Immunoblotting (immunoblot) is a highly specific and highly sensitive reference method confirming the diagnosis for patients with positive or uncertain results of analyzes obtained incl. Using RPGA or IFA .

This method of detecting antibodies to individual pathogen antigens is based on the formulation of an ELISA on nitrocellulose membranes, which in the form of separate bands are prone to specific proteins separated by gel electrophoresis. If there are antibodies against certain antigens - a dark line appears in the appropriate strister of the strister. The uniqueness of the immunoblot consists in its high informativeness and reliability of the results obtained.

Material for research Is serum or blood plasma. To study on the same strip, 1.5-2 ml of blood or 15-25 μl of serum is needed.

According to WHO's recommendation, immunoblotting (western blot) is used in the diagnosis of HIV infection as an additional expert method, which must confirm the results of the ELISA. Usually this method rechecks a positive result for ELFA, since it is considered more sensitive and specific, although more complex and expensive.

Immune blot combines an immuno-immunimal analysis (ELISA) with a preliminary electrophoretic separation of virus proteins in the gel and their transfer to the nitrocellulose membrane. The procedure of the immunoblot consists of several stages. First, the pre-purified and destroyed to composite components of HIV is subjected to electrophoresis, while all the antigens included in the virus are separated by molecular weight. Then, the antigen blotting method is transferred from a gel to a strip of nitrocellulose or a nylon filter, which from now on contains an invisible spectrum of proteins characteristic of HIV. Further, the material (serum, patient blood plasma, etc.) is applied on the strip, and if there are specific antibodies in the sample, they are associated with strictly corresponding strips of proteins-antigens. As a result of subsequent manipulations (similar IFA), the result of this interaction is visualized - it is done visible. The presence of bands at certain parts of the strip confirms the presence of antibodies in the studied serum to strictly defined HIV antigens.

Immune blot is most often used to confirm the diagnosis of HIV infection. WHO considers positive serums in which antibodies to any two HIV shell proteins are detected by the immune blot. According to these recommendations, if there is a reaction with only one of the shell proteins (GP160, GP120, GP41) in combination or without reaction with other proteins, the result is considered doubtful and re-examination is recommended using a set of another series or other firm. If, after this, the result remains doubtful, the research continues every 3 months.

Features

Immunoblot Analysis is a reliable method that allows you to determine the presence of antibodies to the HIV antigens of the first and second type. If a person is infected, an antibodies that can be detected and much later appear in two weeks. The feature of HIV is that the amount of antibodies increases rapidly and is preserved in the patient's blood. Even if they are present, the disease may not manifest itself within two or more years. The ELISA method does not always accurately determine the presence of a disease, therefore confirmation of the results using immunoblotting and PCR, if an immunoassimensional analysis showed a positive result.

Indication for appointment

What is it "Immunoblot" already found out, but who prescribe this study? The reason to pass tests for the immunodeficiency virus (HIV) by the method of immunoblotting becomes a positive result of the ELISA. It is necessary to go through an immunoferment analysis to patients who will be operated. In addition, it is necessary to make an analysis of women planning pregnancy, as well as anyone who leads a messy sex life. Immunoblotting patients with HIV, if the results of the IFA cause doubts.

The following disturbing symptoms may become a reason for appealing to the doctor:

• sharp weight loss;
• weakness, loss of performance;
• intestinal disorder (diarrhea), which continues for three weeks;
• dehydration of the body;
• fever;
• an increase in lymph nodes on the body;
• the development of candidiasis, tuberculosis, pneumonia, toxoplasmosis, exacerbation of herpes.

The patient does not need to be prepared before the surrender of venous blood. 8-10 hours before the study can not be eaten. It is not recommended for a day before the blood is given to use alcoholic and coffee drinks, engage in heavy exercise, waving an excitement.

How is the study?

From the point of view of the patient, immunoblot does not differ from any other analysis: the venous blood is taken, the result is investigated and the result. But if you go a little more details, it is not very simple, but still try to figure it out.

At first, at the reagent plant, the "reference" virus of human immunodeficiency is taken. Then with the help of a special procedure (electrophoresis) in the gel environment, the virus is destroyed to its smallest components: proteins (virus antigens). Then, with the help of blotting itself (from the English), the particles are placed on a special material - nitrocellulose or nylon filter, the indicator is ready for use, the so-called strip. The strip is a strip in which antigens are distributed depending on their molecular weight, in a clear sequence, that is, each millimeter paper corresponds to a certain protein.

As you may know if there are viruses in the blood of a person, the body begins to produce antibodies against their shells (defined proteins) antibodies, and what for each virus has its own individual antigen protein set. Identification of antibodies to antigens proteins in the blood is the basis of the immunoblot method. After all, if the antibody faces an antigen, they certainly interact with each other - "stick".

So, the antigens are on the strip strip and, in the case of the presence in the blood of the underlying suitable antigens, they necessarily interact with each other, and in this place, on the strip strip, the indicator will appear - a flat (as a pregnancy test) will appear. And in a particular place of the strip, thus the doctor will understand whether there is a set of proteins characteristic for a particular virus in the blood.

So, for example, if there is a darkening center in the location of proteins gP160, GP120, GP41hIV is diagnosed, for other viruses it will be a completely different set of proteins.

It should be noted that immunoblot allows you to accurately establish the presence of a virus only if the set of antibodies in the blood is complete, that is, if the proteins GP160, GP120, GP41 are present at the same time, then it is 100% HIV infection. But if at least one is not, for example: GP41 is absent, and there is only GP160, GP120, then the test is regarded as dubious and requires repeat.

FAQ

What stages include immunoblot?

1. Preparation of a strip.Pre-purified and destroyed to composite components, the immunodeficiency (HIV) is subjected to electrophoresis, while the antigens included in the HIV are separated by molecular weight. Then by blotting (an analogue of extrusion on the "wet" excess ink) antigens are transferred to the nitrocellulose strip, which from now on contains an invisible image of an antigenic strip spectrum characteristic of HIV.
2. Sample study.The test material (serum, patient blood plasma, etc.) is applied on the nitrocellulosic strip), and if there are specific antibodies in the sample, they are associated with strictly corresponding to them (complimentary) antigenic stripes. As a result of subsequent manipulations, the result of this interaction is visualized - it is done visible.
3. Interpretation of the result.The presence of bands in certain areas of the nitrocellulosic plate confirms the presence in the studied sera of antibodies to strictly defined HIV antigens.

• Strip A - Positive Control
• Strip in - weakly-bed control
• Strip C - Negative Control
• Strip D is a positive sample (the presence of antibodies to HIV-1 was detected)

How to decipher analysis?

If IFA has shown the presence of all or almost all antibodies to antigens according to this test system, it means a positive HIV analysis. If the answer after the second serological immunoassay analysis is positive, then immunoblot needs to be carried out. The decoding of its results will be more correct. If an enzyme immunoassal analysis gave a positive result, the following analysis of the immunoblot also showed HIV, then the final result is set.

When analyzes are decrypted, it is necessary to know that a positive HIV test is determined by:

• from 60% to 65% 28 days after infection;
• in 80% - after 42 days;
• 90% - after 56 days;
• 95% - after 84 days.

If a positive HIV response is received, it will mean that antibodies to the virus are revealed. To avoid a false-positive response, you must pass again analyzes, it is desirable twice. If the antibodies to the immunodeficiency are revealed during the delivery of two analyzes from two or with the surrender of 3 tests in 2 of them, it is considered that the result is positive.

Antigen R 24 can be revealed in the blood after 14 days from the date of infection. Using the method of immununimal analysis, this antigen is detected from 14 to 56 days. After 60 days there is no longer it in the blood. Only when AIDS is formed in the body, the growth of this protein P24 in the blood is repeated. Therefore, the test systems of immunooperment analysis are used to detect HIV in the first days of infection either in order to determine how disease occurs and monitor the treatment process. High analytical sensitivity of the immunoopermental analysis detects antigen P24 in biological material in HIV of the first subtype at a concentration of from 5 to 10 pkg / ml, with a second subtype HIV of 0.5 ng / ml and less.

Under doubtful The result of an immuno-enzyme analysis is meant that during the diagnosis somewhere was mistaken, as a rule, the medical workers confused something, or in humans there are signs of infection, and the result is negative, which causes suspicion, a person is sent to reassessment.

Under false positive The result is the result when the delivery of blood tests was made in the following patient's states:

• pregnancy;
• if a person has a violated hormonal background;
• with prolonged immunosuppression.

How to decipher the analysis in this case? The false positive result is placed if at least one protein is revealed. Due to the fact that the antigen P24 is very dependent on individual variations, then using this method, in the first period of infection is detected from 20% to 30% of patients.

