diagnostic kits appeared chlamydial infection The reaction of direct immunofluorescence, which is practically not inferior in specificity and sensitivity, but have a significantly lower price.

Material for the study serves strokes-prints prepared in compliance with the rules. Rift smears are prepared on special decolated glasses within a limited area with a diameter of 8 - 10 mm. After that, they are dried in air and fixed, immersing for 5 - 10 minutes in a cold 96% (better absolute) ethanol or influencing 0.1 - 0.15 ml of chilled anhydrous acetone to its full evaporation. Fixed drugs can be explored immediately or, if necessary, to store at room temperature during the day or at -20 ° C for 2 weeks (it is necessary to eliminate moisture access when storing and bringing the drug to room temperature before studying with a polyethylene package and Selikahel).

When spending a straight reef on the drug, a solution of labeled antibodies is caused in the amount required for the total coating of the smear (25 - 30 μl). The drug is incubated in a horizontal position in a wet chamber for 15 minutes at + 37 ° C. The drying of the reagent is unacceptable to avoid false positive results. Next, the smear was washed in running water 30 seconds are ringed in distilled water, dried and prepared for microscopy.

When conducting an indirect reef on the drug, the required amount of solution of antihamdial antibodies is applied and carried out the first incubation under the same conditions as during a direct reef. The preparation is then washed, dried in air and the second reagent is applied - labeled antibodies to chlamydial AT and carry out the second incubation in the wet chamber at + 37 ° C 15 minutes. After washing the drug is dried and prepared for microscopy. Ready drugs are recommended to view immediately. If necessary, it is possible to store painted drugs for 1 to 2 days at + 2-8 ° C in a dark place without moisture.

Microscopy is carried out using an immersion system. Two options for immersion microscopy are possible:

1. Oil Immersion: 20-5 μl of mounted liquid (glycerin, buffered by phosphate buffer with pH 7.2 - 7.6) are applied to the ready-made dried preparation (glycerin), covered with a degreased coating glass, which is then applied with a drop of hydroelectric immersion oil and microscopy lens (marking "L "- Luminescent) with an increase in 90 and an eyepiece with increasing 5.

2. Water immersion: A drop of 20 μl of phosphate buffer with pH 7.4 is applied to a ready-made drug and microscopy lens for an aqueous immersion (marking - white stripe and "l" - luminescent) with an increase of 60 and an eyepiece with increasing 5. Water immersion gives a more even, Bright and clear glow.

Reef allows you to identify antigenic structures of both elementary and reticular taurus. This is preferably extracellular, rounded shape with smooth edges, small (size 1: 100 - 1: 150 in relation to the surrounding cells), a homogeneous structure, have a bright specific emerald green glow, which when microventine can give diffraction rings (rings Diaketonal). The RT is much less common, they are located preferably intracellularly, have less homogeneous structure, more polymorphic, but also with clear edges and have a bright specific emerald green glow. Any other fluorescent material of the irregular shape, uneven fuzzy edges, having a non-rigid green color, or the glow of another color refers to artifacts. The intensity, brightness and shade of a specific glow depends on the pH of the mounted liquid or the buffer used for microscopy. The large intensity of the candle in the alkaline medium increases the sensitivity of the method, but leads to an increase in the number of objects with non-specific luminescence, and, consequently, to false positive results. In an acidic medium, the intensity of the candlestation Fitz falls, which can give a false-negative result. The concomitant microflora, as well as the nucleus of surrounding cells, is nonspecifically stained in various shades of orange-colored with indium bromide, their cytoplasm - in various shades of brick-brown-colored with blue Evans.

To obtain a reliable result, it is recommended to view many fields of view of the drug. The result is considered positive if the drug contains epithelium cells and it is possible to detect at least 6 elementary tales having all the above signs.

The detection of a smaller pathoger makes the result doubtful and requires a re-study, preferably on the background of provocations (food - alcohol, drug-medicated - Pyrogenal injection, mechanical - urethra massage on the buzz). Covering control should be carried out no earlier than two weeks, since it is possible to preserve the non-eliminated antigenic material of a non-viable pathogen, which will give false positive results. Obtaining 3 negative results of the study in men within a month and in women for 3 menstrual cycles, absence clinical manifestations Chlamydial infection testifies to recovery.

