Modern methods for diagnosing HIV infection. Using the Combibest test in HIV diagnosis What can you expect from HIV results

ELISA (enzyme-linked immunosorbent assay, ELISA) came into use practical medicine back somewhere in the 60s of the last century. His initial task was histological studies for scientific purposes, which boiled down to the search and identification of the antigenic structure of cells of a living organism.

The ELISA method is based on the interaction of specific (AT) and related antigens (AG) with the formation of an “antigen-antibody” complex, which is detected using an enzyme. This fact prompted scientists to think that the method can be used for diagnostic purposes to identify specific immunoglobulins of various classes involved in the immune response to a particular infection. And it was a breakthrough in clinical laboratory diagnostics!

The method began to be actively used only in the early 80s, and then mainly in specialized institutions. The first immunoenzyme analyzers were equipped with blood transfusion centers and stations, infectious diseases and venereology hospitals, since the formidable AIDS, born on the African continent, appeared on our horizon and immediately joined the “old” infections, required immediate diagnostic measures and the search for therapeutic drugs affecting him.

Scope of application of the ELISA method

The possibilities of enzyme immunoassay are truly extensive. Now it is difficult to imagine how one can do without such research, which is used in literally all branches of medicine. It seems, what can ELISA do in oncology? It turns out it can. And a lot. The ability of the analysis to find markers characteristic of certain species malignant neoplasms, is the basis early detection tumor, when it is not yet determined in any other way due to its small size.

Modern clinical laboratory diagnostics (CDL), in addition to tumor markers, has a significant arsenal of ELISA panels and uses them to diagnose various pathological conditions (infectious processes, hormonal disorders) and monitoring of pharmaceutical drugs in order to identify their effect on the patient’s body and, by the way, not only on humans. Currently, enzyme immunoassay is widely used in veterinary services, because “our little brothers” are also susceptible to many diseases, from which they sometimes suffer greatly.

Thus, ELISA, due to its sensitivity and specificity, can determine from a blood sample taken from a vein:

• Hormonal status (hormones thyroid gland and adrenal glands, sex hormones);
• The presence of viral and bacterial infection(HIV, B and C, chlamydia, syphilis, and, and, as well as many other diseases caused by pathogenic microorganisms);
• Traces of the vital activity of microorganisms that initiated the infectious process, which successfully ended and moved into the stage of forming an immune response to this pathogen. Such traces, that is, antibodies, in many cases remain circulating in the blood for life, thereby protecting a person from re-infection.

What is the essence of ELISA?

The enzyme immunoassay method allows one to determine not only the presence of the pathogen itself (qualitative analysis), but also its quantitative content in the patient’s blood serum.

The viral or bacterial dose significantly influences the course of the infectious process and its outcome, therefore quantitative analysis plays an important role in the diagnosis and treatment of diseases in various forms and stages.

However, knowing enzyme immunoassay studies as the ELISA method, we don’t even think about how it manages to cover such a wide range of microorganisms inhabiting our planet, many of which pose a direct threat to the health and life of humans and animals. But the fact is that ELISA has many options (non-competitive and competitive - direct and indirect), each of which solves its own problem and, thus, allows for a targeted search.

To identify immunoglobulins of one class or another, a traditional 96-well polystyrene panel (plate) is used, in the wells of which sorbed recombinant proteins are concentrated in the solid phase. Antibodies or antigens that get into the well with blood serum find a “familiar” object and form a complex with it (AG - AT), which, fixed by an enzyme conjugate, will be manifested by a change in the color of the well when reading the results.

Enzyme immunoassay is carried out using test systems of a certain specificity, manufactured in special laboratories and equipped with all the necessary reacting components. Research can be carried out using washing devices (“washers”) and reading spectrophotometers, where for the most part manual labor involved. On full automatic machines, which free the laboratory assistant from monotonous instillation, washing and other routine tasks, of course, it is faster and more convenient to work, but not all laboratories can afford such luxury and continue to work the old fashioned way - on semi-automatic machines.

Interpretation of ELISA results is within the competence of the physician. laboratory diagnostics, in this case, the inherent property of almost all immunochemical reactions to give false-positive or false-negative responses is also taken into account.