How reliable is the positive test result?

Sometimes IFA has false positive results (about 1% of cases), the cause of this result may be pregnancy, various viral infections, as well as a simple accident. After receiving a positive result, a more accurate test - immunoblot, based on the results of which the diagnosis is made. The positive result of the immunoblot after a positive ELISA is reliable by 99.9% - this is the maximum accuracy for any medical test. If the immunoblot is negative, it means that the first test was false positive, and in fact HIV in humans.

What is an indefinite (dubious) result?

If the IFA is positive or negative, then immunoblot can be positive, negative or uncertain. An indefinite result of the immunoblot, i.e. The presence of at least one protein in the immunoblot may be observed if the infection has recently happened and there are fewer antibodies to HIV in the blood, in this case the immunoblot will become positive after a while. Also, an indefinite result may appear in the absence of HIV infection in hepatitis, some chronic metabolic diseases, or during pregnancy. In this case, either immunoblot will be negative, or the cause of an uncertain result will be detected.

How much is the analysis?

Immunoblot on HIV does not apply to cheap research. On average, screening examination with immunoferment methods costs from 500 to 900 rubles. Immunoblotting is a verification study, the cost of which ranges from three to five thousand rubles. More complex methods are much more expensive. For example, for the analysis of the polymerase chain reaction (PCR), it will be necessary to pay about 12,000 rubles.

Where to make analysis?

Where can I pass on HIV? ELISA, immunoblot research is carried out in urban private clinics, the results are issued during the day. Urgent diagnostics is possible. In state medical institutions, IFA tests and immunoblotting are carried out for free, according to the legislation of the Russian Federation. In mandatory, pregnant women are inspected for infectious diseases, as well as patients who have hospitalization or operational intervention.

Immunoblotting -(From English. "Blot" is a stain) - the method of identifying antigens (or antibodies) with known serums (or antigens). It is a combination of gel electrophoresis from ELISA. Initially, bacterial cells or virions are destroyed by ultrasound, and then the electrophoresis method is separating all the antigens of the virus or bacterial cells and receive a commercial reagent on a special nitrocellulose film. When performing immunoblotting on a film with known antigens, a studied patient serum is applied. After incubation and laundering of unrelated antibodies, it is embarked in ELISA - an antiserum is applied to the immunoglobulin of a person, labeled by an enzyme, and a chromogenic substrate, changing the painting when interacting with the enzyme. In the presence of complexes antigen antibody-anti-svelope to the immunoglobulin-enzyme on the carrier, painted stains appear. The immunoblotting method allows you to separately detect antibodies to various pathogen antigens (for example, with HIV infection, immunoblotting reveals antibodies to GP120, GP24 and other virus antigens).

Radioimmune analysis (RIA)

The method is based on the antigen antibody reaction using the antigen marking or an antibody radionuclide. As a label uses 125i, 14C, 3H, 51CR and other radionuclides. The resulting immune complexes are isolated from the system and determine their radioactivity on the counters (β-radiation). The radiation intensity is directly proportional to the number of connected antigen and antibody molecules.

The solid phase version of RIA using labeled antibodies or antigens, sorbed in the holes of polystyrene panels, is widespread.

RIA is used to identify microbes, viruses, various hormones, enzymes, medicinal substances, immunoglobulins, as well as other substances contained in the material under study in minor concentrations 10-12-10-15 g / l.

Control questions

Reaction of immune bacteriolization: what is this reaction; What is an antigen that - antibodies, reaction mechanism, methods of formulation, practical application. Reaction of immune hemolysis: the necessary ingredients, methods of formulation; Controls, practical application. The reaction of local hemolysis in the gel (reaction of yerne): the principle of reaction, methods of formulation, practical application. Complement binding reaction: reaction principle; which is formed in the interaction of immune sera with a specific antigen; What happens to the complement if it is present at the same time? What is the fate of the complement in the event that there are no specific affinity between the antigen and antibodies? With the help of which reaction can be determined what happened with the complement; why this reaction is used; What is the visible positive result of RSK? Why? What is the complement property in the first phase of RSK? In the second phase? If the ultimate RSK is hemolysis, what does this mean - a positive or negative result? Explain the results: RSK + + + +, RSK + + +, RSK + +, RSK +. Name the ingredients of the first RSK system and the ingredients of the second RSK system. Why should the test serum be inactivated? How to titrate complement? Hemolytic serum: what does it contain, how do they get, what is the titer and how to determine it? What animals are used to obtain RSK components? Methodology of RSK in the cold. When setting out of which of the listed reactions is necessary to participate complement: precipitations, flocculation, agglutination, detection of incomplete antibodies, immune bacteriolization, immune hemolysis, yerne, RSK? Immunofluorescence reaction (reef) - specify the sequence of events with a direct reaction of Kuns; Required ingredients. What is an antigen that - antibodies than marked antibodies, as the result of the reaction is taken into account, what does a positive result look like? Practical application - what can be determined by this reaction? Indirect immunofluorescence reaction - specify the sequence of events with this reaction, the necessary ingredients, which is an antigen, which immune serums are applied; practical use; The advantage of indirect reef compared to a direct reaction. Immunoenimen analysis (IFA) - the principle of reaction; necessary ingredients; Specify the sequence of events when reaction formulation in order to detect the antigen in the material under study; necessary ingredients; What happens with a positive result, what does he look like? Specify the sequence of action when performing an ELISA in order to detect antibodies in the test serum; necessary ingredients; What happens with a positive result? Immunoblotting is the principle of reaction; main steps; necessary ingredients; As the result is taken into account; Advantages of the reaction. Radioimmune analysis (RIA) - from which basic stages the reaction is a reaction; What is marked by antibodies or antigens, how does the result take into account? Immunoelectronic microscopy - method principle; main steps; necessary ingredients; than marked antibodies; As the result of the reaction is taken into account. Immobilization reactions - the principle of the method, technician productions, components, accounting results.

Tasks for performing in the process of self-preparation.

Fill in the "Immunity Reaction" table with respect to reactions disassembled within the framework of this topic.

Immunity reactions

Student work in a practical lesson

Work start immediately from setting 1 phase RSK, but to record a notebook later (see below).

1. Reaction of immune hemolysis. View the demonstration reaction of immune hemolysis, sketch in the form of a schema, explain the result in the experienced and in control tubes.

2. Complement binding response

a) Disassemble RSK on the table;

b) draw in the notebook the scheme setting RSK in the form of a table;

c) put the second phase of RSK (the first phase is placed at the beginning of the class);

d) disassemble diagnostic preparations necessary for RSK;

e) take into account the result. Formulate the conclusion about the presence of specific antibodies in the test serum.

3. Immunofluorescence reaction. Explore the table, make a chart of reaction formulation in the notebook; Look at diagnostic serum; Determine - which contains serum, as cooked, for which reaction (direct or indirect reef) it is applied. See a demonstration result of the reef in a luminescent microscope.

4. Immuno enzyme analysis (ELISA). In the notebook, the diagram of reaction formulation in two versions: to detect the antigen in the material under study and to detect serum antibodies. Check out the set of ingredients to diagnose HIV infection and hepatitis V. Determine that it contains each ingredient and for which it is applied.

5. Immunoblotting. Make a reaction scheme in the notebook; Look at the demonstration - the result of the reaction.

6. Radioimmune analysis (RIA). Make a reaction scheme in the notebook.

7. Immune electron microscopy (IEM). Look at the demonstration - the result of the reaction, make a reaction scheme in the notebook, indicate the arms an antigen (virus) and labeled antibodies.

Immunoblotting is a highly sensitive method for detecting proteins, based on the combination of electrophoresis and ELISA or RIA. Immunoblotting is used as a diagnostic method for HIV infection, etc.

In the general sense, immunoblotting understands the analysis of the mixture of proteins transferred to a solid substrate-membrane with which they are associated with covalent bonds, followed by immunodection.

You can analyze a mixture of proteins directly applied to the substrate - dot-blot analysis - or after its preliminary fractionation by methods of electrocusing, disc-electrophoresis or two-dimensional electrophoresis - Western blot analysis (Western blotting).

The antigens of the pathogen are separated by electrophoresis in the polyacrylamide gel, then transfer them from the gel to activated paper or nitrocellulose membrane and manifest themselves using ELISA.

Firms produce such strips with "BLOTS" of antigens. Sick serum is applied to these strips. . Then, after incubation, they are laundered from unreactive antibodies of the patient and serum against human immunoglobulins, labeled by the enzyme . The complex (antigen + antibody + antibody + antibody antigen + antigen + antibody) is detected by the addition of a chromogenic substrate changing the color under the action of the enzyme.

