The immune blot is positive. Immunoblotting hiv positive

Immune blotting (immunoblot, Western Blot, Western blot)- combines enzyme-linked immunosorbent assay (ELISA) with preliminary electrophoretic transfer of virus antigens to a nitrocellulose strip (strip).

In this beautiful scientific name, “blot” most likely translates as “blot”, and “western” as “western” reflects the direction of distribution of this “blot” on paper from left to right, that is, on a geographical map, this corresponds to the direction from west to east. ". The essence of the "immune blot" method is that the immunoenzymatic reaction is carried out not with a mixture of antigens, but with HIV antigens, previously distributed by immunophoresis into fractions located according to molecular weight on the surface of a nitrocellulose membrane. As a result, the main proteins of HIV, carriers of antigenic determinants, are distributed over the surface in the form of separate bands, which appear during the enzyme immunoassay.

Immunoblot includes several stages:

Strip preparation. The immunodeficiency virus (HIV), which has been previously purified and destroyed to its constituent components, is subjected to electrophoresis, while the antigens that make up HIV are separated by molecular weight. Then, by blotting (analogous to squeezing out excess ink on a "blotter"), the antigens are transferred to a strip of nitrocellulose, which now contains a spectrum of antigenic bands characteristic of HIV, invisible to the eye.

Sample study. The test material (serum, blood plasma of the patient, etc.) is applied to the nitrocellulose strip, and if there are specific antibodies in the sample, they bind to the strictly corresponding (complementary) antigenic bands. As a result of subsequent manipulations, the result of this interaction is visualized - made visible.

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

Lane A - Positive control

Lane B - Weak positive control

Lane C - Negative Control

Strip D - Positive sample (antibodies to HIV-1 detected)

Currently, immune blotting (immunoblot) is the main method for confirming the presence of virus-specific antibodies in the test serum. In some cases of HIV infection, before the development of seroconversion, specific antibodies are more effectively detected by the method immune blotting than ELISA. An immune blotting study revealed that antibodies to gp 41 are most often detected in the sera of AIDS patients, and the detection of p24 in persons examined for prophylactic purposes requires additional studies for the presence of HIV infection. Immunoblot test systems based on genetically engineered recombinant proteins proved to be more specific than conventional systems based on purified virus lysate. When using a recombinant antigen, not a diffuse, but a clearly defined narrow band of antigen is formed, which is easily accessible for accounting and evaluation.

Serum of persons infected with HIV-1, detect antibodies to the following major proteins and glycoproteins - structural envelope proteins (env) - gp160, gp120, gp41; nuclei (gag) - p17, p24, p55, as well as virus enzymes (pol) - p31, p51, p66. For HIV-2, antibodies to env are typical - gp140, gp105, gp36; gag - p16, p25, p56; pol-p68.

Among the laboratory methods necessary to establish the specificity of the reaction, the detection of antibodies to HIV-1 envelope proteins - gp41, gp120, gp160, and HIV-2 - gp36, gp105, gp140 has the greatest recognition.

WHO considers sera positive if antibodies to any two HIV glycoproteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (rp 160, rp 120, rp 41) in combination or without reaction with other proteins, the result is considered doubtful and a second test is recommended using a kit from another series or from another company. If after this the result remains doubtful, observation for 6 months is recommended (research after 3 months).

The presence of a positive reaction with the p24 antigen may indicate a period of seroconversion, since antibodies to this protein sometimes appear first. In this case, it is recommended, depending on the clinical and epidemiological data, to repeat the study with a serum sample taken at least 2 weeks later, and this is exactly the case when paired sera are necessary in HIV infection.

Positive reactions with gag and pol proteins without the presence of a reaction with env proteins may reflect the stage of early seroconversion, and may also indicate HIV-2 infection or a non-specific reaction. Individuals with such results after testing for HIV-2 are re-examined after 3 months (within 6 months).

Immunoblot includes several stages:

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

Lane A - Positive control

Lane B - Weak positive control

Lane C - Negative Control

Strip D - Positive sample (antibodies to HIV-1 detected)

Currently, immune blotting (immunoblot) is the main method for confirming the presence of virus-specific antibodies in the test serum. In some cases of HIV infection, prior to seroconversion, specific antibodies are more effectively detected by immunoblotting than by ELISA. An immune blotting study revealed that antibodies to gp 41 are most often detected in the sera of AIDS patients, and the detection of p24 in persons examined for prophylactic purposes requires additional studies for the presence of HIV infection. Immunoblot test systems based on genetically engineered recombinant proteins proved to be more specific than conventional systems based on purified virus lysate. When using a recombinant antigen, not a diffuse, but a clearly defined narrow band of antigen is formed, which is easily accessible for accounting and evaluation.