Video: modern enzyme immunoassay

ELISA results using the example of syphilis

Enzyme immunoassay is suitable for detecting all forms, and, in addition, is used in screening studies. To carry out the analysis use venous blood patient taken on an empty stomach. The work uses tablets with a certain specificity (AB classes A, M, G) or total antibodies.

Considering that antibodies in syphilis are produced in a specific sequence, ELISA can easily answer the question of when the infection occurred and at what stage the process is, and the interpretation of the results obtained can be presented in the following form:

• IgM indicates the duration of the infectious process (may appear during exacerbation of chronic inflammatory diseases);
• IgA states that the infection occurred more than a month ago;
• IgG indicates that the infection is in full swing or that treatment has recently been carried out, which is easily determined by taking an anamnesis.

When testing for syphilis, negative wells (and the negative control) will remain colorless, while positive wells (and the positive control) will show a bright yellow color due to the color change of the chromogen added during the test. However, the color intensity does not always coincide with the control, that is, it may be slightly paler or slightly yellowish. These are dubious results, which, as a rule, are subject to re-examination with mandatory consideration of the quantitative indicators obtained on the spectrophotometer, but in general, the color is directly proportional to the number of immune complexes (associated Ags and ATs).

The most exciting of the enzyme immunoassays is the HIV ELISA

Analysis on is perhaps more interesting than others to a wide range of the population, because it is still impossible to say with certainty that many have disappeared social problems(prostitution, drug addiction, etc.). Unfortunately, HIV affects not only these layers of human society; you can become infected under various circumstances not related to sexual immorality or drug use. But if there is a need for an HIV test, you should not be afraid that everyone around you will know about your visit to such a laboratory. Now HIV-infected people are protected by law, and those who have doubts can turn to anonymous offices where they can solve the problem without fear of publicity and condemnation.

The enzyme-linked immunosorbent method used to diagnose HIV infection is one of the most important standard tests, which, however, requires special conditions, because the topic is very sensitive.

It makes sense to carry out HIV ELISA after sexual contact, blood transfusion, other medical procedures suggesting infection, and at the end of the incubation period (“seronegative window”), but it should be borne in mind that this period of time is not constant. It can end in 14-30 days, or it can last up to six months, so the average value is considered to be an interval from 45 to 90 days. On HIV blood They are given in the same way as for other infections - from a vein on an empty stomach. The results will be ready depending on the accumulation of material in the laboratory and its workload (from 2 to 10 days), although most often laboratories provide an answer on the same day or the next.

What can you expect from your HIV results?

ELISA for HIV infection detects antibodies to two types of the virus: HIV-1 (more common in Russia and other countries of Europe and Asia) and HIV-2 (more common in West Africa).

The task of the HIV ELISA is to search for class G antibodies, which are detected on all test systems, but at a later period, and class A and M antibodies, detected on new generation recombinant test kits, which make it possible to find antibodies on the most early stages (incubation period– “seronegative window”). You can expect the following answers from the ELISA:

1. Primary positive result: the blood must be retested using a test system of the same type, but if possible of a different series and by another person (laboratory assistant);
2. Repeated (+) suggests new fence blood from the patient with its examination similar to the primary analysis;
3. Another positive result is subject to reference analysis, which uses highly specific test kits (2-3 pcs.);
4. A positive result in both (or three) systems is sent for immunoblotting (the same ELISA, but performed in individually for test kits of particularly high specificity).

The conclusion about HIV infection is made only on the basis of immunoblotting. A conversation is conducted with the infected person in complete confidentiality. Disclosure of medical secrets in Russia, as well as in other countries, is subject to criminal punishment.

Tests for chlamydia and cytomegalovirus using the enzyme immunoassay method have also gained particular popularity, due to the fact that they make it possible to determine the time of infection, the stage of the disease and the effectiveness of treatment measures.

