In Russia, at present, the standard procedure for laboratory diagnosis of HIV infection is detection of antibodies to HIVusing enzyme immunoassay followed by confirmation of their specificity in the reaction immune blotting.

Antibodies to HIV appear in 90-95% of those infected within 3 months after infection, in 5-9% - after 6 months from the moment of infection, and in 0.5-1% - at a later date. The earliest time for the detection of antibodies is 2 weeks from the moment of infection.

Detection of antibodies to HIV involves 2 stages. At the first stage the total spectrum of antibodies against HIV antigens is detected using various tests: enzyme immunoassay, agglutination, combined, comb, membrane-filter or membrane-diffuse. At the second stage immunoblotting is used to determine antibodies to individual proteins of the virus. In the work, it is permissible to use only test systems that have permission for use by the Ministry of Health of the Russian Federation. Diagnostic procedures should only be carried out in accordance with approved instructions for the use of the appropriate tests.

Blood sampling is made from the cubital vein into a clean, dry test tube in an amount of 3-5 ml. Cord blood can be taken from newborns. The obtained material (whole blood) is not recommended to be stored for more than 12 hours at room temperature and for more than 1 day in a refrigerator at 4-8°C. The upcoming hemolysis may affect the results of the analysis. Serum is separated by centrifugation or by tracing blood along the wall of the test tube with a Pasteur pipette or glass rod. The separated serum is transferred into a clean (preferably sterile) test tube, vial or plastic container, and in this form it can be stored for up to 7 days at a temperature of 4-8°C. When working, you should follow the safety rules given in the "Instructions on the anti-epidemic regime in AIDS diagnostic laboratories" No. 42-28 / 38-90 dated July 5, 1990.

Determination of total antibodies to HIV.

Upon receipt of the first positive result, the analysis is carried out 2 more times (with the same serum and in the same test system). If at least one positive result was obtained (two positive results out of three ELISA tests), the serum is sent to the reference laboratory.

In the reference laboratory, the primary positive sera (that is, that gave two positive results in the first test system) is re-examined in the ELISA in the second (other) test system selected for confirmation.

Upon receipt of a positive result of the analysis in the second test system, the serum must be examined in the IS.

If a negative result is obtained in the second test system, the serum is re-examined in the third test system.

If a negative test result is obtained in both the second and third test systems, a conclusion is issued on the absence of antibodies to HIV.

When a positive result is obtained in the third test system, the serum is also sent for analysis in immune blotting.

immune blotting.

The principle of the method is to detect antibodies to certain proteins of the virus immobilized on a nitrocellulose membrane. The envelope proteins (env) of HIV-1 are commonly referred to as glycoproteins ("gp" or "gp"), with molecular weights expressed in kilodaltons (cd): 160 kd, 120 kd, 41 kd. In HIV-2 glycoproteins have a weight of 140 kd, 105 kd, 36 kd. Core proteins (gag) (commonly referred to as proteins - "p" or "r") in HIV-1 have a molecular weight of 55 kd, 24 kd, 17 kd, respectively, and HIV-2 -56 kd, 26 kd, 18 kd. Enzymes HIV-1 (pol) have a molecular weight of 66 kd, 51 kd, 31 kd, HIV-2-68 kd.

Immunoblotting results are interpreted as positive, indeterminate, and negative.

positive(positive) are considered samples in which antibodies to 2 or 3 HIV glycoproteins are detected.

negative(negative) are sera that do not detect antibodies to any of the antigens (proteins) of HIV.

Samples that detect antibodies to one HIV glycoprotein and/or any of the HIV proteins are considered dubious(undefined or uninterpretable).

When an indeterminate result is obtained with antibodies to the core proteins (gag) in the immune blot with HIV-1 antigens, a test with HIV-2 antigens is performed.

Upon receipt of positive results of immune blotting, a conclusion is made about the presence of antibodies to HIV in the test material.

Upon receipt of a negative test result, the IB issues a conclusion that there are no antibodies to HIV.

Upon receipt of an indeterminate result (if the p24 antigen was not detected), repeated tests for antibodies to HIV are carried out after 3 months,

and while maintaining indeterminate results after another 3 months. If the p24 antigen was detected, a second examination is carried out 2 weeks after receiving the first indeterminate result.