What is it - immunoblot? This is a common method laboratory diagnostics viral infections person. It is considered one of the most accurate and reliable ways to detect the presence of HIV. In its reliability, it surpasses even the results of the immunoblot are considered irrefutable and final.

general information

Immunoblot - what is it? In order to recognize an HIV infection in a person, it is necessary to undergo a laboratory test of blood serum for the presence of antibodies. The Western blot technique is also called Western blot. It is used to detect human viral infections as an additional expert method. It is necessary to confirm the ELISA - a laboratory test that allows you to determine the presence of HIV antibodies in the blood. A positive ELISA analysis is rechecked by immunoblotting. It is considered the most sensitive, complex and expensive.

purpose

What is it - immunoblot? This is a technique for laboratory testing of blood serum for the presence of antibodies to the virus. During the study, the specialist pre-separates the proteins of the virus in the gel and transfers them to a nitrocellulose membrane. The immunoblotting procedure is intended to determine HIV for different stages. At the first stage, the purified virus from its constituent parts is subjected to electrophoresis, and the antigens that make up its composition are separated by molecular weight.

It reproduces in a living cell, embedding its genetic information into it. At this stage, a person becomes a carrier of the HIV virus if he was infected. The specificity of the disease is that it may not manifest itself for a long time. The virus destroys lymphocytes, so the human immunity is reduced, and the body becomes unable to resist infections. If HIV is treated competently and on time, the patient will live to a ripe old age. Lack of therapy inevitably leads to death. From the moment of infection, but without treatment, the maximum life span is no more than ten years.

Peculiarities

Immunoblot analysis is a reliable method that allows you to determine the presence of antibodies to HIV antigens of the first and second types. If a person is infected, antibodies appear within two weeks, which can be detected much later. The peculiarity of HIV is that the number of antibodies increases rapidly and remains in the blood of the patient. Even if they are present, the disease may not manifest itself for two or more years. The ELISA method does not always accurately determine the presence of the disease, therefore, confirmation of the results using immunoblotting and PCR is required if the enzyme immunoassay is positive.

Indications for appointment

What is this "immunoblot" has already been found out, but who is prescribed this study? The reason to take tests for the method of immunoblotting is a positive ELISA result. It is necessary to go through enzyme immunoassay for patients who will be operated on. In addition, an analysis should be made for women planning a pregnancy, as well as for everyone who is promiscuous. sexual life. Assign immunoblotting to patients with HIV, if the results of ELISA are in doubt. The following alarming symptoms may be the reason for going to the doctor:

sharp weight loss;
weakness, loss of working capacity;
bowel disorder (diarrhea) that lasts for three weeks;
dehydration of the body;
fever;
increase lymph nodes on the body;
development of candidiasis, tuberculosis, pneumonia, toxoplasmosis, exacerbation of herpes.

The patient does not need to prepare before donating venous blood. 8-10 hours before the study, you can not eat. It is not recommended to drink alcoholic and coffee drinks, to engage in heavy physical exercises, to experience excitement a day before blood donation.

Where to do the analysis?

Where can I get tested for HIV? ELISA, immunoblot studies are carried out in urban private clinics, the results are issued within a day. Immediate diagnosis is also possible. In state medical institutions, ELISA tests and immunoblotting are carried out free of charge, in accordance with the legislation of the Russian Federation. Pregnant women, as well as patients who are to be hospitalized or undergo surgery, are required to be tested for infectious diseases.

How is the research done?

How is ELISA performed? Immunoblot positive/negative confirms or refutes the results of enzyme immunoassay. The research procedure is quite simple. The specialist conducts a venous blood sampling, in time it takes no more than five minutes. After sampling, the injection site should be disinfected and sealed with a plaster. The sampling is carried out on an empty stomach, so after the procedure it does not hurt to eat a bar of dark chocolate or drink a sweet hot drink.

In order to get a referral for a free analysis in the state medical institution you need to visit a general practitioner. In general, the immunoblot does not differ from other blood tests in terms of the method of sampling. The research methodology is simple. If a virus is present in a person's blood, the body begins to produce antibodies to destroy it. Each virus has its own set of antigen proteins. The detection of these antibodies is the basis of the Western blotting technique.

Price

How much does the analysis cost? Immunoblot for HIV does not apply to cheap research. On average, a screening examination by enzyme immunoassay costs from 500 to 900 rubles. Immunoblotting is a verification study, the cost of which is from three to five thousand rubles. More complex methods are much more expensive. For example, you will need to pay about 12,000 rubles for it.