During implementation, one can also observe the appearance of antibodies of various classes. in different phases of the pathological condition caused by an infectious agent:

• IgM can be detected as early as seven days after infection;
• IgA indicates that the infection has been living in the body for more than a month;
• IgG confirms the diagnosis of chlamydia and helps monitor treatment and determine its effectiveness. It should be noted that class G antibodies remain and circulate in the body regardless of the duration of the disease, therefore, to correctly interpret the analysis, you need to take into account the reference values ​​(norms), which, by the way, are different for each CDL: taking into account the brand of the test system and the specificity of the reagents included in the set. The normal values ​​are entered in the form next to the ELISA result.

As for , it’s a little different here: Class M antibodies appear after about a month and a half, that is, the result (IgM+) becomes positive in the phase of primary infection or during reactivation hidden infection and remains so from 4 months to six months.

The presence of class G AT is characteristic of the onset of primary acute infection or reinfection. The analysis states that the virus is present, but does not provide information on what stage the infectious process is at. Meanwhile, determining the normal IgG titer also causes difficulties, since it entirely depends on immune status specific person, which, however, is established by identifying class G immunoglobulins. Given this behavior of antibodies, when diagnosing CMV, there is a need to assess the ability of class G antibodies to interact with CMV in order to then “neutralize” it (AT avidity). On initial stage IgG diseases bind very poorly to viral antigens (low avidity) and only then begin to show activity, therefore, we can talk about an increase in the avidity of antibodies.

We can talk a lot about the advantages of enzyme immunoassay, because this method has managed to solve many diagnostic problems using only venous blood. There is no need for long waits, worries and problems with collecting material for research. In addition, test systems for ELISA continue to be improved and the day is not far off when the test will give a 100% reliable result.

Video: educational film of Moscow State Medical University named after. Sechenov on the basics of ELISA

Owners of patent RU 2283497:

The invention relates to the field of biotechnology and medicine. The enzyme-linked immunosorbent test system for identifying the spectrum of antibodies to the human immunodeficiency virus (HIV) of the first and second types, the first type of group O and identifying the antigen to the human immunodeficiency virus of the first type p24 includes an immunosorbent based on human immunodeficiency virus antigens representing gp41 (env HIV-1 and HIV-2 group O), gp120 (env), p24 (gag), p31 (pol), gp36 (env HIV-2), antibodies to HIV antigen 1 p24, and detection reagents, the above HIV antigens and HIV antibodies sorbed in different wells of plates for enzyme-linked immunosorbent assay, and 96-well polystyrene collapsible or non-separable plates are used for sorption. The invention provides increased sensitivity, simplification, and elimination of subjectivity in evaluating results. 1 salary files, 10 tables, 1 ill.

The invention relates to the field of biotechnology and medicine. Use: differentiated detection of all classes of specific antibodies to human immunodeficiency virus proteins 1 and 2, HIV 1 group O and HIV 1 p24 antigen.

Essence: obtaining an enzyme-linked immunosorbent test system for identifying the spectrum of antibodies of all classes to individual proteins and HIV 1 and 2, HIV 1 group O and HIV 1 p24 antigen in blood serum (plasma), immunoglobulins and human blood products in order to identify the spectrum of antibodies to HIV 1 and 2, HIV 1 group O, detection of HIV 1 p24 antigen and confirmation of positive or questionable screening results for antibodies to HIV 1 and 2, HIV 1 group O and HIV 1 p24 antigen.

DESCRIPTION OF THE INVENTION

Laboratory diagnosis of HIV infection is based on three areas: a) indication of HIV and its components; b) detection of antibodies to HIV; c) determination of changes in the immune system. Among existing methods The most common laboratory diagnostics are serological - detection of antibodies to virus antigens.

To detect antibodies in HIV infection, enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) are mainly used. ELISA is based on the immobilization of viral antigens on plates to which the patient’s antibodies bind, and the antigen-antibody complex is detected using horseradish peroxidase-conjugated mouse monoclonal antibodies to human immunoglobulins G and M. The method is quite specific and sensitive and allows the detection of virus-specific antibodies in 95% of patients. The remaining 5% of cases occur in the early stages of infection, when there are still few antibodies in the blood serum, or in the terminal phases of the disease, when the body is no longer able to synthesize antibodies due to severe depletion immune system. False-positive ELISA results are also possible, mainly in patients with autoimmune and oncological diseases, as well as in cases of infection caused by the Eshptain-Barr virus. In this case, a cross-reaction of antibodies to rheumatoid factor occurs, Epstein-Barr virus or to antigenic determinants that are similar to proteins of the major histocompatibility complex class 1 and 2 (HLA-4 and DQW3) and have the ability to bind to HIV antigens. False-positive results are common in pregnant women and the elderly. Hemolysis, lipemia, and bacterial contamination of serum may also cause unreliable results.