Such a methodology is also used to select bacteria, phages or viruses expressing target cloned gene products.

Transfer of proteins per membrane is carried out either passively or using equipment for electricity. The effectiveness of protein transfer to the membrane is influenced by many factors, such as molecular weight of proteins, gel porosity, transfer time and composition of the buffer solution used (trans buffer).

Depending on the tasks and conditions of the experiment, the transfer conditions are selected providing the best results. Nitrocellulosic, polyvinylidendyluoride (PVDF) or positively charged nylon membranes are usually used as substrates. Nitrocellulose can bind to 80 - 100 μg of protein per 1 cm2.

Low molecular weight proteins (with a molecular weight of less than 20 kDa) may be lost as a result of washing the ability to pre-explore the polymorphism of certain genetic loci on the lengths of the corresponding restriction fragments of DNA.

In addition, using hybridization according to the Sarew, it is easy to find out whether the target gene has a portion of hydrolysis of a certain restriction in its inner part, which allows you to choose an optimal strategy for cloning a studied genome area.

By a similar scheme of an agarose gel on a nitrocellulosic filter, the RNA molecules can be transferred. This method was named by the Northern Blotting (Northern Blotting) as opposed to the Southern blotting blotting, since the Southern Southern Language Surname means "South".

Transfer to filters from protein gel was respectively called Western blotting (Western blotting). Large proteins (more than 100 kDa), denatured in sodium dodecyl sulfate solution (SDS), may be weakly transferred to the membrane if ethanol is present in the trans buffer. The alcohol significantly improves the transfer of proteins with an SDS polyacrylamide gel, but narrows pores in the gel, which leads to a delay of large proteins.

The PVDF membrane is optimized for immunodection and is able to retain specifically associated proteins up to 160 μg / cm2 at a very low level of non-specific binding.

Immunoblotting

An important property of this membrane is the possibility of its repeated use. ZETA-PROBE nylon membranes effectively bind SDS proteins in the absence of alcohol, and this binding is resistant to subsequent treatments. Low molecular weight proteins are also detected effectively. Due to the high binding capacity - about 480 μg of protein by 1 cm2 - Zeta-Probe membranes allow detecting trace amounts of protein in the analyzed mixtures.

After the antigen turns out to be immobilized on the membrane, the remaining binding centers block the gelatin solutions or bovine serum albumin, or skimmed milk.

The membrane is then incubated in a solution of polyclonal or monoclonal antibodies to the anti-gene under study. After washing unbound antibodies, the membrane is incubated in a solution of secondary antibodies, which are an alkaline phosphatase enzyme conjugate (Alkaline phosphatase, AR) or horseradish peroxidase (HORSERADISH PEROXIDASE, HRP) with anti-love antibodies (goat antibodies to the immunoglobulin of rabbit, mouse or humans) or proteins (Staphylococcus aureus protein) or G (protein Streptococcus Sp.), having high affinity to the FC region of immunoglobulins.

The detection of educated immune complexes is carried out with a chemical or chemiluminescent way. Substrates for a chemical reaction using alkaline phosphatase conjugates are 5-bromine-4-chlorine-3-indolylphosphate (BCIP) or Tetrazolium Blue (NBT), and when using krena peroxidase conjugates - 4-chlorine-1-naphthol and hydrogen peroxide.

As a result of enzymatic reactions on the membrane, a painted strip or spot is formed at the localization site of the antigen antibody complex.

The sensitivity of this method is 100 GHG protein when using the AR and 100-500 GH conjugates when using HRP conjugates. Chemiluminescent detection of immune complexes allows you to determine less than 5 PG antigen. The principle of this method lies in the fact that with HRP reaction with hydrogen peroxide and cyclic diacylhydrazinyluminol, the emission of light of a wavelength of 428 nm occurs, which can be fixed on the photosensitive film.

The immunobloting reaction (RI) is developed on the basis of ELISA. It is the most specific and sensitive method of immunochemical analysis. Immunoblotting (from the English blot - glow, stain) combines IFA with electrophoresis. It is used to identify non-complex antibodies to HIV, and antibodies to its separate structural proteins (proteins-P24, glycoprotein-GR120, GR 41, etc.). Refer to expert (supporting) reactions of diagnosis of HIV infection.

The reaction is carried out in several stages:

The virus is destroyed by components - antigens (P24, GR120, GR 41, etc.), which are subjected to electrophoresis in a polyacrylamide gel, that is, the separation of antigens on the molecular weight fractions.

2. The gel is covered with a nitrocellulose membrane and the antigens fractions are transferred to it using electrophoresis. Nitrocellulose behaves like watering paper. The membrane is cut on strips (strips). Firms produce such strips with "BLOTS" of antigens.

Immunoblotting - additional indirect method

Streaps with HIV antigens deployed on it are immersed in the serum of the examined and then wash away from the unbiased material.

4. Streaps are incubated with antihylobulin serum, labeled peroxidase, washed.

Substrate is added and the number of painted fractions (spots), which correspond to the Localization zone of the AG-AT complex, is noted.

The presence of bands at certain parts of the strip confirms the presence of antibodies in the studied serum to strictly defined HIV antigens. The result of immunoblotting is considered positive if the membrane shows the bands corresponding to any two of the three HIV antigens - P24, GP41 and GP 120 (Fig. 37).

Pictures

List of used literature

Main literature

Medical microbiology, virology and immunology: textbook for students honey. universities 2nd ed., University. and add. - 702 p. Ed. A.A. Sparrow. M.: Mia, 2012.

2. Microbiology, virology and immunology: manual for laboratory classes: studies. Victims / (V.B.Subacov, etc.); Ed. VB Subachakova, M.M. Karapatsa. - M.: Gootar Media, 2014.- 320С.: IL.

3. Medical Microbiology, Immunology and Virology [Electronic resource]: Tutorial for honey.

universities - 760 p. - Access mode: http://www.studmedlib.ru/book/isbn9785299004250.html Kijiyev A.I., Babichev S.A. St. Petersburg: Speclit, 2010.

4. Medical microbiology, virology and immunology [Electronic resource]: Tutorial: in 2 t. / T. 1. - 448 p. - Access mode: http://www.studmedlib.ru/book/isbn97859704142241.html Zverev V.V., Boychenko M.N.

M.: Gootar Media, 2010.

5. Medical Microbiology, Virology and Immunology [Electronic resource]: Tutorial: 2 tons. T. 2. - 480 p. Access mode: http://www.studmedlib.ru/book/isbn97859704142242.html Zverev V.V., Boychenko M.N. M.: Gootar Media, 2010.

additional literature

1. Immunodiagnostic reactions: Tutorial / Cost: G. K. Davletshina, Z.G. Gagabidullin, A.A. Aahtarieva, M.M.Tuigunov, A.K. Bulgakov, T.A. Savchenko, R.F .

Husnarizanova, Yu.Z. Gabidullin, M.M. Elsynbaev - Ufa: Publishing House of GBOU VPO BGMU of the Ministry of Health of Russia, 2016. - 86c.

2. Features of some properties that determine the pathogenic potential of the eNterobacter, Citrobacter, Serratia, E. Coli, Proteus, SERRATIA, E. Coli, Proteus: Scientific Edition / Yu. Z. Gabidullin, R. S. Sufiyarov, I. I. Dolgushin - Ufa, 2015. - 250 s.

Home »Immunoblot - What is it? Immunoblot in the diagnosis of infectious diseases

Immunoblot - what is it? Immunoblot in the diagnosis of infectious diseases

What is immunoblot? This is a common method of laboratory diagnostics of human viral infections. It is one of the most accurate and reliable ways to detect HIV.

For reliability, it is even more than an enzyme-connected Assay immunosorbent (ELISA). Immunoblot results are considered an irrefutable and final.

Immunoblot - What is it? In order to recognize a person HIV, you must pass the laboratory test method of blood serum for antibodies.

The Western blot method sections are also called Western blot (Western blot). It is used to detect human viral infections as an additional expert method. This is necessary to confirm the ELISA - laboratory study, which allows to determine the presence of antibodies to HIV in the blood. Immunoblot renounce positive ELISA.

It is considered the most sensitive, complex and expensive.

purpose

What is immunoblot? This method of laboratory test serum tests for the presence of antibodies to the virus.

In the course of special research, the total virus proteins in the gel and nitrocellulose membranes.

Immunoblotting (detection of antibodies in serum patients to certain pathogen antigens)

Designed procedure for Western-blot sections to determine HIV infection at different stages. At the first stage, the purified virus from component parts is subjected to electrophoresis and antigens included in it divided into molecular weight.

The human immunodeficiency virus breeds in living cells implemented in its genetic information. At this stage, a person becomes a carrier of HIV virus if you were infected.