Interpretation of the result

The most common methods for diagnosing HIV infection are enzyme immunoassay and immunoblot. They are used to determine the serum antibodies to the immunodeficiency virus. The presence of infection is usually confirmed by two tests: screening and confirmatory. The interpretation of the results of the study should be carried out by a doctor, he also makes a diagnosis and prescribes treatment. If the immunoblot is positive, this means that a virus is present in the human body.

A positive result should not be a reason for self-treatment, since each patient may have his own picture of the disease. Qualitative Analysis includes screening and verification. If the patient does not have a virus, then the result is indicated as “negative”. When detected by the screening method, an additional verification study is carried out. An immunoblot is an analysis that confirms or refutes a screening. If darkening appears on the test strip in certain areas (sites of protein localization), a diagnosis of HIV is made. If the results are in doubt, the tests are carried out within three months.

You can prevent infection with the immunodeficiency virus if you follow certain rules: avoid casual sex, use a condom during contacts, do not take drugs. If the disease is detected in a pregnant woman, it is important to strictly follow the recommendations of the attending physician, do not forget to undergo examinations for the presence of the virus.

In the practice of laboratory diagnostics of infectious diseases, there is sometimes a need to determine antibodies not to a pathogen in general, but to certain of its proteins (antigens), that is, a spectrum of specific antibodies. If for this purpose the method of enzyme-linked immunosorbent assay is used, then in this case it is necessary to isolate and purify the necessary antigens from the pathogen culture. The resulting proteins are applied separately to the solid phase. In the case of using a 96-well plate - in each well, one type of antigen. The specific antibodies are then determined by an indirect method.

By the presence of a positive reaction in the well with one or another antigen, one can judge the presence of the corresponding specific antibodies. This kind of enzyme immunoassay test systems are offered by manufacturers, however, due to the greater information content and ease of execution of the study itself, the method of immune blotting (Western blot) has become widespread.

Immune blotting makes it possible to determine antibodies in the blood serum simultaneously and at the same time differentially to all diagnostic tests. important proteins pathogen. Translation from English Western blot means western transfer (literally - blot). The history of this unusual term is as follows.

A scientist by the name of Southern (E. Southern) in 1975 first proposed a method for transferring electrophoretically separated DNA fragments from a gel to a membrane. According to the author, the method was called Southern blot, which means “southern transfer”. The method of transferring RNA molecules, in turn, was nicknamed Northern blot by specialists - “northern transfer”. At first as a joke, and then this name was fixed in the official scientific literature.

G. Toubin in 1979 published the results of the first experiments on protein blotting. In continuation of the traditions of "geographical" names of methods for the transfer of biological macromolecules, this method became known as "Western" transfer - Western blot.

At the first stage of this method, electrophoretic separation of a mixture of pathogen proteins in a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS) is carried out. SDS, being superficial active substance, uniformly envelops the protein molecules and gives them all a negative charge of approximately equal magnitude. Therefore, molecules move in an electric field in one direction, and the speed of movement depends only on the size of the molecule (molecular weight) of the protein.

As a result of the electrophoretic procedure, a gel plate is obtained, in the thickness of which proteins are located in the form of separate thin linear zones. In the direction of movement, they are divided in the following order: proteins of large molecular weight, about 120-150 kDa, are closer to the start, and proteins with a mass of 5-10 kDa have advanced further to the finish line. At the second stage, the gel plate is placed on a sheet of nitrocellulose and this structure is placed between the electrodes of the DC source. Under the action of an electric field, the proteins flow from the porous gel to a denser membrane, where they are firmly fixed.

The resulting blot is treated with a blocking solution containing antigenically indifferent proteins and/or non-ionic detergents (Tween 20), which block antigen-free sites on the membrane. The membrane sheet is then cut into narrow strips so that each strip contains all of the antigenic fractions. The described steps are carried out by the manufacturer.

Commercial test systems for the detection of antibodies by immune blotting contain blots (strips, or strips) ready for analysis. The user determines the entire spectrum of specific antibodies to pathogen proteins according to the scheme of the indirect method. As a chromogen for carrying out a color (enzymatic) reaction, a soluble colorless substance is used, the product of which acquires a color, becomes insoluble and settles (precipitates) on nitrocellulose.

As a result of sequential immune and enzymatic reactions, in the presence of antibodies to pathogen proteins in the test sample, dark transverse stripes appear on the blot, the location of which is in the zone of certain pathogen proteins. Each such band indicates the presence of specific antibodies to the corresponding antigen. The result of a study conducted by the method of immune blotting is issued in the form of a listing of antibodies to specific proteins of the pathogen. For example: "antibodies to p17 and p24 proteins were detected."