In this regard, a number of methods have been proposed and used to test the specificity of antibody detection results. Of these methods, the most commonly used reaction is “immune blotting” in the modification “Western Blot”. The essence of the method is as follows: at the first stage, HIV proteins are separated by molecular weight using polyacrylamide gel electrophoresis. Then electrophoretic transfer from the polyacrylamide gel to the surface is carried out nitrocellulose membrane. Antigens transferred in this way are detected on the membrane using indirect analysis: the membrane is incubated with the test material; the contained antibodies bind to HIV antigens transferred to a nitrocellulose membrane, then the membrane strips are incubated with the conjugate; When an antigen-antibody complex is formed, the conjugate attaches to it; after washing from the conjugate and incubation with the substrate, staining occurs in those areas of nitrocellulose where the formation of the antigen-antibody-conjugate complex occurred. The drawing shows examples of positive, weakly positive and negative immunoblot results.

Antibodies to the following proteins (p) and glycoproteins (gp) are detected in the serum of persons infected with HIV-1 and HIV-2: Table A.

Table B presents the criteria for assessing the results of immunoblotting recommended by WHO and the Russian Center for the Prevention and Control of AIDS.

Based on WHO criteria, sera in which antibodies to any two HIV-1 envelope proteins are detected by the IB method are considered positive. If there is a reaction with only one of the envelope proteins (gp160, gp120, gp41) in combination with or without a reaction with other proteins, the result is considered doubtful. According to the Federal Scientific and Methodological Center of the Ministry of Health of the Russian Federation for the Prevention and Control of AIDS, it is possible to interpret sera as positive even in the presence of antibodies to only one membrane protein.

The detection of antibodies to the p24 antigen may indicate the period of onset of seroconversion, since antibodies to this protein appear first. Positive reactions with gag and pol proteins without the presence of a reaction with env proteins may reflect the stage of early seroconversion and also indicate the presence of HIV-2 infection or a nonspecific reaction.

The use of immunoblotting reaction as an expert method for diagnosing HIV has a number of significant disadvantages:

1. The impossibility of confirming the detection of HIV p24 antigen 1 in the case of using tests that simultaneously detect antigen and antibodies to HIV, which makes it inappropriate (meaningless) to use screening tests for the simultaneous detection of antigen and antibodies to HIV (Order of the Ministry of Health of the Russian Federation No. 292 of July 30. 2001: “to examine donors at blood transfusion stations, it is necessary to use test systems that simultaneously detect antigen and antibodies to HIV”).

2. Detection of antibodies of the IgG class only. It is impossible to fully confirm the positive results obtained using 3rd and 4th generation tests (detecting IgG antibodies and IgM).

3. Subjectivity in the interpretation of test scores, especially in cases of “doubtful” and “uncertain” results and in initial stages seroconversion (the detected bands on nitrocellulose membranes in these cases are unclear, barely visible to the naked eye and when assessing by different persons there are often disagreements).

4. Impossibility of automated quantification analysis results.

5. Lower sensitivity compared to ELISA.

6. Short shelf life (during storage, strips of nitrocellulose membrane fade and cannot be an objective confirmation of the detection or non-detection of HIV in controversial cases).

7. Difficulty in setting up the reaction and storing (nitrocellulose membrane strips are very fragile and often break).

8. High cost of information security kits.

A known reagent is a kit for the simultaneous determination of antigens and antibodies to the same pathogen, including HIV (DE 4236189 F1, 04/28/1994). However, a test system that allows diagnosing HIV infection at various stages is not disclosed.

The objective of the present invention is to obtain a test system with which it is possible to confirm positive or questionable results of screening tests for antibodies to HIV 1 and 2, HIV 1 group O and HIV 1 p24 antigen when using tests for the simultaneous detection of antigens and antibodies.