If, 6 months after the first examination, indeterminate results are again obtained, and the patient does not have risk factors for infection and clinical symptoms of HIV infection, the result is regarded as a false positive. (If there are epidemiological and clinical indications, serological studies are repeated as prescribed).

Immune blotting using recombinant virus-specific polypeptides "HIV blot" is distinguished by the fact that it does not use the viral proteins themselves, but recombinant polypeptides - analogues of HIV antigens ("Env1", "Gag1", "Poll", "Env2"). The recombinant Env1 polypeptide detects antibodies directly to HIV-1 gp120 and gp41, the Gag1 polypeptide to the p17 and p24 antigens, the Po11 polypeptide to the p51 antigen, the Env2 polypeptide to the HIV-2 gp110 and gp38 antigens. Serum is considered positive if it reacts with either Env1 or Env2 or both Env (HIV type 1 and 2 dual infection). Reaction with only Poll and Gag is considered as an indeterminate result, in which case the follow-up is carried out similarly to the cases of indeterminate results of a classic immunoblot using HIV lysate.

Peculiarities of serological diagnosis of HIV infection in children born from HIV-infected mothers is that both infected and uninfected children in the first 6-12 months of life have antibodies to HIV of maternal origin, which can then disappear. The criterion indicating the presence of HIV infection in a child is the detection of antibodies to HIV at the age of 18 months or more. The absence of antibodies to HIV in an 18-month-old child born to an HIV-infected mother is a criterion against HIV infection.

Description

Method of determination Immunoblot.

Material under study Serum

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Antinuclear antibodies are a family of autoantibodies that bind to ribonucleic acids and their associated proteins. They occur in more than 90% of patients with diffuse connective tissue diseases, and are also often observed in autoimmune liver diseases and a number of other conditions. To date, about 200 varieties of this family of autoantibodies have been characterized, but not all of them can be used in clinical practice.

The immunoblot of antinuclear antibodies allows simultaneously conducting a study of 15 main types of antinuclear antibodies in one test, which ensures the differential diagnosis of the main systemic rheumatic diseases. Each type of autoantibody detected by immunoblot is usually observed in patients with a characteristic clinical picture, so the spectrum of autoantibodies allows not only to diagnose the disease, but also to establish the risk of developing certain clinical manifestations.

An immunoblot of antinuclear antibodies is advisable to use at the second stage of a serological examination with a positive result of other tests indicating the presence of antinuclear antibodies in the serum of the subject. These tests include the determination of antinuclear antibodies (ELISA screening), the detection of antinuclear factor (ANF) on Hep2 cells (), antinuclear antibodies and antibodies to the extractable nuclear antigen (ENA,).

The immunoblot method of antinuclear antibodies in the diagnosis of systemic rheumatic diseases is characterized by high clinical specificity. But the specificity of antinuclear antibodies, even at high titers of ANF (), is not always possible to establish, since a number of antinuclear antibody antigens still remain uncharacterized. A negative immunoblot result in this case does not exclude the diagnosis of systemic rheumatic diseases. A number of antinuclear antibodies can be detected using an immunoblot - a panel of myositis-specific autoantibodies () and an immunoblot - a panel of autoantibodies in scleroderma ().

Literature

1. Lapin S.V. Totolyan A.A. Immunological laboratory diagnostics of autoimmune diseases / Publishing house "Chelovek", St. Petersburg - 2010. 272 ​​p.
2. Nasonov E.L., Aleksandrova E.N. Modern standards for laboratory diagnosis of rheumatic diseases. Clinical guidelines / BHM, M - 2006.
3. Conrad K, Schlosler W., Hiepe F., Fitzler M.J. Autoantibodies in Organ Specific Autoimmune Diseases: A Diagnostic Reference/ PABST, Dresden - 2011. 300 p.
4. Conrad K, Schlosler W., Hiepe F., Fitzler M.J. Autoantibodies in Systemic Autoimmune Diseases: A Diagnostic Reference/ PABST, Dresden - 2007. 300 p.
5. Gershvin ME, Meroni PL, Shoenfeld Y. Autoantibodies 2nd ed./ Elsevier Science - 2006. 862 p.
6. Shoenfeld Y., Cervera R, Gershvin ME Diagnostic Criteria in Autoimmune Diseases / Humana Press - 2008. 598 p.
7. Reagent Kit Instructions.