Nitrocellulose blots after development can be stored for a long time in a dried form. However, the color intensity is significantly weakened. Wet blots can be photographed or, using scanners, their graphic image can be entered into the memory of personal computers. Special computer programs allow you to process the results and quickly track the dynamics of the spectrum of antibodies during dynamic monitoring

Immunoblotting -(from the English "blot" - spot) - a method for identifying antigens (or antibodies) using known sera (or antigens). It is a combination of gel electrophoresis with ELISA. Initially, bacterial cells or virions are destroyed using ultrasound, and then all antigens of the virus or bacterial cells are separated by electrophoresis and a commercial reagent is obtained on a special nitrocellulose film. When setting up immunoblotting, the test serum of the patient is applied to the film with known antigens. After incubation and washing of unbound antibodies, they proceed to ELISA - an antiserum to human immunoglobulins labeled with an enzyme and a chromogenic substrate that changes color when interacting with the enzyme are applied to the film. In the presence of antigen-antibody-antiserum to immunoglobulin-enzyme complexes, colored spots appear on the carrier. The method of immunoblotting allows you to separately detect antibodies to various antigens of the pathogen (for example, in HIV infection, immunoblotting detects antibodies to gp120, gp24 and other antigens of the virus).

Radioimmunoassay (RIA)

The method is based on the antigen-antibody reaction using an antigen or antibody label with a radionuclide. 125I, 14C, 3H, 51Cr and other radionuclides are used as a label. The resulting immune complexes are isolated from the system and their radioactivity is determined on counters (β-radiation). The radiation intensity is directly proportional to the number of bound antigen and antibody molecules.

The solid-phase version of RIA using labeled antibodies or antigens adsorbed in the wells of polystyrene panels is widely used.

RIA is used to detect antigens of microbes, viruses, various hormones, enzymes, medicinal substances, immunoglobulins, as well as other substances contained in the test material in minor concentrations of 10–12–10–15 g/l.

test questions

Immune bacteriolysis reaction: what kind of reaction is it; what is an antigen, what is an antibody, reaction mechanism, staging methods, practical use. Immune hemolysis reaction: necessary ingredients, method of setting; controls, practical application. Reaction of local hemolysis in gel (Yerne reaction): principle of the reaction, method of formulation, practical application. Complement fixation reaction: reaction principle; what is formed when the immune serum interacts with a specific antigen; what happens to complement if it is present in this interaction? What is the fate of complement if there is no specific affinity between antigen and antibodies? What reaction can be used to determine what happened to the complement; why this reaction is used; what is the visible positive result of RSK? Why? What property of complement is used in the first phase of CSC? In the second phase? If the end result of RSK is hemolysis, does that 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 does the test serum need to be inactivated? How is complement titrated? Hemolytic serum: what does it contain, how is it obtained, what is a titer and how is it determined? What animals are used to obtain RSC components? The technique of setting RSC in the cold. When staging which of the following reactions, the participation of a complement is necessary: precipitation, flocculation, agglutination, detection of incomplete antibodies, immune bacteriolysis, immune hemolysis, Jerne, CSC? Immunofluorescence reaction (RIF) - indicate the sequence of events in the direct Coons reaction; the necessary ingredients. What is an antigen, what is an antibody, how are antibodies labeled, how is the result of the reaction taken into account, what does a positive result look like? Practical application - what can be determined using this reaction? Indirect immunofluorescence reaction - indicate the sequence of events in this reaction, the necessary ingredients, what is the antigen, what immune sera are used; practical use; advantage of indirect RIF over direct reaction. Linked immunosorbent assay(ELISA) - reaction principle; necessary ingredients; indicate the sequence of events when setting up a reaction in order to detect an antigen in the test material; necessary ingredients; what happens with a positive result, what does it look like? Specify the sequence of actions during ELISA in order to detect antibodies in the test serum; necessary ingredients; what happens with a positive result? Immunoblotting - the principle of the reaction; main steps; necessary ingredients; How is the result taken into account? reaction benefits. Radioimmunoassay (RIA) - what are the main stages of the reaction; what are antibodies or antigens labeled with, how is the result taken into account? Immunoelectron microscopy - the principle of the method; main steps; necessary ingredients; What are antibodies labeled with? how the result of the reaction is taken into account. Immobilization reactions - the principle of the method, the technique of setting, components, accounting for results.

Tasks to perform in the process of self-training.

Complete the Immunity Reactions table for the reactions covered in this topic.

Immune reactions

Student's work in a practical lesson

Start work immediately with the formulation of the 1st phase of the RSC, but write it down in a notebook later (see below).

1. The reaction of immune hemolysis. View a demonstration reaction of immune hemolysis, draw it as a diagram, explain the result in the experimental and control tubes.