Proposed technical solution is achieved by an enzyme-linked immunosorbent test system for identifying the spectrum of antibodies to the human immunodeficiency virus (HIV) of the first and second types, the first type of group O and identifying the p24 antigen to the human immunodeficiency virus of the first type p24, characterized in that it includes an immunosorbent based on human immunodeficiency virus antigens , representing gp41 (env HIV-1 and HIV-2 group O), gp120 (env), p24 (gag), p31 (pol), gp36 (env HIV-2), antibodies to HIV 1 antigen p24, and detection reagents , while the above-mentioned HIV antigens and HIV antibodies are sorbed in different wells of plates for enzyme-linked immunosorbent assay.

In addition, 96-well polystyrene collapsible or non-dismountable plates for enzyme immunoassay are used for sorption.

The technical result achieved by the present invention is the possibility of confirming positive results for antibodies to HIV 1 and 2, HIV 1 group O and HIV 1 p24 antigen, high sensitivity, the possibility of automated interpretation of results, which eliminates the subjectivity of the assessment, ease of performing the test, less than with existing cost information security sets.

This technical solution differs from the known ones:

1. Using a plate for immunological reactions as a solid-phase carrier.

2. Simultaneous use of antibodies to HIV p 24 and a set of HIV antigens as a sorbent.

The invention is illustrated by the following example.

The active principles of the developed test system "DS-ELISA-ANTI-HIV 1,2-SPECTRUM + AG p24 HIV 1" are:

Immunosorbent - recombinant antigens similar to the structural proteins of HIV-1: gp41 (env HIV-1 and HIV 1 group O), gp120 (env), p24 (gag), p31 (pol), HIV-2: gp36 (env) and monoclonal mouse antibodies to HIV 1(p24) antigen, adsorbed separately on strips of a polystyrene collapsible plate.

To prepare immunosorbent use:

1. HIV-1 gp41 is a protein produced by E. Coli strain No. AHIV 103.

2. HIV-1 gp120 is a protein produced by E. Coli strain No. AHIV 109.

3. HIV-1 p24 is a protein produced by E. Coli strain No. AHIV 105.

4. HIV-1 p31 is a protein produced by E. Coli strain No. AHIV 108.

5. HIV-2 p36 is a protein produced by E. Coli strain No. AHIV 106.

Conjugate 1, lyophilized or liquid, is a mouse monoclonal antibody to the HIV1 p24 antigen conjugated with biotin.

1. Conjugate 2, lyophilized or liquid, is a mixture of recombinant antigens similar to the structural proteins of HIV-1: gp41 (env HIV-1 and HIV 1 group O), gp120 (env), p24 (gag), p31(po1); HIV-2: gp36 (env), conjugated to biotin;

2. Conjugates 3, 4 - lyophilized or liquid - streptavidin, labeled with horseradish peroxidase;

During preliminary studies, the design of the test system was selected, the technology for preparing its components was developed, and the conditions for conducting the enzyme immunoassay reaction were optimized.

When performing ELISA in the test system "DS - ELISA - ANTI-HIV 1,2-SPECTRUM + Ag p24 HIV 1", 25 μl of conjugate-1 is added to the wells of the plate with sorbed antibodies to p24, and 25 μl of conjugate-1 is added to the wells with sorbed antigens. 25 µl of conjugate-2. The reaction scheme is shown below. Next, 25 μl of the test sample is added to each well. At the same time, in the wells with antigens the color changes from orange to pink, and in the well with antibodies - from green to gray. The mixture is incubated for 45 minutes at 37°C on a shaker (or 1 hour at 37°C in a thermostat). Then, without washing, add 50 μl of conjugate-3 into the wells of the plate for determining the p24 antigen, and 50 μl of conjugate-4 into the wells for determining antibodies. After incubation for 20 minutes at 37°C on a shaker (or 30 minutes at 37°C in a thermostat), the plate is washed and developed with a substrate mixture. Total reaction time 1 hour 25 minutes (or 1 hour 50 minutes). The p24 antigen present in the test sample binds to monoclonal antibodies to p24, and specific antibodies form a complex with recombinant antigens on the plate. The resulting immune complex of anti-p24 with p24 is detected with anti-p24-biotin conjugates, then with streptavidin-peroxidase, and the Ag-HIV immune complexes with At-HIV are detected with an Ag-biotin conjugate, then with streptavidin-peroxidase.