The specifics of this disease lies in the fact that it is not manifested for a long time. The virus destroys lymphocytes, thus, a person decreases immunity and the body becomes unable to deal with infections.

If HIV is correct and timely treated, the patient will live to a deep old age. Lack of treatment inevitably leads to death. Since infection, but without treatment, the maximum period of no more than ten years.

Features

An analysis of immunoblot is a reliable method that allows you to determine the presence of antibodies to HIV antigens of the first and second type.

If a person is infected, after two weeks of antibodies that can be discovered significantly later. A feature of HIV is that the amount of antibodies increases rapidly and remains in the patient's blood. Even if they are present, the disease may not manifest themselves for two or more years. The ELISA method does not always accurately indicate a disease that requires confirmation of the results from PCR and Western blot sections, if an immuno-immimensional analysis showed a positive result.

Indications K.

What kind of "immunoblot" already found out, but which are introduced into the study?

The reason for the inspection of the human immunodeficiency virus (HIV) immunoblot will become a positive result of the ELISA. It is necessary to go through solid-phase immunoassay analysis in patients who have to have an operation. In addition, you must make the analysis of the women's pregnancy, as well as those who are raising a messy life. Western blot sections are prescribed to patients with HIV infection if the ELISA results are dubious.

The following disturbing symptoms can be a reason for accessing a doctor: Fast weight loss; weakness, loss of functions; intestinal disorders (diarrhea), which lasts three weeks; dehydration; fever; increase in lymph nodes in the body; the development of candidosis, tuberculosis, pneumonia, toxoplasmosis, The exacerbation of herpes.

The patient does not need to be prepared before surrendering venous blood.

For 8-10 hours before the test, do not eat. It is not recommended a day before the blood of drinking alcohol and coffee beverages, heavy exercise, taking excitement.

Where to make analysis?

Where can I pass HIV testing?

ELISA, immunoblot analysis conducted in urban private clinics, results are given during the day. Urgent diagnostics is possible. In public institutions, ELISA medical tests and Western blot section for free, in accordance with the legislation of the Russian Federation.

Mandatory inspection on infectious diseases of pregnant women, and patients who need hospitalization or surgery. How does this study take place?

How to hold an ELISA? Immunoblot positive / negative confirms or refutes the results of ELISA. The procedure is quite simple. The specialist takes the fence of venous blood, it takes less than five minutes by time.

After sampling, the place of injection should be disinfected and stuck with the plaster. The fence is conducted on an empty stomach, so after the procedure it will not hurt to eat a bitter chocolate tile or a sweet hot drink.

To get a referral for free analysis in the State Medical Institution, you need to visit the therapist.

In general, immunoblot does not differ from other blood studies by fence. The study methodology is simple. If a virus is present in the blood of a person, the body for its destruction begins to produce antibodies. For each virus there are many protein antigens. The detection of these antibodies is the basis of the Western blot section sections. Cost

How much analysis? Immunoblot HIV refers to cheap research.

On average, screening methods of immunoassay is from 500 to 900 rubles. Western blots of sections is to study the inspection, the cost of which ranges from three to five thousand rubles. More complex methods are much more expensive. For example, to analyze the polymerase chain reaction (PCR), it will be necessary to pay about 12,000 rubles.

Interpretation of results

The most common methods of diagnosing HIV infection is an immuno-immimensional analysis and immunoblot.

They are for determining antibodies to the human immunodeficiency virus. The infection is typically confirmed by two tests: screening and confirming. The interpretation of the results should be done by the doctor, it diagnoses and prescribes treatment. If the immunoblot is positive, it means that in the human body the virus.

A positive result should not be a reason for self-treatment, since each patient can have its own diseases of the disease.

Qualitative analysis includes screening and certification. If the patient does not detect the virus, the result shows "negatively". When this certificate was detected, screening is carried out additional research. Immunoblot Analysis that confirms or refute screening. If the test strips appear in the darkening of certain areas (protein localization), the diagnosis of HIV is diagnosed.

If the results are doubtful, the tests are carried out within three months.

To prevent the infection of the human immunodeficiency virus, it is possible to comply with certain rules: avoid random sex contacts, use condoms when contacting, do not use drugs.

If the disease is detected in a pregnant woman, it is important to follow the recommendations of the attending physician, do not forget about the tests for the presence of a virus.

What is western blotting?

The identification of the protein of cardiac mixtures or extracts of various tissues is one of the most common tasks. Using such a tool as specific antibodies, you can define the studied protein with a minimum of temporary and financial costs.

In the Western blotting method (Western blotting) at the first stage, a mixture of proteins is separated by electrophoresis in the presence of sodium dodecyl sulfate (DSN), then transferred to the nitrocellulose membrane by the method of electroblotting.

The essence of this method is that the gel after electrophoresis is placed on the nitrocellulose membrane between the layers of filter paper. The "sandwich" assembled in this way is placed in the electric field so that protein-DSN complexes are moving across the plate plate and are immobilized (as a result of non-specific sorption) on the surface of the nitrocellulose membrane.

In the binding of the protein-dsn complex with a nitrocellulose membrane, they take part in the main force of electrical nature, and this interaction is multipoint and leads to "molding" proteins on the surface of the membrane. Thus, after the electric barrel, we get a gel replica on nitrocellulose with proteins, located in the same way as in the polyacrylamide gel.

After conducting dsn - electrophoresis, electric trail and sorption of proteins from the gel on a nitrocellulose membrane, the tertiary conformation of the protein is strongly changed, if generally correctly talk about the existence of a tertiary structure for a protein after such rigid processing. Therefore, for the immunochemical detection of the protein under study, only mono- or polyclonal antibodies, specificity of the protein molecule, are usually used.

51. Immuno-immunimal analysis, immunoblotting. Fur, components, use.

Antibodies specific to conformational epitopes (or areas including intersubating contacts) are usually not suitable for use in the Western blot method.

After transfer of proteins, the membrane is incubated sequentially with antibodies specific to the protein under study, and then with secondary antibodies, specific to Fc fragments of primary antibodies conjugated with an enzyme (or other) label (Fig.

1 A). In the case when the label is conjugated directly primary, specific antibody antigen, the secondary antibodies are not required (Fig. 1 b). The immune complexes "manifest themselves" formed in the localization of the studied protein using a chromogenic substrate (depending on the type of tag).

The sensitivity and specificity of the method is greatly dependent on which antibodies are used during research.

The antibodies used should be specific to the unique, characteristic of the studied protein of the amino acid sequence. Otherwise, the interaction (especially in the case of coarse protein extracts) of antibodies with several protein molecules is possible, which in turn will lead to the appearance of several colored strips on the membrane.

The identification of the protein under study in this case is often difficult or impossible at all.

The second important factor that should be remembered when choosing antibodies is affinity. The higher the affinity of the antibodies used, the brighter and more clearly the protein strips are scored, the higher the sensitivity of the method. When using highlyaphief antibodies, it is possible to achieve a sensitivity of 1 ng and even higher.

To visualize the result of the interaction of the membrane of the antigen and antibodies, the secondary antibodies conjugated with agents capable of giving a certain signal under certain conditions are used.

Usually, the enzyme (peroxidase or phosphatase) is used as such an agent, the product of the reaction is painted and falls on the membrane as an insoluble precipitate.

Also in this method it is possible to use fluorescent tags.

Fig. 1. The scheme of immunochemical staining of the protein under study: a - using secondary antibodies conjugated with an enzyme label; B - Primary antibody directly conjugated with an enzyme label.

Protocol:

I. Preparation of gel and membrane and protein electric

The polyacrylamide gel after electrophoresis is placed in a Blotting Buffer Bath (25 mM Tris, pH 8.3, 192 mm glycine, 10% ethanol).

Two sheets of filter paper, cut on the shape of a cassette for blotting and moistened with spuffer for blot, are placed on that part of the cassette that will be addressed to the anode. Then the nitrocellulosic membrane is placed on the filter paper, which follows the air bubbles between the membrane and paper.

After that, the gel should be carefully placed on the membrane, turning special attention to the absence of air bubbles between the gel and the membrane. The formation of a sandwich is completed two layers of moistened filter paper, which are placed on the gel surface (Fig. 2). The resulting sandwich is clamped in the cassette and is placed between the electrodes so that the membrane is facing the anode.

Fig. 2. Scheme of electric proteins on the membrane.

II. Electricity

Electricity proteins on a nitrocellulose membrane are carried out in a buffer containing 25 mM Tris, pH 8.3, 192 mm glycine, 10% ethanol for 30-50 min at a constant voltage of 100 V.