Reef In proper preparing the patient, compliance with the rules for thering of material and the reaction formulation is a highly sensitive and specific method for the diagnosis of urogenital chlamydia and allows you to identify the pathogen in 90 - 95% of patients. This method is relatively cheap, simple to perform, highly informative, does not require special expensive equipment, it allows you to quickly get the result (0.5 - 1 hour) and visually control the quality of the taking material for the study.

Methods using the principles of molecular biology

The group includes the methods of DNA probes and a polymerase chain reaction (PCR), which allow you to identify the genetic material of the pathogen in the studied biomaterial. Sets for diagnosing chlamydia DNA probes are still under development and clinical trials.

PCR is actively introduced into the practice of laboratory diagnostics. The method is based on the selection of a specific sequence of DNA or RNA of the causative agent using complementary primers, subsequent multiple copying and accumulation for further detection of conventional detection methods (electrophoresis or IFA). This method has high specificity and sensitivity, practically approaching culture and makes it possible to detect single pathogens in the material under study. The PCR method requires special expensive equipment, a separate specially equipped laboratory, appropriate training and high qualifications. medical personnel. At the same time, the lack of certification of primers used in Russia and sufficient experience in the application of the PCR method, the specificity of the material under study, frequent contamination of the accompanying microflora (which can produce false positive results) does not allow to unambiguously judge its value in the diagnosis of urogenital chlamydia.

Thus, currently the most affordable, simple and at the same time highly informative method for the diagnosis of urogenital chlamydia and the establishment of curability is the reaction of direct immunofluorescence (reef). Control over the dynamics of the course of the disease and the effectiveness of treatment should be carried out by determining the titer of antihammodium antibodies in blood serum by the method of enzyme immunoassay analysis (IFA).

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

Used for rapid diagnostics 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 antihyglobulin serum with antigen-fixed antigen, glowing antigen complexes are formed, which are found in luminous microscopy.

22. Immuno enzyme analysis - Laboratory immunological method of high-quality or quantitative determination of various compounds, macromolecules, viruses, etc., based on a specific antigen antibody reaction. The identification of the resulting complex is carried out using the enzyme as a label to register a signal.

Classification:

Competitive (in the system at the same time there is an analyzed compound and its analogue)

Non-competitive (if only the analyzed compound is present in the system and the corresponding binding centers (antigen and specific antibodies))

Direct and indirect

1. Available containing a mixture of AT is incubated with AG fixed on a solid substrate.

2.At that does not bind AG are removed by multiple washing.

3. Enter by the enzyme-labeled antiserism to AT, binding

4. Determined the number of enzyme marker connected with AT

Indirect:

At-positive serum

1.Specific AT in the referee serum bind to hypertension, fixed on a solid substrate

2. Specific AT, labeled by the enzyme, do not interact with the associated ag-content of the marker in the substrate low

At-negative serum

1. Nonspecific AT in the test serum do not associate AG, fixed on a solid substrate

2. Specific AT, labeled by the enzyme, interact, with a fixed AG-Southern Marker High

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. As a solid medium, polystyrene micropanes are used. When determining the antibodies in the wells with a sorbed antigen, a serum labeled by an enzyme is sequentially added, and a mixture of solutions for enzyme and chromogen. 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 artized antigen is introduced into the wells with sorbed antibodies, an immune serum is added against an antigen, labeled by an enzyme, and then mixture of substrate solutions for enzyme and chromogen.

Application:to diagnose diseases caused by viral and bacterial causative agents.

23. Serological reaction - The reaction by which the antigen reaction is investigated (microbial, virus, alien protein) with serum antibodies.

Serological studies - These are the methods of studying certain antibodies or antigens in the blood serum patients based on immunity reactions. With their help, antigens of microbes or tissues are revealed to identify them.

The detection in serum of the patient of the antibody to the causative agent of infection or the corresponding antigen allows you to establish the cause of the disease.

Serological studies are also used to determine antigens of blood groups, tissue antigens and humoral levels of immunity.

Serological studies include various serological reactions:

1. The reaction of agglutination.

2. Precipient reaction.

3. Neutralization reaction.

4. Reaction involving complement.

5. Reaction using labeled antibodies or antigens.

Immunofluorescence reaction (reef) - serological reactionallowing to identify antibodies to known antigens. The method lies in microscopy of painted smears.