Training

It is preferable to withstand 4 hours after the last meal, there are no mandatory requirements.

Indications for appointment

The test is indicated for the diagnosis and differential diagnosis of the following conditions:

• systemic lupus erythematosus;
• subacute cutaneous lupus and other types of cutaneous lupus;
• mixed connective tissue disease;
• Sjögren's syndrome and associated diseases;
• diffuse and localized scleroderma, CREST syndrome;
• inflammatory myopathies (polymyositis and dermatomyositis);
• juvenile chronic arthritis;
• autoimmune hepatitis;
• primary biliary cirrhosis and sclerosing cholangitis;
• the use of this test is indicated when detecting high titers of antinuclear factor, antinuclear antibodies, antibodies to extractable nuclear antigen, antibodies to DNA, antibodies to nucleosomes and antiphospholipid antibodies.

Interpretation of results

The interpretation of test results contains information for the attending physician and is not a diagnosis. The information in this section should not be used for self-diagnosis or self-treatment. An accurate diagnosis is made by the doctor, using both the results of this examination and the necessary information from other sources: history, results of other examinations, etc.

Units of measurement: qualitative test, the result is presented in the form of "detected" or "not found".

When a band characterizing the presence of any type of antibody is detected, the color intensity of the band is additionally described by the number of pluses ("crosses") for each of the identified types of antibodies. An increase in the degree of positivity indirectly reflects the content and affinity of autoantibodies.

Reference values: antibodies to Sm, RNP/Sm, SS-A (60 kDa), SS-A (52 kDa), SS-B, Scl-70, PM-Scl, PCNA, CENP-B, dsDNA, Histone, Nucleosome , Rib P, AMA-M2, Jo-1 were not found.

The result of the determination of autoantibodies is presented in "crosses" for each corresponding antigen. An increase in the degree of seropositivity indirectly reflects the content and affinity of autoantibodies. The seropositivity score options are listed below:

1. Antibodies were not found.
2. +/- - borderline result;
3. + - low content of autoantibodies to a specific antigen;
4. ++ is the average content of autoantibodies to a specific antigen;
5. +++ - high content of autoantibodies to a specific antigen.

The main diseases associated with the detection of antinuclear antibodies:

Antigen	Meaning
Sm (Smith)	Specific marker for systemic lupus erythematosus (included in the 10th criterion for SLE of the American College of Rheumatology, ACR)
SS-A (Ro52)	It is noted in various autoimmune diseases, more often in systemic lupus erythematosus and its skin forms, systemic rheumatic diseases, rheumatoid arthritis, autoimmune liver diseases, etc.
SS-A (Ro60)	Systemic lupus erythematosus, cutaneous forms of lupus erythematosus, photosensitivity in systemic lupus erythematosus, high risk of congenital lupus erythematosus and fetal heart disease. The main serological indicator in Sjögren's syndrome. It is often noted together with antibodies to the SS-A (Ro52) antigen.
SS-B	Sjögren's syndrome, systemic lupus erythematosus.
PCNA	Systemic lupus erythematosus, risk of lupus nephritis.
Ribosomes (Ribo P)	Systemic lupus erythematosus, risk of damage to the central nervous system.
Nucleosomes	Systemic lupus erythematosus, high risk of lupus glomerulonephritis.
double stranded DNA	Specific marker of systemic lupus erythematosus (included in the 10th criterion of SLE ACR), high risk of lupus nephritis.
snRNP/Sm	Mixed connective tissue disease, systemic lupus erythematosus with a low risk of kidney damage, scleroderma.
Histones	Systemic lupus erythematosus, drug-induced lupus, scleroderma.
Scl-70	Systemic sclerosis with diffuse lesions of the skin and internal organs.
PM-Scl	Scleroderma with polymyositis.
CENP-B	CREST syndrome with sclerodactyly, telangiectasias, subcutaneous calcifications, Raynaud's syndrome, esophagitis.
Jo-1	Polymyositis in the form of antisynthetase syndrome.
AMA-M2	Primary biliary cirrhosis, Sjögren's syndrome.