The electric barrel time depends on the size of the portable proteins, the more protein, the longer the electric barrel is carried out. The quality of the electricity and the location of the protein bands is estimated, staining the nitrocellulose membrane with a 0.3% solution of Ponceau S in 1% acetic acid. Before conducting immunochemical staining, the membrane should be flushed several times weakly alkaline aqueous tris solution to remove the dye-tied with proteins.

III. Immunochemical staining of proteins immobilized on nitrocellulose membrane

To block the nonspecific binding places of antibodies, the membrane is incubated with constant stirring at room temperature for 30 minutes to PBST (for better lock, a PBST solution containing 10% dry skimmed milk can be used).

After blocking, the membrane is incubated for an hour at room temperature and constant stirring in PBST containing 1-10 μg / ml of specific antibodies.

The optimal concentration of antibodies is selected empirically and depends on the affinity of the interaction of antibodies with an antigen.

At the end of incubation, the membrane washed 5 times PBST and transfer to a solution of secondary antibodies conjugated with horseradish peroxidase. The conjugate breeding is usually indicated by the manufacturer on the package, or is selected by the researcher empirically. In the solution of secondary antibodies, the membrane is incubated for 1 hour with constant stirring.

After a thorough wash (minimum 5 - 6 times, the PBST membrane is transferred to a solution of a chromogenic substrate, containing 3 mg of diaminobenzidine (DB) and 10 μl of 30% of hydrogen peroxide in 10 ml of 0.1 m tris-HCl, pH 7.6.

Incubation is carried out with stirring 5 - 10 minutes. Membrane After the end of incubation with the substrate should be rinsed with PBST, dry, glowing with filter paper, and immediately make an electronic copy, scanning in color. If the membrane is completely dried, the scratched protein strips are pale, and the image is less bright and contrast.

Note: DUB is a toxic substance and potential carcinogen. Work only in rubber gloves!

His name AIDS Vyacheslav Zalmanovich Tarantul

Immunoblotting - additional indirect method

In addition to IFA, in certain cases, the procedure called "immunoblotting" or "immune blot" is used to test HIV infection (sometimes called "Western blot"). On the WHO recommendation, immunoblotting is used in the diagnosis of HIV infection as an additional expert method, which must confirm the results of the ELISA. Usually this method rechecks a positive result for ELFA, since it is considered more sensitive and specific, although more complex and expensive. But before signing the final sentence to the patient, the doctor must ensure completely in the correctness of the diagnosis. Therefore, the question of complexity and high cost here cannot be decisive.

Program, as well as in the method of execution to the immune blotting, a well-known expression "bring to the wet" is well suited. Immune blot combines an immunoassay analysis (ELISA) with a preliminary electrophoretic separation of virus proteins in the gel and their transfer to the nitrocellulose membrane ("Wrought"). The procedure of the immunoblot consists of several stages (Fig. 27). First, the pre-purified and destroyed to composite components of HIV is subjected to electrophoresis, while all the antigens included in the virus are separated by molecular weight. Then by blotting (an analogue of extrusion on the "wet" excess ink) antigens are transferred from a gel to the nitrocellulose or nylon filter strip, which from now on contains an invisible eye of the spectrum of proteins characteristic of HIV. Further, the strip is appreciated the material (serum, the patient's blood plasma, etc.), and if there are specific antibodies in the sample, they are associated with strictly corresponding strips of proteins-antigens. As a result of subsequent manipulations (similar IFA), the result of this interaction is visualized - it is done visible. Ultimately, the presence of bands at certain parts of the Strepa confirms the presence in the studied sera of antibodies to strictly defined HIV antigens.

Immune blot is most often used to confirm the diagnosis of HIV infection. WHO considers positive serums in which antibodies to any two HIV shell proteins are detected by the immune blot. According to these recommendations, if there is a reaction with only one of the shell proteins (GP160, GP120, GP41), in combination or without reaction with other proteins, the result is considered doubtful and re-study using a set of another series or other firm. For safety praise if

Fig. 27. For immunoblotting at the first stage, proteins contained in serum, separated in the gel by their molecular weight and charge using an electric field (by electrophoresis in gel). Then the gel is applied to the nitrocellulose or nylon membrane and "wedging" (this is Blotting). This is carried out in a special chamber that allows you to carry out a complete perit of the gel material on the membrane. The result is the picture of the location of the proteins, which was on the gel, is reproduced on the membrane (blot), from which it is further easily manipulated. Initially, the membrane is treated with antibodies to the desired antigen, and after washing unrelated material, radioactively labeled conjugate is added, which specifically binds to antibodies (as in ELISA). The location of the resulting complex "Antigen antibody - labeled conjugate" is determined using autoradiography using X-ray film. After its manifestation, it becomes clear whether there are antigens in the blood or there are no them and after that the result remains doubtful, the subsequent observation is recommended for six months (the research continues every three months).

The present in the Russian Federation for use in the diagnosis of HIV infection is recommended to use five test systems, among which are both Russian and foreign ones.

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When the tests passed on the STD, I decided to pass on HIV. On the day, I received ready-made tests on Ethia except HIV in the end I found chlamydia. Herpes and micramplasmosis. But HIV did not come. I call me after 3 days and they say you need to appear in the AIDS Center to Pass Rather I was building on Imubabot IMUNUBOTI showed positive. Can immanublot be false positive in diseases of chlamydia micraplasmosis HPV Herpes

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don't want to scare you but when they cause the center almost always positive as do not cross, good luck to you, the main thing to take therapy and live long

One friend lives with HIV for ten years, saves himself.
It is said that the years after three may find a medicine.
In any case, do not despair and do not distribute it, treated

No, IB cannot be false positive and no STDs affect this analysis, if it is positive, then alas, you have HIV, you need to follow the immune cells and start therapy in time.

there are no alas ((if called to the center, then for sure HIV

As far as I know, the first and even subsequent results of immunoblot may be dubious. Not false positive, namely, dubious. And then prescribe repeated analysis. I give text from the site:
"Immune blotting is most often used to confirm the diagnosis of HIV infection. WHO considers positive serums in which antibodies to any two HIV shell proteins are detected by the immune blot. According to these recommendations, if there is a reaction with only one of the shell proteins (GP160, GP120, GP41) in combination or without reaction with other proteins, the result is considered doubtful and re-examination is recommended using a set of another series or other firm. If, after this, the result remains doubtful, the research continues every 3 months. "
you can google. If so, I hope you do not confirm HIV. But if you confirm, know that today they live with this diagnosis and live as much as healthy people and children give birth. The main condition is discipline in treatment. Health to you!

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Decoding analysis - Immunoblot

Help please decrypt analysis
ENV (GP160) +
ENV (GP110 / 120) +
ENV (GP41) +
GAG (P55) +
GAG (P40) +
GAG (P25) +
POL (P68) +
POL (P52) +
POL (P34) +

Hello! 2.5 years ago I handed over HIV analysis, there was such immunoblot:
GP-160 +, GP-110/120 +, GP-41 +, P17 +, P25 +, P31 +, P34 +, P52 +, P55 +, P68 +. But Imunnoblot from 30.05.18 g.: GP-160 +, GP-41 +, GAG 1 -, Poll +, Env2-. It is not clear what ROll + mean? Is he there only one or not revealed? And where are the rest of the proteins?

POL and ENV genes that encode protein groups. Consider that just a record is different. The question is different. And what are we waiting for? Why do you consider blot? Everything is pretty clear. Do something you need.

Already bounced with what I have HIV. And ready for therapy.
Just it is interesting to listen to your opinion. This, it turns out, fresh infection or I do not understand something? Or sometimes it happens that immunoblot is slowly very revealed?

Today I looked at my analyzes in my personal case (we were made in the SC):
IFA on the first test system - put
IFA on the second test system is negative (how is it?)

Next, IB (made in Invitro and SC month ago):
immunoblot on the first test system: GP 160+, GP 41+
immunoblot on another test system: GP41 +, P24 +, P17 +
immunoblot on the third test system: GP160 +, GP120 +, GP41 +, P24 +

According to my forecasts, I could infect a year ago (the acute stage was in April somewhere), or 9 months ago, but in general - xs when) always prevented and sex without condoms was just one time in the fall of 2017.

Today she once passed on VN and IP. Thera will begin in a month, as the order comes.

Put the dates to the tests mentioned.

First Blot before IFA? Does not be fighting. There is no moment that would allow me to say that there is a very likely fresh infection, or the opposite.
It will remain a mystery, if you do not find a random source and do not compare.