This reaction is used in immunology, virology and microbiology. It allows you to determine the presence of viruses, bacteria, mushrooms, simplest and ICC. Reef is very widely used in diagnostic practice to detect viral and bacterial antigens in infectious material. The method is based on the ability of fluorochrome to communicate with proteins without disturbing their immunological specificity. It is mainly used in the diagnosis of urinary tract infections.

The following methods of the immunofluorescence reaction are distinguished: direct, indirect, with complement. The direct method consists in staining the material with fluorochromas. Thanks to the ability of microbes or tissue antigens or tissue in the UV-ray of a luminescent microscope, they are defined as cells with brightly colored border color.

The indirect method is to determine the antigen + antibody complex. For this, the experimental material is treated with antimicrobial rabbit whey antibodies intended for diagnosis. After antibodies contact with microbes, they are separated from non-contacted and treated with anti-cancer serum marked with fluorochroma. After that, the microb complex + animicrobial antibodies + anitic and the antibodies is determined using an ultraviolet microscope as well as with the direct method.

Immunofluorescence reaction is indispensable in the diagnosis of syphilis. Under the influence of fluorochrome, the syphilis causative agent is defined as a cell with a yellow-green border. The absence of glow means that the patient is not infected with syphilis. This analysis is often prescribed with a positive reaction of Wasserman. This method is very effective in diagnosing, as it allows to determine the pathogen on early stages Diseases.

In addition, the reef allows you to diagnose syphilis, it is also used to determine the presence of such pathogens as chlamydia, mycoplasma, trichomonas, as well as causative agents of gonorrhea and genital herpes.

For analysis, strokes or venous blood are used. The macale taking procedure is completely painless and no danger. To the delivery of this analysis must be prepared. Twelve hours before him, it is not recommended to use hygiene tools, such as little or gels. Sometimes, according to the testimony of the doctor, a provocation is carried out. For this, it is recommended to receive acute food or alcohol or an injection of provoking substance is carried out, such as a goganovakcision or pyrogenal. In addition, the gap between the reception antibacterial drugs And the surcharge must be at least fourteen days.

When evaluating the results, it should be considered the fact that the glow is observed not only in living bacteria, but also the dead, this is especially true of chlamydia. After the course of antibiotics, dead chlamydia cells also possess a glow.

With proper preparation of the patient and compliance with the taking machine, this analysis allows you to identify diseases in the early stages, which is very important for timely treatment. Positive aspects This method is a short time to obtain the result, simplicity of execution and a small cost of analysis.

The disadvantages include the fact that for the analysis it is necessary to have a sufficiently large amount of material under study. In addition, only an experienced specialist can be evaluated.

For the first time, COOMBS was proposed in 1942. Reef is based on the detection of antigens in clinical material, blood cells and other drugs with monoclonal antibodies or sera labeled with fluorochroma (direct reef). The first (diagnostic) antibodies can be detected by anti-immoglobulin serum labeled with fluorochromas (indirect reef). There are modifications of reef to detect antibodies to serum infectious agents or serum antibodies.

The popularity of reef is explained by the cost-effectiveness. wide spectrum diagnostic sets, speed of receipt. Today, this reaction is used both polyclonal serums and monoclonal antibodies, labeled fluorescene isothiocyanate (FITC). To reduce the non-specific abandonment of the background, we use the processing of drugs by bovine serum albumin, labeled by rhodamine or blue evans.

Most often, the reef is used to quickly detect the pathogen in pathological material. In this case, strokes on the subject glass are prepared from the material under study, as for conventional microscopy. The drug is fixed with methyl alcohol, acetone or another chemical retainer. Laundered serum phytz or monoclonal antibodies are applied to the surface of a fixed smear (in the case of an indirect reef, the drug is first treated with serum against the desired antigen, and then labeled antibodies to immunoglobulins used in the first stage). Since the reef is a type of heterogeneous analysis, one stage is separated from another washing.

Accounting for the results of the reaction is carried out using a luminescent microscope, to the optical system of which a set of light filters is installed, providing lighting the drug with ultraviolet or blue-purple light with a given long wave. The researcher evaluates the nature of the glow, form, size of objects and their mutual location.