Immunoblotting (immunoblot) is a highly specific and highly sensitive reference method that confirms the diagnosis for patients with positive or indeterminate test results obtained incl. using RPHA or ELISA .

This method of detecting antibodies to individual antigens of the pathogen is based on ELISA on nitrocellulose membranes, on which specific proteins are applied in the form of separate bands, separated by gel electrophoresis. If there are antibodies against certain antigens, a dark line appears at the corresponding strip locus. The uniqueness of the immunoblot lies in its high information content and reliability of the results obtained.

Research material is human serum or plasma. For research on one strip, 1.5-2 ml of blood or 15-25 µl of serum is required.

According to the WHO recommendation, immunoblotting (western blot) is used in the diagnosis of HIV infection as an additional expert method, which should confirm the results of ELISA. This method is usually used to double-check a positive ELISA result, as it is considered more sensitive and specific, although more complex and expensive.

Immunoblotting combines enzyme immunoassay (ELISA) with preliminary electrophoretic separation of virus proteins in a gel and their transfer to a nitrocellulose membrane. The immunoblot procedure consists of several stages. First, pre-purified and destroyed to its constituent components, HIV is subjected to electrophoresis, while all the antigens that make up the virus are separated by molecular weight. Then, by blotting, the antigens are transferred from the gel to a strip of nitrocellulose or a nylon filter, which now contain a spectrum of proteins that is characteristic of HIV, invisible to the eye. Next, the test material (serum, blood plasma of the patient, etc.) is applied to the strip, and if there are specific antibodies in the sample, they bind to the strips of antigen proteins that strictly correspond to them. As a result of subsequent manipulations (like ELISA), the result of this interaction is visualized - made visible. The presence of stripes in certain areas of the strip confirms the presence in the studied serum of antibodies to strictly defined HIV antigens.

Immunoblotting is most commonly used to confirm the diagnosis of HIV infection. The WHO considers sera positive if antibodies to any two of the HIV envelope proteins are detected by immunoblotting. According to these recommendations, if there is a reaction with only one of the envelope proteins (gp160, gp120, gp41) in combination or without reaction with other proteins, the result is considered doubtful and a second study is recommended using a kit from a different series or from another company. If after that the result remains doubtful, the studies continue every 3 months.

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 to whom is this study prescribed? The reason to take tests for the human immunodeficiency virus (HIV) by 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 sexually promiscuous. 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;
• 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. 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.

How is the research done?

From the point of view of the patient, the immunoblot is no different from any other analysis: venous blood is taken, examined and the result is obtained. But if you go into a little more detail about the technique, then it is not very simple, but still try to figure it out.

First, a “reference” human immunodeficiency virus is taken at the reagent manufacturing plant. Then, using a special procedure (electrophoresis) in a gel medium, the virus is destroyed to its smallest components: proteins (virus antigens). Then, using the blotting itself (from the English wetting), the particles are placed on a special material - nitrocellulose or a nylon filter, a ready-to-use indicator, the so-called strip, is obtained. A strip is a strip in which antigens are distributed depending on their molecular weight, in a clear sequence, that is, a certain protein corresponds to each millimeter of paper.

As you may know, if viruses are present in human blood, then the body begins to produce antibodies against their shells (certain proteins), and each virus has its own individual set of antigen proteins. The detection of antibodies to antigen proteins in the blood is the basis of the immunoblot method. After all, if an antibody collides with an antigen, then they will certainly interact with each other - they “stick”.

So, the antigens are on the strip strip and if there are suitable antigens in the blood of the test subject, they will necessarily interact with each other, and in this place, on the strip strip, an indicator will appear - a flat will appear (like a pregnancy test). Moreover, in a specific place of the strip, in this way the doctor will understand whether there is a set of proteins characteristic of a particular virus in the blood.

So, for example, if there is a darkening on the strip in the places of protein localization gp160, gp120, gp41 HIV is diagnosed, for other viruses it will be a completely different set of proteins.