If you specify, then

Purchased to Invitro.
First IFA - May 11th.
Then the same serum went on blot and had a positive analysis, where two proteins revealed.
gP 160+, GP 41+

Next, I have already passed into the SC again.
Blood passed on May 29th.
And there he was driven over several test systems, where the first showed a negative, the second positive. Negative IFA is funny yet.
Next, the same serum goes to blot, where the data came from:

First Test System: GP41 +, P24 +, P17 +
Second test system: GP160 +, GP120 +, GP41 +, P24 +

But do not essence.
A new analysis came - 754. It pleases. VN is not ready yet. As it will come, probably, I will start the theer.

The fact that infection was a year ago - I am confident of 80%. And the fact that blot is slowly revealed and the IFA is negative - this is strange. Or is it within the normal range?

Negative IFA is funny yet. Know the name of the system still to record in a black notepad. Although, this moment, if you do not take the theory with marriage, strongly for early infection.
The fact that infection was a year ago - I am confident of 80%. Blot is poor for that time. Not impossible, but unlikely.

At some point, I even began to doubt the results of HIV tests - therefore I wait
There is already a last point in the question.

The fact that IS for the month grew by 200 copies without terra - is it also within the normal range? I take Ursosan now from the polyp in a bile - in the liner it is written that the drug enhances immunity.

It is necessary to evaluate with relative content, and take into account the data of one laboratory with one counting method. Because - God knows what's from CD4.

In general, CD4 - 780 cells / μl. VN - 250 copies / ml.
This is no therapy. That is, the CD4 of 486 per month has rushed to 780.
VN fell from 550 to 250 copies.

Yet it is not strange or such jumps within the normal range? Theory is time to start?)

Hello! I will hand over IFA three times, each time +, from SC came immunoblot P24 + P18 +. The potential risk was over six months ago. P24 indicates that this is all the same HIV?

Blot dubious, repeat after 6-8 weeks. Most likely false alarm.

Good day!
The results of analyzes came, including blot:

Dangerous contact: 04/24/2018
Analysis on HIV IFA 23.05.2018 (4 weeks from dangerous contact or 29 days) Result "+"
Analysis on HIV IFA 05/28/2018 (5 weeks from dangerous contact or 34 days) Result "Remember"
Analysis on HIV IFA 06/01/2018 (5 weeks from dangerous contact or 38 days) Result "+"

Blot came from first analysis (05.23.18):
GP160 SL.
P24 sl.

New tests have been commissioned 06.06.2018 ELISA and PCR RNA HIV in local AIDS center. Results are still waiting.

Blot questions:
1. If P24 is present, is it necessary an antigen of HIV or such protein can be detected with other diseases?
2. What does the abbreviation "Sl" mean next to the protein? Usually in the described fleet have + or -
3. What does the fleet deployment depend on? From the term or from the individual characteristics of the body?
4. With such a blot on the 4th week from dangerous contact there is a chance that this is not HIV?

Have a nice day and thank you.

1. No, it can just be a similar protein of other nature. 2. Weak. those. Doubtful. 3. Terms and implement an individual feature, but on average, everything is very close. 4. The question is incorrect, there is always a chance until the diagnosis is set.

Hello!
Help, please navigate the results of the tests and understand how to behave correctly, so that repeated analyzes are correct.

Analyzes handed over 05/25/2018
IB HIV
New Lav Blot 1 - Understand. from 05/29/2018
IB HIV markers
gP 160 +.
gP 120 -
gP 41 -
p55 +.
p40 -
p 24 +.
p18 -
p 68 -
p 52 -
p 34 -
IFA HIV
Advia Centaur HIV AG-AB Reactivity IFA 12.00 Position (05/28/2018)
Recommended Analysis Repeat in 2 weeks.

In November 2017 there was NAP, after which tests were handed over to the HIV AG \\ AB COMBO ABBOTT Architect test system, then the result was negative.

Parallel handed over a general blood test. Limphocytes are slightly elevated, Eosinophils are exactly 0 (but I had such indicators for eosinophils before.

The main question is:
what medications can and can not be taken in these two weeks? (I want nothing "Phonil")
I periodically pass attacks of allergies, I usually drink Tueguil. Should I refuse it?
Is it possible to take before analysting antibiotics? (if they are prescribed a doctor for the treatment of other infections and diseases)

Immune blot in the diagnosis of HIV

Immune Blotting (Immunoblot, Western Blot, Western Blot) - Combines an immuno-enzyme analysis (ELISA) with preliminary electrophoretic transfer to the nitrocellulose strip (strip) of the virus antigens.

In this beautiful scientist name "blot" translates most likely as "blots", and "Western" - as "Western" reflects the direction of the spread of this "blots" on paper from left to right, that is, on the geographical map, this corresponds to the direction from the West to the East. " The essence of the "Immune blot" method is that the immuno-immunimal reaction is carried out not with a mixture of antigens, but with HIV antigens, pre-distributed by immunoforesis using fractions, located according to molecular weight over the surface of the nitrocellulose membrane. As a result, the main proteins of HIV, carriers of antigenic determinants are distributed over the surface in the form of individual bands, which manifest themselves when conducting an immunoferment reaction.

Immunoblot includes several stages:

Preparation of a strip. Pre-purified and destroyed to composite components, the immunodeficiency (HIV) is subjected to electrophoresis, while the antigens included in the HIV are separated by molecular weight. Then by blotting (an analogue of extrusion on the "wet" excess ink) antigens are transferred to the nitrocellulose strip, which from now on contains an invisible image of an antigenic strip spectrum characteristic of HIV.

Sample study. The test material (serum, patient blood plasma, etc.) is applied on the nitrocellulosic strip), and if there are specific antibodies in the sample, they are associated with strictly corresponding to them (complimentary) antigenic stripes. As a result of subsequent manipulations, the result of this interaction is visualized - it is done visible.

Interpretation of the result. The presence of bands in certain areas of the nitrocellulosic plate confirms the presence in the studied sera of antibodies to strictly defined HIV antigens.

Currently, immune blotting (immunoblot) is the main method for confirming the presence of virus specific antibodies in the test serum. In some cases, HIV infection, prior to the development of seroconversion, specific antibodies are more efficiently detected by the method of immune blot than ELISA. In the study by the method of immune blotting, it was found that antibodies to GP 41 are detected in serums in serums, and the detection of P24 in persons surveyed with a prophylactic goal requires additional studies for their HIV infection. The test system for immune blotting based on recombinant proteins obtained by genetic engineering turned out to be more specific than conventional systems based on purified viral lysate. When using a recombinant antigen, not diffuse, but a clearly pronounced narrow antigen strip, easily accessible for accounting and evaluation.

Serums of persons infected with HIV-1 are detected by antibodies to the following main proteins and glycoproteins - the structural shell proteins (ENV) - GP160, GP120, GP41; Kernels (GAG) - P17, P24, P55, as well as virus enzymes (POL) - P31, P51, P66. For HIV-2 typical antibodies to ENV - GP140, GP105, GP36; GAG - P16, P25, P56; POL - P68.

Among the laboratory methods necessary to establish the specificity of the reaction, the greatest recognition has the detection of antibodies to the proteins of the HIV-1-GP41 shell protein, GP120, GP160, and HIV-2 - GP36, GP105, GP140.

WHO considers positive serums in which antibodies to any two HIV glycoproteins are found by the method of immune blotting. According to these recommendations, in the presence of a reaction with only one of the shell proteins (GP 160, GP 120, GP 41), in combination or without reaction with other proteins, the result is considered doubtful and re-examination is recommended using a set of another series or other firm. If, after this, the result remains doubtful, observation is recommended for 6 months (studies after 3 months).

The presence of a positive reaction with an antigen of P24 may indicate for the period of serocaurus, as antibodies sometimes appear to this protein first. In this case, it is recommended, depending on clinical and epidemiological data, repeat the study with a serum sample, taken at least in 2 weeks, and this is the case when with HIV infection it is necessary to study pair serums.

Positive reactions with GAG and POL proteins without a reaction with ENV proteins can reflect the step of early seroconversion, and may also indicate the HIV-2 infection or nonspecific response. Persons with such results after testing on HIV-2 are re-examined after 3 months (within 6 months).

Question: Repeated HIV Analysis?

He was examined for infections received by sexual way (no symptoms - wanted confidence in relationships with a woman). HIV test was positive. Next ultrasound showed a tumor on the kidney, removed (turned out to be malignant).
I read the book Sazonova I.M., it says that a malignant tumor can give a positive result of HIV tests.
Could it be that way, or hope not to?