2. Complement binding reaction

a) disassemble the RSC according to the table;

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

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

d) disassemble the diagnostic preparations necessary for the RSK;

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

3. Immunofluorescence reaction. Study the table, draw up a scheme for setting up the reaction in your notebook; see diagnostic sera; determine what the serum contains, how it is prepared, for which reaction (direct or indirect RIF) it is used. Look at the demonstration result of the RIF in a fluorescent microscope.

4. Enzyme immunoassay (ELISA). In your notebook, draw up a reaction scheme in two versions: for the detection of an antigen in the test material and for the detection of antibodies in the serum. Review the HIV and Hepatitis B Diagnosis Ingredient Kit. Determine what each ingredient contains and what it is used for.

5. Immunoblotting. Make a diagram of the reaction in your notebook; see the demo - the result of the reaction.

6. Radioimmunoassay (RIA). Draw the reaction scheme in your notebook.

7. Immune electron microscopy (IEM). Watch the demonstration - the result of the reaction, draw up a reaction scheme in your notebook, indicate the antigen (virus) and labeled antibodies with arrows.

Immunoblotting is a highly sensitive protein detection method based on a combination of electrophoresis and ELISA or RIA. Immunoblotting is used as diagnostic method with HIV infection, etc.

In a general sense, immunoblotting is understood as the analysis of a mixture of proteins transferred to a solid support-membrane, with which they bind by covalent bonds, followed by immunodetection.

It is possible to analyze a mixture of proteins directly deposited on a substrate (dot blot analysis) or after its preliminary fractionation by electrofocusing, disk electrophoresis, or two-dimensional electrophoresis (Western blotting).

Pathogen antigens are separated by polyacrylamide gel electrophoresis, then transferred from the gel to activated paper or nitrocellulose membrane and developed by ELISA.

Firms produce such strips with "blots" of antigens. The patient's serum is applied to these strips. . Then, after incubation, the patient is washed from unbound antibodies of the patient and serum against human immunoglobulins labeled with the enzyme is applied. . The complex formed on the strip (antigen + antibody of the patient + antibody against human Ig) is detected by adding a chromogenic substrate that changes color under the action of the enzyme.

This methodology is also applied to the selection of clones of bacteria, phages or viruses expressing the products of the target cloned genes.

Transfer of proteins to the membrane is carried out either passively or using electrotransfer apparatus. The efficiency of protein transfer to the membrane is affected by many factors, such as the molecular weight of the proteins, the porosity of the gel, the transfer time, and the composition of the buffer solution (trans buffer) used.

Depending on the tasks and conditions of the experiment, transfer conditions are selected that provide the best results. The substrates commonly used are nitrocellulose, polyvinylidene difluoride (PVDF), or positively charged nylon membranes. Nitrocellulose can bind up to 80-100 micrograms of protein per 1 cm2.

Low molecular weight proteins (with a molecular weight of less than 20 kDa) can be lost as a result of washings, which makes it possible to preliminarily study the polymorphism of certain genetic loci by the lengths of the corresponding restriction DNA fragments.

In addition, using Southern hybridization, it is easy to find out whether the target gene has a site of hydrolysis by a certain restriction enzyme in its internal part, which allows you to choose the optimal strategy for cloning the region of the genome under study.

According to a similar scheme, RNA molecules can also be transferred from agarose gel to a nitrocellulose filter. This method has been called Northern blotting as opposed to Southern blotting, since the surname Southern in English language means "southern".

The transfer to filters from the protein gel was accordingly called Western blotting. Large proteins (greater than 100 kDa) denatured in sodium dodecyl sulfate (SDS) solution may be poorly transferred to the membrane if ethanol is present in the trans buffer. Alcohol significantly improves the transfer of proteins from the SDS-polyacrylamide gel, but narrows the pores in the gel, which leads to the retention of large proteins.

The PVDF membrane is optimized for immunodetection and is able to retain specifically bound proteins up to 160 µg/cm2 with 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 retained effectively. With a high binding capacity of approximately 480 μg of protein per 1 cm2, Zeta-Probe membranes allow the detection of trace amounts of protein in assay mixtures.

After the antigen is immobilized on the membrane, the remaining binding sites are blocked with solutions of gelatin, or bovine serum albumin, or skim milk.

Then the membrane is incubated in a solution of polyclonal or monoclonal antibodies to the tested antigen. After washing off unbound antibodies, the membrane is incubated in a solution of secondary antibodies, which are a conjugate of alkaline phosphatase enzymes (alkaline phosphatase, AP) or horseradish peroxidase (HRP) with anti-species antibodies (goat antibodies to rabbit, mouse or human immunoglobulins) or proteins A (Staphylococcus aureus protein) or G (Streptococcus sp. protein) having a high affinity for the Fc region of immunoglobulins.