It should be noted that the immunoblot allows you to accurately determine the presence of the 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 this is 100% HIV infection. But if at least one is missing, for example: gp41 is absent, but there are only gp160, gp120, then the test is regarded as doubtful and requires repetition.

FAQ

What are the stages of immunoblot?

1. 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 that is invisible to the eye.
2. 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.
3. 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
• Stripe D - Positive sample (antibodies to HIV-1 detected)

How to decipher the analysis?

If the ELISA showed the presence of all or almost all antibodies to antigens according to this test system, this indicates a positive HIV test. If the response after the second serological enzyme immunoassay is positive, then an immunoblot should be performed. The interpretation of his results will be more correct. If the enzyme-linked immunosorbent assay gave a positive result, the next immunoblot analysis also showed the presence of HIV, then the final result is put.

When analyzes are deciphered, you need to know that a positive HIV test is determined by:

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

If the response to HIV is positive, this will mean that antibodies to the virus have been detected. To avoid a false positive answer, it is necessary to re-test, preferably twice. If antibodies to immunodeficiency are detected when passing two tests out of two or when passing 3 tests in 2 of them, then it is considered that the result is positive.

The p24 antigen can be detected in the blood as early as 14 days from the date of infection. Using the enzyme immunoassay method, this antigen is detected from 14 to 56 days. After 60 days, it is no longer in the blood. Only when AIDS is formed in the body does the re-growth of this p24 protein in the blood occur. Therefore, enzyme immunoassay test systems are used to detect HIV in the first days of infection or to determine how the disease progresses and monitor the treatment process. The high analytical sensitivity of enzyme immunoassay detects the p24 antigen in the biological material for HIV of the first subtype at a concentration of 5 to 10 pg/ml, for HIV of the second subtype from 0.5 ng/ml or less.

Under dubious the result of an enzyme immunoassay implies that the diagnosis was mistaken somewhere, as a rule, medical workers mixed up something, or the person has signs of infection, and the result is negative, which causes suspicion, the person is sent for re-testing.

Under false positive The result is understood as the result when blood tests were taken under the following conditions of the patient:

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

How to decipher the analysis in this case? A false positive result is given if at least one protein is detected. Due to the fact that the p24 antigen is very dependent on individual variations, using this method, from 20% to 30% of patients are detected in the first period of infection.

How reliable is a positive test result?

Sometimes ELISA has false positive results (in about 1% of cases), the reason for this result can be pregnancy, various viral infections, as well as a simple accident. After receiving a positive result, a more accurate test is needed - an immunoblot, according to the results of which a diagnosis is made. A positive immunoblot result after a positive ELISA is 99.9% reliable - this is the maximum accuracy for any medical test. If the immunoblot is negative, then the first test was false positive, and in fact the person does not have HIV.

What is an indeterminate (doubtful) result?

If the ELISA is positive or negative, then the immunoblot can be positive, negative, or indeterminate. Indeterminate immunoblot result, i.e. the presence in the immunoblot of at least one protein to the virus can be observed if the infection has occurred recently and there are still few antibodies to HIV in the blood, in which case the immunoblot will become positive after a while. Also, an indeterminate result may appear in the absence of HIV infection with hepatitis, some chronic metabolic diseases, or during pregnancy. In this case, either the immunoblot will become negative or the cause of the indeterminate result will be found.

How much does the analysis cost?

Immunoblot for HIV does not apply to cheap research. On average, a screening examination by immunoenzyme methods 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, for the analysis of the polymerase chain reaction (PCR), you will need to pay about 12,000 rubles.

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.

hypotension, tachycardia, shortness of breath, cyanosis. In severe cases of the disease, bleeding, vomiting with an admixture of blood are possible. The liver and spleen are enlarged. Note oliguria. The temperature remains consistently high for 3–10 days. In the peripheral blood - neutrophilic leukocytosis with a shift of the formula to the left. In addition to the described general manifestations of the plague, lesions characteristic of individual clinical forms of the disease develop.