Scheme for setting up the reaction.

The results are taken into account spectrophotometrically at two wavelengths: 450/620-680 nm with the device configured “by air”. Let us take into account the results at one wavelength of 450 nm.

The results of the analysis are taken into account if the average values ​​of optical density (OD) in wells with K- are no more than 0.2, in wells with K+ - no less than 1.0. OP crit. calculated by the formula:

OP crit. gp41 = avg. meaning OP K-(gp41)+0.15

OP crit. gp120 = avg. meaning OP K-(gp120)+0.15

OP crit. p24 = avg. meaning OP K-(р24)+0.15

OP crit. p31 = avg. meaning OP K-(р31)+0.15

OP crit. gp36 = avg. meaning OP K-(gp36)+0.15

OP crit. Ag p24 = avg. meaning OP k- (Ag p24)+0.04

where 0.15 and 0.04 are coefficients established by statistical processing at the manufacturer. During the development of the test system the following were used:

1. Blood serum samples from healthy donors (n=610).

2. Blood serum samples from patients with various infectious diseases not related to HIV (acute respiratory infections, pneumonia, tonsillitis, herpetic and cytomegalovirus infection, syphilis, chlamydia, viral hepatitis A, B and C (n=224)).

3. Blood serum samples from patients with various non-communicable diseases- injuries, diseases of cardio-vascular system, oncology (n=35).

4. Blood serum samples from pregnant women (n=40).

5. Blood serum samples seropositive in ELISA and confirmed in immunoblot (n=428).

6. Serum samples with a positive result obtained in enzyme immunoassay test systems for the simultaneous determination of HIV 1.2 antibodies and p24 antigen, and an indeterminate result by immunoblot (n=123).

7. Internal panel of sera containing and not containing antibodies to HIV 1,2, tested on enzyme immunoassay test systems and immunoblot registered by the Ministry of Health of the Russian Federation (n=21).

8. Standard "HIV 1 ANTIGEN STANDARD", "BIO RAD", France, cat. No. 72217, which is an antigen obtained from viral lysate.

9. Internal standard of the enterprise. A sample containing p 24 antigen at a concentration of 200 pg/ml, obtained from the viral lysate and titrated according to the "HIV 1 ANTIGEN STANDARD" company "BIO RAD", France, cat. No. 72217.

10. Standard panel of sera containing antibodies to human immunodeficiency virus type 1 (HIV 1) - OSO 42-28-212-93-02P, "Medical and Biological Union", Novosibirsk.

11. Standard panel of sera containing antibodies to human immunodeficiency virus type 2 (HIV 2) - OSO-42-28-216-02P, "Medical and Biological Union", Novosibirsk.

12. Standard panel of sera that do not contain antibodies to human immunodeficiency virus type 1 and 2 (HIV 1, 2) - OSO-42-28-214-94-02P, "Medical and Biological Union", Novosibirsk.

The sensitivity of the test system for detecting the p24 antigen was assessed using the “HIV I ANTIGEN STANDARD”, “BIO RAD”, and the enterprise’s Internal Standard. Using the Internal Standard, 4 serial 2-fold dilutions from 40 pg/ml to 5 pg/ml were prepared with normal donor plasma, free of HIV antibodies 1, 2, as a diluent. The smallest amount of detectable antigen was taken as the sensitivity criterion. The obtained data are presented in Table 1.

To assess the sensitivity of the test system in detecting specific antibodies, standard panels of samples containing antibodies to the human immunodeficiency virus (HIV 1, 2) were used (OSO 42-28-212-93-02P p.012, OSO 42-28-216- 02P (HIV 2) p.003).

The obtained data are presented in tables 3, 4.