Detection of the formed immune complexes is carried out by chemical or chemiluminescent method. Substrates for chemical reaction when using alkaline phosphatase conjugates are 5-bromo-4-chloro-3-indolylphosphate (BCIP) or tetrazolium blue (NBT), and when using horseradish peroxidase conjugates - 4-chloro-1-naphthol and hydrogen peroxide.

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

The sensitivity of this method is 100 pg of protein when using AP conjugates and 100-500 pg when using HRP conjugates. Chemiluminescent detection of immune complexes can detect less than 5 pg of antigen. The principle of this method is that when HRP reacts with hydrogen peroxide and cyclic diacylhydrazineluminol, light is emitted at a wavelength of 428 nm, which can be recorded on a photosensitive film.

The immunoblotting reaction (RI) was developed on the basis of ELISA. It is the most specific and sensitive method of immunochemical analysis. Immunoblotting (from the English blot - to get wet, spot) combines ELISA with electrophoresis. It is used to detect not complex antibodies to HIV, but antibodies to its individual structural proteins (proteins-p24, glycoproteins-gp120, gp 41, etc.). Refer to expert (confirmatory) reactions for the diagnosis of HIV infection.

The reaction is carried out in several stages:

The virus is destroyed into components - antigens (p24, gp120, gp 41, etc.), which are subjected to electrophoresis in a polyacrylamide gel, that is, the separation of antigens into fractions by molecular weight.

2. The gel is covered with a nitrocellulose membrane and antigen fractions are transferred to it by means of electrophoresis. Nitrocellulose behaves like blotting paper. The membrane is cut into strips (strips). Firms produce such strips with "blots" of antigens.

Immunoblotting - an additional indirect method

Strips with HIV antigens applied to it are immersed in the serum of the subject and then washed from unbound material.

4. The strips are incubated with peroxidase-labeled antiglobulin serum and washed.

A substrate is added and the number of colored fractions (spots) is noted, which correspond to the localization zone of the AG-AT complex.

The presence of stripes in certain areas of the strip confirms the presence in the studied serum of antibodies to strictly defined HIV antigens. The result of immunoblotting is considered positive if bands corresponding to any two of the three HIV antigens - p24, gp41 and gp 120 are visible on the membrane (Fig. 37).

DRAWINGS

LIST OF USED LITERATURE

Main literature

Medical microbiology, virology and immunology: a textbook for medical students. universities 2nd ed., corrected. and additional — 702 p. Ed. A.A. Vorobyov. M. : MIA, 2012.

2. Microbiology, virology and immunology: a guide to laboratory studies: study guide / (V.B. Sboychakov et al.); ed. V.B.Sboychakova, M.M.Karapats. – M.: GEOTAR-Media, 2014.- 320 pp.: ill.

3. Medical microbiology, immunology and virology [Electronic resource]: a textbook for honey.

universities - 760 p. — Access mode: http://www.studmedlib.ru/book/ISBN9785299004250.html Korotyaev A.I., Babichev S.A. St. Petersburg: Spetslit, 2010.

4. Medical microbiology, virology and immunology [Electronic resource]: textbook: in 2 volumes / V. 1. - 448 p. — Access mode: http://www.studmedlib.ru/book/ISBN97859704142241.html Zverev V.V., Boychenko M.N.

M. : Geotar Media, 2010. .

5. Medical microbiology, virology and immunology [Electronic resource]: textbook: in 2 volumes. Vol. 2. - 480 p. Access mode: http://www.studmedlib.ru/book/ISBN97859704142242.html Zverev V.V., Boychenko M.N. M. : Geotar Media, 2010.

additional literature

1. Immunodiagnostic reactions: tutorial/ compiled by: G.K. Davletshina, Z.G. Gabidullin, A.A. Akhtariyeva, M.M.

Khusnarizanova, Yu.Z. Gabidullin, M.M. Alsynbaev - Ufa: Publishing House of GBOU VPO BSMU of the Ministry of Health of Russia, 2016. - 86p.

2. Features of some properties that determine the pathogenic potential of co-cultivated variations of Enterobacter, Сitrobacter, Serratia, E. coli, Proteus bacteria: scientific publication / Yu. Z. Gabidullin, R. S. Sufiyarov, I. I. Dolgushin - Ufa, 2015. - 250 s.

Home » Immunoblot - what is it? Immunoblot in diagnostics infectious diseases

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

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

For reliability, it is even larger than the enzyme-coupled immunosorbent (elisa) assay. Immunoblot results are considered conclusive and conclusive.General Information

Immunoblot - what is it? In order to recognize a person as HIV positive, you must undergo a laboratory test to test for the presence of antibodies in the blood serum.