The cutaneous form is rare (3–5%). A spot appears at the site of the entrance gate of infection, then a papule, a vesicle (conflict), filled with serous hemorrhagic contents, surrounded by an infiltrated zone with hyperemia and edema. Flikten is characterized by severe pain. When opened, it forms an ulcer with a dark scab at the bottom. A plague ulcer is characterized by a long course, heals slowly, forming a scar. If this form is complicated by septicemia, secondary pustules and ulcers occur. Perhaps the development of a regional bubo (skin-bubonic form).

The bubonic form occurs most often (about 80%) and is characterized by a relatively benign course. From the first days of the disease, a sharp pain appears in the region of the regional lymph nodes, which makes it difficult to move and makes the patient take a forced position. The primary bubo, as a rule, is solitary; multiple buboes are less commonly observed. In most cases, the inguinal and femoral lymph nodes are affected, axillary and cervical lymph nodes are somewhat less common. Bubo sizes vary from a walnut to a medium-sized apple. Bright features are sharp pain, dense consistency, adhesion to the underlying tissues, smoothness of the contours due to the development of periadenitis. Bubo begins to form on the second day of illness. As it develops, the skin over it turns red, shiny, often cyanotic. At the beginning it is dense, then it softens, fluctuation appears, the contours become fuzzy. On the 10-12th day of illness, it opens - a fistula, ulceration is formed. With a benign course of the disease and modern antibiotic therapy, its resorption or sclerosis is observed. As a result of hematogenous introduction of the pathogen, secondary buboes can form, which appear later and are small in size, less painful and, as a rule, do not suppurate. A terrible complication of this form can be the development of a secondary pulmonary or secondary septic form, which sharply worsens the patient's condition, up to death.

Primary pulmonary the form is rare, during periods of epidemics in 5-10% of cases and is the most epidemiologically dangerous and severe clinical form of the disease. It starts sharply, violently. Against the background of a pronounced intoxication syndrome, a dry cough, severe shortness of breath, cutting pains in the chest appear from the first days. The cough then becomes productive, producing sputum that may vary in amount from a few spittles to huge quantities, rarely absent at all. The sputum, at first frothy, glassy, ​​transparent, then acquires a bloody appearance, later becomes purely bloody, contains a huge amount of plague bacteria. Usually it is a liquid consistency - one of the diagnostic signs. Physical data are scarce: a slight shortening of the percussion sound over the affected lobe, during auscultation, non-abundant fine bubbling rales, which clearly does not correspond to the general serious condition of the patient. The terminal period is characterized by an increase in shortness of breath, cyanosis, the development of stupor, pulmonary edema, and TSS. Blood pressure falls, the pulse quickens and becomes threadlike, heart sounds are muffled, hyperthermia is replaced by hypothermia. In the absence of treatment, the disease is fatal within 2-6 days. With early use of antibiotics, the course of the disease is benign, differs little

The MPBA-Blot-HIV-1, HIV-2 reagent kit is designed to confirm the detection of antibodies to individual proteins (antigens) of HIV-1 and/or HIV-1 group O and/or HIV-2 in human serum or plasma by the method immune blot.

Distinctive features:

• The set of reagents "MPBA - Blot - HIV-1, HIV-2" contains purified lysate viral proteins of HIV 1 and a peptide - antigenic determinate gp36 of HIV-2;
• Provides detection of antibodies to HIV-1, HIV-1 group O, HIV 2 on one strip;
• A simple procedure for preparing and conducting analyzes;
• Internal quality control of the reaction*
• The maximum speed of the analysis (3 hours);
• A small volume of the test sample - 20 µl;
• Does not require additional equipment for research;
• The quality of the kit is guaranteed by the use of Russian and international standard samples**

* Internal quality control is ensured by the presence of:

• internal control strips, provides control of the introduction of a serum or plasma sample;
• control negative serum (K-);
• control positive serum (K+), which allows to identify the bands detected on the strip;
• control weakly positive serum (K + cl), which provides control of the sensitivity of the reagent kit.

**Quality assurance:

The characteristics of the MPBA-Blot-HIV-1, HIV-2 reagent kit were determined by testing samples from a random sample of donors, patients diagnosed with HIV infection, commercial seroconversion panels, standard panels, and samples with "potentially interfering with the determination" components.