When setting the reef to detect antibodies, strokes from the reference strain of the pathogen are prepared. The test serum is applied to the smear. If it presents the desired antibodies, they bind to microbial cell antigens. Washing the drug with a buffer solution allows you to remove unrelated antibodies. The preparation is then treated with labeled serum against human immunoglobulins. In the case of a positive result of the reaction during microscopy, a specific glow of reference culture is observed in a luminescent microscope.

The main disadvantage of reef is its subjectivity.

Classical criteria for the specificity of this reaction are:

· Characteristic morphology, sizes and location of the pathogen in a smear;

· Peripheral nature of object luminescence;

· Fluorescence color;

· Fluorescence intensity.

In the study of large objects (trichomonas, human cells, cells affected by bacteria or viruses) these criteria allow you to get a reliable result. At the same time, the elementary tales of chlamydia and mycoplasma have dimensions lying on the limit of the resolution of the fluorescent microscope. In this case, the assessment of the morphology of microorganisms is difficult, and the glow loses peripheral character. The remaining criteria is clearly not enough for confident identification of the observed microorganism. In connection with the foregoing, the subjective nature of the reaction accounting makes special requirements for personnel qualifications conductive.

2.2. Fluorescent Immunoanalysis with temporary resolution (FIA BP, Etkins R. et Wallac O., 1984)

This type of FIA is based on the principles of sorption of one of the solid phase reagents and the use of Sandwich technology, i.e. Double recognition, like a typhoid. However, the important difference of the method is the use of lanthanide chelate as a label (rare-earth elements of Europe, Samaria, Terbium and Disrosion). The advantages of FIA BP are high sensitivity, formulation technology, such an ELISA, and the potential for significant gain of the beneficial signal due to a very high signal-to-noise ratio. A specific fluorescent label fluorescents is immeasurably stronger and longer than background fluorescence. In addition, the label has the ability to restore the ability to the luminescence (pulsed excitatory radiation with a period of 1C - more than 1000 pulses is used for accounting), which leads to accumulation (gain) of the beneficial signal. The described system is implemented by Perkinelmer, USA, called Delfia and has a sensitivity of more than 10-17 m in the definition of antigens.

2.3. Flow cytofluorimetry

Opened in 1942 by Kuns, the immunofluorescence reaction is not a new research method. However, the emergence of hybridoma technologies that allowed to obtain monoclonal antibodies, gave the "second life" of this reaction, since their use allowed several times to increase the sensitivity of this reaction and its specificity.

And today we will tell you in detail about the reaction of direct and indirect immunofluorescence (reef) as a method for diagnosing kuns for adult men and women during pregnancy.

What is the immunofluorescence reaction

Introducing an excellent opportunity to quickly obtain an accurate diagnosis, the immunofluorescence reaction makes it possible to determine the presence of a causative agent of the disease in pathological material. For this, a stroke of a material is used, which is specially processed using labeled fitz (fluorescene isothiocyanate), and is studied as heterogeneous analysis.

To obtain the result, a luminescent microscope is used, in its optical system there is a set of light filters to provide a preparation with a blue-purple or ultraviolet light having a predetermined wavelength. This condition allows the fluorochrum to be removed at a given range. The researcher is estimated by the properties of the glow, its character, the size of objects and their interpretation.

To whom it is prescribed

The immunofluorescence reaction can be assigned in the diagnosis of a set of viral diseases. In particular, it is prescribed when comprehensive examination To identify the following factors:

• the presence in the body of the virus;
• salmonella infection;
• existence in the body of certain antigens;
• the probability of infection with chlamydia, mycoplasmas and other microorganisms that have the ability to initiate human virus diseases are detected;
• diagnosis of viral diseases in animals.

These indications allow to use the immunofluorescence reaction when identifying viral diseases in human and animal organism.

Targets

Since this diagnostic method has many advantages to which its high performance, the speed and obtaining the result, as well as the absence of a large number of contraindications, is determined by its help viral infections. Therefore, it can be assigned to this analysis both for the formulation and to clarify the diagnosis, on the basis of which the treatment scheme is appointed.