The method of Western blot sections is 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 - a laboratory test that allows you to determine the presence of antibodies to HIV in the blood. Immunoblot recheck positive ELISA.

It is considered the most sensitive, complex and expensive.

Target

What is an immunoblot? This technique laboratory research blood serum for the presence of antibodies to the virus.

During special studies of the total viral proteins in the gel and on nitrocellulose membranes.

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

The Western blot section procedure is intended to determine HIV infection at different stages. In the first step, the purified virus from its constituent parts is subjected to electrophoresis and the antigens included in it, divided by the molecular weight.

The human immunodeficiency virus replicates in living cells embedded in its genetic information. At this stage, the person becomes a carrier of the HIV virus if you have been infected.

The specificity of this disease is that it does not manifest itself for a long time. The virus destroys lymphocytes, thus, a person's immunity decreases and the body becomes unable to fight infections.

If HIV is treated correctly and in a timely manner, the patient will live to a ripe old age. Lack of treatment inevitably leads to death. From the moment of infection, but without treatment, the maximum period is not more than ten years.

Peculiarities

Immunoblot analysis 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, which can be detected much 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 itself for two or more years. The ELISA method does not always accurately indicate the presence of the disease, requiring confirmation of the results from PCR and Western blot sections if the enzyme immunoassay showed a positive result.

Indications for

What kind of “immunoblot” has already been found out, but which is being introduced in the study?

The reason for testing for the human immunodeficiency virus (HIV) immunoblot will be a positive result ELISA. It is necessary to go through an enzyme-linked immunosorbent assay in patients who are about to undergo surgery. In addition, you should make an analysis of women planning pregnancy, as well as those who are promiscuous. Western blot sections are given to patients with HIV infection if elisa results are questionable.

The following alarming symptoms may be the reason for going to the doctor: rapid weight loss; weakness, loss of function; intestinal disorders (diarrhea), which lasts three weeks; dehydration; fever; swollen lymph nodes in the body; development of candidiasis, tuberculosis, pneumonia, toxoplasmosis, exacerbation of herpes.

The patient does not need to prepare before donating venous blood.

Do not eat for 8-10 hours before the test. It is not recommended the day before blood donation to drink alcohol and coffee drinks, heavy exercise to experience excitement.

Where to do the analysis?

Where can I get tested for HIV?

ELISA, an immunoblot analysis carried out in urban private clinics, gives results within a day. Immediate diagnosis is also possible. in public institutions, medical tests elisa and Western blot section for free, in accordance with the legislation of the Russian Federation.

Mandatory screening for infectious diseases of pregnant women, and patients requiring hospitalization or surgery. How is this study carried out?

How to conduct an ELISA? Immunoblot positive/negative confirms or refutes elisa results. The procedure is quite simple. The specialist takes venous blood, in time it takes less than five minutes.

After sampling, the injection site should be disinfected and sealed with a plaster. The sampling is carried out on an empty stomach, so after the procedure it does not hurt to eat a bar of dark chocolate or a sweet hot drink.

To get a referral for a free analysis at a public medical institution, you must visit a therapist.

In general, the immunoblot does not differ from other blood tests by sampling. The research methodology is simple. If a virus is present in a person's blood, the body begins to produce antibodies to destroy it. There are many protein antigens for each virus. The detection of these antibodies is the basis of the Western blot sectioning method. Price

How much analysis? Immunoblot HIV refers to cheap research.

On average, screening immunoassay methods range from 500 to 900 rubles. Western blot sections is a study check, 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), you will need to pay about 12,000 rubles.

Interpretation of results

The most common methods for diagnosing HIV infection are enzyme immunoassay and immunoblot.

They are for the determination of serum antibodies to the human immunodeficiency virus. Infection is usually confirmed by two tests: screening and confirmatory. The interpretation of the results should be made by a doctor, he diagnoses and prescribes treatment. If the immunoblot is positive, it means that human body virus.

A positive result should not be a reason for self-treatment, since each patient may have his own picture of the disease.

Qualitative analysis includes screening and certification. If the patient does not have the virus, the result is “negative”. When this certificate is found, additional screening tests are carried out. Immunoblot analysis that confirms or refutes screening. If the test strips appear in the darkening of certain areas (protein localization), the diagnosis is "HIV".

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

To prevent infection with the human immunodeficiency virus, it is possible if you follow certain rules: avoid casual sexual contact, use condoms during contact, 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 tests for the presence of the virus.

What is Western Blotting?

Protein identification in complex mixtures or extracts of various tissues is one of the frequently encountered problems. Using such a tool as specific antibodies, it is possible to determine the protein under study with a minimum of time and financial costs.