The kit of reagents does not give false positive results in the study of sera of the standard panel that do not contain antibodies to HIV 1.2 and HIV-1 antigen ("Standard AT (-) HIV", No. FSR 2007/00953 dated 10.25.2007). Specificity - 100%.

Diagnostic specificity was determined by examining a random sample of 200 donors from various blood centers and clinics with a preliminary confirmed absence of HIV-1, HIV-2 infection. Specificity in the study of a random sample of donors was 100%;

The specificity of the reagent kit was determined in the study of 250 samples, including serum or plasma samples obtained from pregnant women, hospitalized patients, patients with hepatitis C and E, and samples with "potentially interfering determination" components. When using the MPBA-Blot-HIV-1, HIV-2 kit, no false-positive results were found for these samples.

Diagnostic sensitivity was determined using:
- Plasma samples from the Boston Biomedica, Inc HIV-1 panel (WWRB 301) from different regions containing different subtypes of HIV-1: group M (subtypes A, B, C, D, E, F), and group O; the sensitivity of the reagent kit was 100%;

The sensitivity of the reagent kit was determined in the study of international seroconversion panels Boston Biomedica, Inc (SeraCare Life Sciences), cat. nrs. PRB 903, PRB 904, PRB 909, PRB 912, PRB 916, PRB 917, PRB 918, PRB 919, PRB 921, PRB 923, PRB 924, PRB 927, PRB 928, PRB 932, PRB 940

The kit of reagents detects antibodies to HIV-1 in the sera of a standard panel containing antibodies to HIV-1 (“Standard AT (+) HIV-1”, No. FSR 2007/00953 dated October 25, 2007), detects antibodies to HIV-2 in the sera of the standard panel containing antibodies to HIV-2 ("Standard AT (+) HIV-2", No. FSR 2007/00953 of 10/25/2007). Sensitivity - 100%.

Registration certificate No. FSR 2010/07958 dated July 13, 2011 (validity is not limited)

Compound:

• Immunosorbent. White nitrocellulose membrane strips with individual HIV-1 proteins (gp160, gp120, p66, p55, p51, gp41, p31, p24, p17) adsorbed on them by the method of electrotransfer and applied to the strip with a synthetic HIV-2 peptide, an analog of the gp36 protein and anti-IgG human (internal control) - 18 pcs;
• K- - control negative serum. Human blood serum that does not contain antibodies to HIV-1,2, HCV, HIV antigen, HBsAg, is inactivated by heating at 560C; transparent light yellow liquid - 1 test tube (0.08 ml). Contains preservatives: thimerosal and sodium azide;
• K+ - control positive serum. Human blood serum containing antibodies to HIV-1,2 (titer not less than 1:10000), not containing HBsAg, HIV antigen, antibodies to HCV, inactivated by heating at 560C; transparent light yellow liquid - 1 test tube (0.08 ml). Contains preservatives: thimerosal and sodium azide;
• K+sl - control weakly positive serum. Human blood serum containing antibodies to HIV-1,2 (titer not more than 1:200), not containing HBsAg, HIV antigen, antibodies to HCV, inactivated by heating at 560C; transparent light yellow liquid - 1 test tube (0.08 ml). Contains preservatives: thimerosal and sodium azide;
• RROKk (x10) - solution for dilution of samples and conjugate. Concentrate - Tris buffer containing pre-treated normal goat serum; opaque gray liquid - 1 vial (10 ml). Contains preservative: thimerosal;
• PRk (x20) - washing solution. Concentrate - Tris buffer containing Tween-20; clear colorless liquid - 1 vial (70 ml). Contains preservative: thimerosal;
• Conjugate. Goat antibodies to human IgG, conjugated with alkaline phosphatase; clear colorless liquid - 1 test tube (0.06 ml);
• Substrate (coloring solution). Solution of 5-bromo-4-fluoro-indolyl-phosphate (BCIP) and nitrosine tetrazolium (NBT); transparent light yellow liquid - 1 vial (50 ml);
• Powder for immune blotting. Skimmed milk powder - amorphous white or light yellow powder - 5 pack x 1g;
• A tablet with a lid for setting up a reaction - 2 pieces;
• Plastic tweezers - 1 piece.