The diagnostic efficiency of the test system "DS-ELISA-ANTI-HIV 1,2-SPECTR+AGr24 HIV 1" was compared with the test systems "Jenskrin-HIV-Ag/At" (Bio-Rad), "DIA-HIV 1 /2" (Diaprof-Med), "Vironostika HIV Uni-form II Ag/At" (Biomerioux), "Recombinant-HIV1,2 DSM" (MBS), "Amercard Anti-HIV-1,2 K" (Amercard) , "HIV-1, HIV-2-ELISA-Avicenna" (Avicenna). For this purpose, serum samples from the internal panel were tested in all specified tests. The results presented in Table 5 indicate the high diagnostic efficiency of “DS-ELISA-ANTI-HIV 1,2-SPECTRUM+AGr24 HIV 1”.

Sensitivity studies of the test system "DS-ELISA-ANTI-HIV 1,2-SPECTRUM+AGr24 HIV 1" were also carried out with blood sera from patients confirmed by immunoblotting as HIV positive. A total of 428 samples of such sera were tested. Several options have been obtained for detecting antibodies to different HIV proteins and the p24 antigen. The results are presented in Table 6.

When testing HIV 1 positive samples, 3% of sera demonstrated positive reaction with gp36 (HIV env 2). OP/OPcrit. in these samples did not exceed 2.0. Our data on the cross-reactivity of the outer shell proteins of HIV 1 (gp41) and HIV 2 (gp36) coincide with the literature data.

Interpretation of results

Analysis of testing of HIV-positive sera and samples from standard and internal panels in "DS-ELISA-ANTI-HIV 1,2-SPECTR+AGr24 HIV 1" allowed us to develop criteria for interpreting the results of this test. Recommended criteria are given in Table 7.

Based on these criteria, all samples of HIV positive sera (n=428) were determined to be positive in the “DS-ELISA-ANTI-HIV 1,2-SPECTRUM+AGr24 HIV1”. Of the 16 samples of the standard panel containing antibodies to HIV 1, 14 were determined to be positive and 2 were determined to be indeterminate. All 8 samples from the standard panel containing antibodies to HIV 2 were determined to be positive. Of the 10 samples of the inner panel containing antibodies to HIV 1, 7 were determined to be positive and 3 were determined to be indeterminate.

Sensitivity studies of the test system "DS-ELISA-ANTI-HIV 1,2-SPECTRUM+AGr24 HIV 1" were also carried out with blood sera from patients who had a positive result in enzyme-linked immunosorbent test systems that simultaneously detect antibodies and p24 antigen, and indeterminate in immunoblot . A total of 123 such samples were tested. The data is presented in Table 8.

When testing sera with an indeterminate immunoblot result (n=123) in “DS-ELISA-ANTI-HIV 1,2-SPECTRUM+AGr24 HIV 1”, 72 samples (58.5%) showed a positive result. Of these, 26 sera (21.1%) were positive only for p24, 20 (16.3%) - only for antibodies, 16 (13%) - for p24 with antibodies to one of the proteins, 10 (8.1%) - on p24 with antibodies to two or more of the proteins. Thus, testing only for antibodies (without detecting p24) would identify 30 samples (24.4%) as positive. The detection of p24 antigen in the test allowed an additional 42 samples (34.1%) to be identified as positive.

To assess the specificity of the test system, a standard panel of sera that did not contain antibodies to HIV 1, 2 (n=20) was used; blood serum samples from healthy donors (n=610), blood serum samples from patients with various infectious diseases (n=224) not related to HIV; blood serum samples from patients with various non-infectious diseases - trauma, diseases of the cardiovascular system, oncology (n=35); blood serum samples from pregnant women (n=40). A total of 929 samples were tested. One sample from the blood sera of healthy donors showed a false positive result - antibodies to gp41 and p24 were detected. 7 samples of blood sera from healthy donors and 2 samples of blood sera from patients with infectious diseases not related to HIV showed a false positive result for p24.

To assess the specificity of the test system, a standard panel of negative samples was used (OSO 42-28-214-94-02p, p. 009). Specificity was 100%.

Thus, the studies have shown that the specificity of the test system “DS-ELISA-ANTI-HIV 1,2-SPECTR+AGr24 HIV 1” is 99% when examining blood serum samples from normal donors and patients with various infectious and somatic diseases.