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

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

In the binding of the protein-SDS complex to the nitrocellulose membrane, mainly electrical forces are involved, and this interaction is multipoint and leads to the "spreading" of proteins on the membrane surface. Thus, after electrotransfer, we obtain a replica of the gel on nitrocellulose with proteins arranged in the same way as in the polyacrylamide gel.

After SDS - electrophoresis, electrotransfer and sorption of proteins from the gel onto a nitrocellulose membrane, the tertiary conformation of the protein is greatly changed, if it is generally correct to speak of the existence of a tertiary structure for the protein after such a harsh treatment. Therefore, for the immunochemical detection of the protein under study, only mono- or polyclonal antibodies specific to the linear regions of the protein molecule are usually used.

51. Enzyme immunoassay, immunoblotting. Mechanism, components, application.

Antibodies specific for conformational epitopes (or sites involving intersubunit contacts) are generally not suitable for use in the Western blot method.

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

1 A). In the case when primary antibodies specific to the studied antigen are directly conjugated with the label, secondary antibodies are not required (Fig. 1 B). The immune complexes formed at the site of the studied protein localization are “manifested” with the help of a chromogenic substrate (depending on the type of label).

The sensitivity and specificity of the method is highly dependent on which antibodies are used in the study.

The antibodies used must be specific to a unique amino acid sequence specific only to the protein under study. Otherwise, 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 bands on the membrane.

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

The second important factor to keep in mind when choosing antibodies is affinity. The higher the affinity of the antibodies used, the brighter and clearer the protein bands stain, the higher the sensitivity of the method. When using high affinity antibodies, sensitivity of 1 ng and even higher can be achieved.

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

Usually, an enzyme (peroxidase or phosphatase) is used as such an agent, the reaction product of which has a color and precipitates on the membrane in the form of an insoluble precipitate.

also in this method fluorescent labels may be used.

Rice. 1. Scheme of immunochemical staining of the studied protein: A - using secondary antibodies conjugated with an enzyme label; B - the primary antibody is directly conjugated with an enzyme label.

Protocol:

I. Gel and membrane preparation and protein electrotransfer

The polyacrylamide gel after electrophoresis was placed in a bath with blotting buffer (25 mM Tris, pH 8.3, 192 mM glycine, 10% ethanol).

Two sheets of filter paper, cut to the shape of the blotting cassette and moistened with blotting buffer, are placed on the part of the cassette that will face the anode. Then, a nitrocellulose membrane pre-moistened with the same buffer is placed on the filter paper, making sure that there are no air bubbles between the membrane and the paper.

After that, the gel should be carefully placed on the membrane, again turning Special attention for the absence of air bubbles between the gel and the membrane. The sandwich is completed by two layers of wetted filter paper, which are placed on the surface of the gel (Fig. 2). The resulting sandwich is clamped in the cassette and placed between the electrodes so that the membrane faces the anode.

Rice. 2. Scheme of electrotransfer of proteins to the membrane.

II. electrotransfer

Electrotransfer of proteins to a nitrocellulose membrane is 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 electrotransfer time depends on the size of the transferred proteins, the larger the protein, the longer the electrotransfer takes. The quality of electrotransport and the arrangement of protein bands is assessed by staining the nitrocellulose membrane with a 0.3% solution of Ponceau S in 1% acetic acid. Before immunostaining, the membrane should be washed several times with a mildly alkaline aqueous Tris solution to remove protein-bound dye.

III. Immunochemical staining of proteins immobilized on a nitrocellulose membrane

To block non-specific antibody binding sites, the membrane is incubated with constant stirring at room temperature for 30 min in PBST (for better blocking, a PBST solution containing 10% skimmed milk powder can be used).

After blocking, the membrane is incubated for one hour at room temperature with 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 the antigen.

At the end of the incubation, the membrane was washed 5 times with PBST and transferred to a solution of secondary antibodies conjugated with horseradish peroxidase. The dilution of the conjugate is usually indicated by the manufacturer on the packaging, or is selected empirically by the researcher. Incubate the membrane in a solution of secondary antibodies for 1 hour with constant stirring.

After thorough washing (at least 5-6 buffer changes), the PBST membrane is transferred into a chromogenic substrate solution containing 3 mg diaminobenzidine (DAB) and 10 µl of 30% hydrogen peroxide in 10 ml of 0.1 M Tris-HCl, pH 7.6.

Incubation is carried out with stirring for 5 to 10 minutes. After the end of incubation with the substrate, the membrane should be washed with PBST, dried by blotting with filter paper, and immediately made an electronic copy by scanning in color. If the membrane dries out completely, the dyed protein stripes fade, and the image is less bright and contrasty.

Note: DAB is toxic and a potential carcinogen. Work only with rubber gloves!