You need to conduct a control analysis for HIV. If the first HIV test was determined by the ELISA, then the result can be false positive. Its reliability can be checked by a more sensitive diagnostic method - PCR (polymerase chain reaction), which defines the DNA of the virus in the blood.

help. 12/16/10 IFA (+) IB (+) Then from 03/23/11 to 19.05.11, nine negative ELISA (-) and PCR quantitative. will not be determined. In 2002, during pregnancy, IFA is (+) that (-) but IB is always (-). From 2004 to 2008 I handed over 2 times a year of IFA (-). But on April 30, 2007, IFA (+) and ib-uncertain. Then again every 2 months I always handed it out (-). And since December 2010, it is written above. I have never been called this, my husband always has an IFA (-). SD4 980 cells. And even blood on syphilis from 29.04 gave 3 +++. And then three times. Deculs. Thread every 10 days. Hepatitis all (-). Was someone like that. Thank you.

Please specify whether Ribt has been carried out (the reaction of the immobilization of pale treponam), if so, what are the results of this study.

no, no one suggested to make such an analysis. And what will he show? I hope you realized that I was talking about HIV tests. Thank you. In your practice there were similar cases? Yes, by the way about IB in 2008 was indefinite because There was a protein P24 / 25. In 2010, IB (+) proteins GP160.41.120 p24.17.31. Then when IFA was again 3rd (-) sent to IB on April 4. The result came positive, but the GP 120 and 41 proteins are crossed out with red paste and at the bottom of red IB repeat. But PCR is denied from the same number. After on April 4, I passed the ELISA for 4 times. All in AIDS center, including antigen and antibody. Now I am waiting for repeated IB and high-quality PCR. that's it. Very tired to think and wait. Hoping for the best. THANK YOU. Waiting for a response very much.

If you ask any question, please try to formulate it more specifically, with the clarification of the diagnosis. Ribt is used to confirm the diagnosis of syphilis. To accurately diagnose HIV infection, the definition of antibodies to HIV in the blood of the IFA method and the Immunoblot method is carried out. The diagnosis is confirmed only if both of these results are positive.

sorry that inaccurately formulated the question. I wrote that in December, IFA and Imunoblot on HIV came positive. But since March of the month of IFA on HIV is negative 9 times. If I was registered in AIDS in AIDS, then does this in principle happens. HIV either always put either negative. And how does the IFA decide on HIV be rejected by the IFA? Then everything will deny the IFA to check on Imhunoblot, so if it turns out? In our AIDS center can not answer anything to me. So I turned to you. Thank you.

Unfortunately, both ELISA and immunoblot can give false-positive results. That is why the diagnosis of HIV is considered final, only with the simultaneous detection of HIV with the help of IFA and the Immunoblot method.

hello. Today received the results of PCR for HIV HIV HIV. The result was not detected and repeated immunoblot on HIV. The result was indefinite because of the protein 41. In the AIDS center, they would most likely have no HIV, and in my body there are bodies similar to HIV. And what do you think, if you consider my questions from 15 and 16 June (see) HIV or not. THANK YOU.

In this case, the diagnosis of HIV infection is doubtful.

you write that only with the simultaneous detection of HIV with the help of ELISA and immunoblot, the diagnosis of HIV is considered final. And how then in my case be? After all, all PCR will deny. And blot and IFA will jump all the time. For 9 years. Tell me if the virus was in my blood, then its RNA and DNA for so many years could be defined. And can the period of incubation or "windows" last so many years? Are the results of PCR on HIV sick, taking into account such a period of time? Yes, I forgot to say that the express HIV tests for HIV passing by me are always negative. And they should not rely on them too? Thank you.

In this case, PCR diagnostics is not the main method of identifying the process dynamics - more informative serological methods. In this case, the likelihood of false negative results is high. Express HIV tests have a high sensitivity threshold, therefore can also give a false negative result.

sorry. I definitely wrote not there. Please answer HIV or not HIV. Thank you.

In the event that you have not received a notification about receiving an answer on your mail, you can view the answer to the question you specified at this address http://tiensmed.ru/news/answers/vich-Ili-ne-vich-.html

Hello! Please tell me what to register in the LCD (now 10 weeks of pregnancy) passed the tests for HIV, a couple of days ago called the doctor and said that the preliminary tests for HIV positive (the first was made in Kirovograd, and there is no official result from Kiev yet ) on the same day did in our city laboratory two express tests of the company "Pharmasko" Cito Test HIV 1/2, both results are negative, the laboratory assistant that these tests are reliable and I can not worry, because during pregnancy it happens, And those tests could simply confuse. The doctor said to pass the blood again and I twice passed my blood for analysis in different hospitals (no one of the three results still do not). I am very worried, I am not a drug addict, there was no dubious sexual relationship, if it's very rare, other analyzes are fine. Is it possible to trust express tests? Does it really happen during pregnancy? It hurts a lot of doctor scared me. Thank you

You need to calm down first and not think about the bad. Sometimes during pregnancy, there are false positive results. It is necessary to re-recall the blood for HIV and wait for the results of the survey.

hello! The fact is that I had sexual contact with a girl 2 months ago (we still meet). After 1.5 weeks, the temperature of 37.4 rose. Soon he slept. For confidence, we have passed the analysis of the EIF in 2 weeks and re-after 1.5 months. Answers both negative. But I still have the temperature and cough with a variable improvement. Tell me, please, is it possible to risk? In addition, I worked for a long time without weekends and a week ago was on sick leave (ORVI). Blood test and lungs in order. Thank you.

This temperature may be associated with the transferred viral disease, the body has not recovered yet, or chronic overwork. In the event that the organic pathology is excluded, a general analysis of blood and urine within the norm, as well as the data of fluorographic research as well as within the norm, it is necessary to exclude sexually transmitted diseases: chlamydia, mycoplasmosis, ureaplasmosis that can cause inflammation of the small organs Pelvic and urethra and as a result, an increase in body temperature. Read more about the reasons for increasing body temperature. Read by clicking on the link: High temperature.

Hello. There was such a business - more than a year ago there was unprotected sexual contact with a walking girl. She assured that it was not sick, but I can't believe her 100 percent. She also assured that he was a medical examination before the device to work (she worked as a seller) and everything was fine. 7 months after contact, I still passed an analysis of HIV in the laboratory, the city abstract turned out to be negative. But lately I often became sick, for now 3 week, as I have a red inflamed throat and I can't cure him. Again began to be afraid, and suddenly still picked up then? Tell me, is it possible, and whether it is worth trusting the analysis from Sitilab? Once again I'm afraid, the nerves will not stand it ..

In the event that the result is negative, then most likely you are not sick and not infected with HIV / AIDS. However, to clarify the diagnosis, it is recommended to re-analyze in specialized laboratories under government agencies, this examination is conducted anonymously. In the event that independently the treatment does not bring the desired result. It is recommended to consult with a otolaryngologist, for adequate examination and appointing appropriate treatment. Read more about the examination for HIV, read the cycle of the articles by clicking on the link: HIV.

Tell me, can you give any characteristic of a satilab laboratory? All the same, it is not always possible to pass the analysis at the state institution. And what is the probability in percent for a man to get infected with unprotected contact?

Unfortunately, we do not give a comparative assessment of laboratories and private medical institutions. In the event that you doubt the reliability of the results, conduct a survey in another center and first ask the license to provide data from medical services, whether this center has the right to carry out this survey and whether all the standards adopted. The risk of infection is the same for both sexes with unprotected intercourse. Read more about the examination for HIV, read the cycle of the articles by clicking on the link: HIV.

Good day! The child was 8 months old, the analysis of HIV by the IFA method in the blood was detected by GP160 + and P25 + the rest of everything minus, the conclusion of ib-dubious. How judge on these analyzes it turns out that the child +? GP160 + GP110 / 120 - P68 - P55 - P52 - GP41 - P34 - P25 + P18 -

Unfortunately, on the basis of the data obtained, it is impossible to diagnose with 100 percentage probability, since the false positive result is not excluded. To form an accurate diagnosis, a number of surveys will need to pass, including to repeat this analysis by the IFA method, as well as pass the analysis according to the PCR technique. After that, you should contact a specialized medical institution, where the doctor infectious player will be able to estimate the results obtained in the complex. You can learn more about the manifestations of HIV infections in the thematic section of our site by clicking on the link: HIV

Can show a false positive result with the "ORZ" or more acutely occurring infectious diseases? Somewhere I read that at 58 diseases or even above can show "+" including vaccination from hepatitis B if the kidneys suffer from etc.?

The probability of a false positive result is, so I recommend you to do as follows: Make a re-analysis - by the IFA method and the PCR method, after which it is re-visited by an infectious doctor. Read more about the diagnosis of HIV infection you can learn from the thematic section: HIV

Good day! Immunoblot is unspecified due to protein P25. What is the probability of HIV?