The procedure does not cause unpleasant sensations, it is necessary to obtain material for analysis, which is taken from any body fluid: saliva, sputum, scraping from the surface of the mucous membranes. Also may have blood for analysis. The immunofluorus reaction frequency assigns a doctor who needs to obtain data on the dynamics of processes occurring in the body.

Since the harm for both the body and for general well-being of a person does not carry this analysis, it may be appointed as needed.

Types of such a procedure

Today, several varieties of this analysis are applied, each of which has a number specific features and allows you to get the most detailed picture of the processes occurring in the body.

The varieties of immunofluorescence reaction include:

1. - One of the most rapidly developing types of diagnostics, this analysis makes it possible to obtain quantitative data without the use of serial dilutions. Due to the use of obtained measurements of the optical density of the fluid, it turns out to accurately determine the level of concentration of the desired component. The wide possibilities of this type of analysis are used when used for its implementation of monoclonal antibodies, which allows to determine the phase of the infectious process, its sharpness;
2. DNA diagnostics - This method is based on complementary binding of nucleotides, for which such fluids can be used, both saliva, blood, liquor, watering, vocal, bioptats, blood. This method most effectively allows you to identify the presence of papilloma viruses in the body, but many modern test systems may occasionally give false-positive and false negative results. The reason for them can be contaminated fluid samples for analyzing specific DNA, the presence of which can have a nest or total nature;
3. immunochromotography - The specificity of this method of determining the presence in the body of the pathological environment and viruses is to be used during the reaction of labeled antibodies. This method of diagnostic is used to identify and degree of activity of the process of infection with streptococci groups A, as well as chlamydia of the following types: Clamikit R Innotech International, Clearview TM Chlamydia Oxoid. Possessing the highest possible sensitivity, test systems that are based on this research methodology. Usually applied as an indicative test.

These varieties have features and specific characteristics Results, but all of them are aimed at obtaining data on the presence of pathological microorganisms and viruses in the body, as well as about the degree of their reproduction and activity.

Indications for holding

The immunofluorescence reaction can be assigned to identify in the body of any kind of pathological environment.

Chlamydia, Trichomonas, Gonokokki and, as well as giardia of all kinds are determined during this type of diagnosis. And, and other diseases also require reef. The appointment of a doctor for its implementation is necessary.

Contraindications for holding

Since there is no type of body fluid as a studied material to carry out the material under study, it is usually not difficult to take difficulties and contraindications to implement the immunofluorescence reaction. However, during pregnancy and children under 6 months, the taking material for the study is carried out with maximum precautions.

The absence of contraindications allows you to carry out this type of diagnosis when appointing a doctor to all patients. Its safety is guaranteed by using a disinfect tool and disposable syringes.

Preparation for the procedure

There are no features to take a material for this analysis. Blood for him is taken on an empty stomach so that there is no increased content in it substances that can change true indications And give a false picture.

How is the assays

Since no special preparation for analysis is required, only meal is excluded 12 hours before it and the lack of application medicinal preparationsThe taking of the test material as the usual process of taking the body fluid on the analysis is carried out.

Subjective sensations during the procedure may vary depending on sensitivity.

Decoding results

The use of modern test systems allows you to get the most accurate analysis results. The following data applies to decrypt the result:

• the degree of intensity of fluorescence;
• shade of fluorescence;
• the peripheral nature of the object of the object of the object;
• the characteristics of morphology, the location of the causative agent in the smear of the material under study and its dimensions.

During the study of objects with large sizes (for example, Gorderell, Trichomonas, cells that are already amazed by viruses) listed above the criteria make it possible to obtain the most reliable results. However, the elementary bodies of mycoplasma and chlamydia have dimensions lying at the limit of resolution of the luminescent microscope, which

obtaining an accurate result, since the peripheral glow loses part of its intensity. The remaining criteria are not sufficient to accurately identify the studied microorganisms. For this reason, special requirements are presented to those skilled in the art. this species Research: The level of their qualifications should be sufficient for operating data.

For this reason, only a doctor with an appropriate level of qualification can be dealt with decoding the analysis. Read the price for the reef method below.

average cost

The price of the immunofluorescence reaction depends on the place of its holding and the level medical institution, and Also qualifications of a conductive analysis of a specialist. Today, the cost ranges from 1280 to 2,160 rubles.

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