In this situation, a thorough study of the study protocols in the complex with other indicators is necessary, since on the basis of these data it is not possible to make an assumption possible. Presumably the result can be considered doubtful and re-study is required after 3 months. Read more in the section of our website: HIV

Good day.
You can comment on IFA on HIV
1 serum +3,559 K \u003d 13.3
+2,121 k \u003d 4.9
r 24 Otr.
2 Serum +3,696 K \u003d 13.9
+2,477 k \u003d 5.7

In this case, a false positive result is not excluded, given that the IFA method is indirectly, so I recommend you to pass the analysis on another, more sensitive method - immune blotting. You can learn more detailed information on this issue in the relevant section of our site by clicking on the following link: HIV

Good afternoon, tell me what to configure? A year ago with her husband, when planning a child, all tests passed, including HIV (treated very seriously and correctly), I was examined in the Kyrgyz Republic. Husband In Kiev, he had a negative response to me that I did not work out what the reagent must be reused in the center AIDS in Kiev. Running the analysis in the center response came negative for me too. Now I'm in position for 14 weeks i.e. I get registered in passing all the tests and again the answer was the answer for HIV uncertain, re-passed in the clinic and went to the "Dovira" an express test for soothing, but did not reassure the express test showed a positive result (the second strip was less pronounced), immediately after all this Procedures I did not lose time turned into the AIDS Center also passed the analysis, I expect the result. (I can't calm down) I ask you to tell how to trust express analyzes and why is there no answer to HIV analysis from the first time? (My husband and I behave a healthy lifestyle and love each other). Thank you.

It is not necessary to panic ahead of time - express diagnostics is not a reason for the diagnosis of HIV, it allows you to identify groups of patients who need a more in-depth study. In such situations, it is recommended to conduct immune blotting and personally consult with an infectious doctor. You can learn more detailed information on this issue in the thematic section of our site by clicking on the following link: HIV. For more information, you can also get in the next section of our website: laboratory diagnostics

Hello, lay in an infectious time, only today was discharged when careing the doctor called me and explained that I had a positive ELISA, first when I entered the hospital, he was negative, then when he crossed him positive, they sent research on Immunoblot to Sokolniki Mount said will be ready to march the week, lay in the hospital with the angina and viruses of paragrippa, arrive in a shock, I still don't understand how to regard it, an extract for my clinic was also made up with an indication that IFA was found and lower than the Immunoblot in work, if tomorrow is discharged in In this discharge, everything will be labeled in this discharge, as far as the probability of HIV is present? Can it be due to the fact that I was treated from the angina of a paragripping virus show positive results on the ELISA?

The probability of a false positive result is very large. The presence of one positive result does not provide grounds for raising the diagnosis of HIV, so we recommend that you wait for the result of immunoblotting, after which it personally consult with the doctor infectious player on further surveys and observation. Angina, Paragripp and other colds of significant influence on the results of the analysis do not have.

i want to believe it, but at the end of August I had an ailment, the temperature rose, 37.5-38 was a liquid chair for about 4 days, it was on vacation where there was a lot of disco, I drank water from under the crane as much as many others, because that was a very expensive glass of water cost 300r, I connected a liquid chair with such a temperature with some kind of intestinal infection with a picked in the water, not remembering exactly but there was a small rash in the upper part of the body, coming home with a temperature called a doctor, she wrote a rotavirus infection, After 5 days of the physician, I volunteered to leave him and go to work where the run in a few days with a sinusitis, (at that time a period of time, I needed to be on the street) I tied it that a big temperature difference from vacation and poisoning praises my immunity and therefore came up once again with the sinusitis, and this is again sick-sized, as directed by Laura, Collid CP 500 was proposed within 10 days, passed, came out again to work after 3 weeks was on a business trip in Arch country for 3 days. Air conditioners in transport and the hotel were merciless and on returning home, in the plane I had a temperature already 39.5.The at home with a temperature of 40, called a doctor to the house wrote ARVI and said the thoughts of Pts Red, I have a chronicle tonsillitis and told it to Laura herself wrote to drink an antibiotic Levieveth R. Skoring Because I didn't leave the pace of 40 and did not fall, the hospitalization was not offered, on the next day the same History did the antipyretic injection and left. On the third time I insisted on hospitalization, barely barely In the infectious hospital, where the mixture infection was infected with paragrippa and adenoviral infection, but if the doctor was discharged, the branch reported that I had a positive HIV positive and what they did twice, I'm shocked, I don't know what to do I can not eat and drink . She said that I have a clearly expressed acute HIV infection and for checking they sent an analysis of my blood to Immunnoblot in AIDS Center,
now I conduct an analogy of events that happened to me for the last time, as well as 3 hospital sheets to a row, all the symptoms for ourselves and I'm terrified from what could be, after discharge on the same day, I went to pass an analysis in Invitro Ananino and On the next day the result of IFA was also +
i forgive forgiveness for such a detailed information But I'm in a shatter and killed, I drink severe sedatives and I don't have an appetite and I don't have it almost lost much
i still have such a question that the doctor with a discharge from the hospital pointed out the result of HIV on IFA was discovered and lower than the Imunnoblot in the work, but as I close the BL in my clinic at the place, everything will be written there. How can I be? This will not be confidential. I asked a doctor to do not write in the discharge this analysis to what she was refused to me, how much my rights about Nespras information are followed.

Unfortunately, the results of studies conducted in the hospital fit into an extract, since the attending district doctor should have information about your health status in full. In this situation, we are not talking about disclosing information, since it is only transmitted to another doctor who will further observe you.

Hello! I handed over HIV tests. Since I needed a certificate for the FMS, the tests did not give a couple of weeks later they were invited to the head and issued the result with the result, they took a bunch of receipts and sent a lot of AIDS to the regional center for further redemption. I want to pass in another clinic and then already go to the regional or it makes no sense? Just do not understand why they have so long they never carefully, the doctor said that allegedly they did some kind of analysis and I still have 4T rubles. How should I do it in addition to Daliba certificate detailed information about the disease?

In this situation, it should not be panicing ahead of time - the receipt of one positive result does not allow to judge reliably about possible infection, since false positive results are not excluded. We recommend that you pass the analysis again and if there is a positive result, you will need another survey - immunoblotting. As a rule, in the laboratory for detailed information about the results do not give, which is normal and ordinary practice. For all the questions that arise, you can answer the attending doctor after inspection on personal consultation.

i forgot to add that from the beginning of June until mid-September, I did an anabolic steroid course, namely Suston250 is a mixture of testosterone and Stanozolol with Primabolan, wanted to prepare himself for the summer and holiday, could they knock down my immunity and everything happened to me.

Immunity impairment, as well as the presence of autoimmune diseases, can give false positive results of HIV analysis. That is why in the case of receipt of 2 positive results, the IFA method recommends that immunobloting is recommended, which will allow you to answer the question with accuracy, there is infection or not.

what does autoimyluine disease mean? What are they?
in general, I can say that I often sick from early childhood and even a couple of three years ago, I asked the attending physician to do my immunity, because there was constant fatigue and often sick, mostly ear throat, but all the time there were negative results on HIV, I have all the time They passed with sufficient ease without thinking.

The false positive result of HIV analysis may be after a recently transferred viral infection, vaccination against hepatitis B, with tuberculosis, hepatitis, herpes, and on the background of autoimmune diseases, such as: rheumatoid arthritis, systemic red lupus, dermatomyomyomy, sclerodermia, connecting tissue diseases and etc.

I want to add to my question, my immunoblot came negative, but the doctor said that since two IFA were + when I was lying in an infectious hospital, then I still need to recall the analysis, but a little later

In this case, medical tactics are justified - we recommend after 1.5-2 months to pass the immunoblot again.

What is the probability of: 2 ELISA + the difference between blood fences is about 2 days, immunoblot -; I was lying in an infectious hospital with adenoviral infection and paragripp, where it was a blood fence, immunoblot sent to AIDS

Good day! I got registered in the LCD, passed all the tests of the doctor says that I have herpes in my blood, then call from AIDS and say that I need to rebound, I have been told, and I have been told that I have a positive HIV, I went to donate in a panic The analysis passed and I have an ELISA and immunoblot + her husband -, it was in a month again. I have + my husband - now I am on the 23 week of pregnancy!

In this situation, unfortunately, there is the likelihood of HIV infection, but the final diagnosis is impossible to put even with positive immunoblot, given the condition of pregnancy. In this situation, the elimination of false positive results is required, so we recommend to pass the analysis to re-and personally consult with the infectious doctor.

if immunoblot showed a positive result on HIV, and the screening is negative what result to believe?

Immunoblot is a more accurate study, so with this study, if a positive result was received, it is necessary to continue the study and personally visit the infectious examist.