The data obtained show the high diagnostic efficiency of the developed test system "DS-ELISA-ANTI-HIV 1,2-SPECTR+AGr24 HIV 1". The test system can be used to detect antibodies to individual proteins of HIV types 1 and 2 and the HIV1 p24 antigen in order to confirm a positive screening result with test systems that detect antibodies, or test systems that simultaneously detect antibodies and HIV1 p24 antigen. The test system can be used as an alternative immunoblot test to confirm positive results, as well as to study the dynamics of HIV antibodies and p24 antigen on different stages infections.

ADVANTAGES OF THE TEST

1. Confirmatory enzyme immunoassay test in tablet format

2. Confirmatory test combining determination of the HIV 1, 2 antibody spectrum and HIV 1 p24 antigen

3. Detection of antibodies of all Ig classes

4. Sensitivity for detecting p24 is at least 5 pg/ml

5. Reduces indeterminate results compared to immunoblotting

6. Analysis time - 1 hour 25 minutes (immunoblot - from 3 to 20 hours)

7. Visual assessment of the addition of all components and samples.

1. An enzyme-linked immunosorbent test system for identifying the spectrum of antibodies to the human immunodeficiency virus (HIV) of the first and second types, the first type of group O and identifying the p24 antigen to the human immunodeficiency virus of the first type p24, characterized in that it includes an immunosorbent based on immunodeficiency virus antigens human, representing gp41 (env HIV-1 and HIV-2 group O), gp120 (env), p24 (gag), p31 (pol), gp36 (env HIV-2), antibodies to HIV 1 antigen p24, and detecting reagents, while the above-mentioned HIV antigens and HIV antibodies are sorbed in different wells of plates for enzyme-linked immunosorbent assay.

2. Enzyme immunoassay test system according to claim 1, characterized in that 96-well polystyrene collapsible or non-separable plates for enzyme immunoassay are used for sorption.

Timely diagnosis of HIV infection becomes an extremely important measure, since earlier initiation of treatment can largely determine further development disease and prolong the patient's life. IN last years There has been significant progress in identifying this terrible disease: Old test systems are being replaced by more advanced ones, examination methods are becoming more accessible, and their accuracy is significantly increasing.

In this article we will talk about modern methods for diagnosing HIV infection, knowledge of which is useful for timely treatment of this problem and maintaining a normal quality of life for the patient.

HIV diagnostic methods

In Russia, a standard procedure is carried out to diagnose HIV infection, which includes two levels:

• ELISA test system (screening analysis);
• immunoblotting (IB).

Other methods can also be used for diagnosis:

• rapid tests.

ELISA test systems

At the first stage of diagnosis, a screening test (ELISA) is used to detect HIV infection, which is based on HIV proteins created in laboratories that capture specific antibodies produced in the body in response to infection. After their interaction with the reagents (enzymes) of the test system, the color of the indicator changes. Next, these color changes are processed using special equipment, which determines the result of the analysis performed.

Such ELISA tests can show results within a few weeks after the introduction of HIV infection. This test does not determine the presence of the virus, but detects the production of antibodies to it. Sometimes, in the human body, the production of antibodies to HIV begins after 2 weeks of infection, but in most people they are produced for more later, after 3-6 weeks.

There are four generations of ELISA tests with varying sensitivities. In recent years, third and fourth generation test systems have been increasingly used, which are based on synthetic peptides or recombinant proteins and have greater specificity and accuracy. They can be used to diagnose HIV infection, monitor HIV prevalence, and ensure safety when testing donated blood. The accuracy of generation III and IV ELISA test systems is 93-99% (tests produced in countries are more sensitive Western Europe – 99%).

To perform an ELISA test, 5 ml of blood is taken from the patient’s vein. At least 8 hours must pass between the last meal and the analysis (usually it is performed in the morning on an empty stomach). It is recommended to take such a test no earlier than 3 weeks after the suspected infection (for example, after unprotected sexual intercourse with a new sexual partner).

ELISA test results are obtained in 2-10 days:

• negative result: indicates absence of HIV infection and does not require contacting a specialist;
• false negative result: can be observed in the early stages of infection (up to 3 weeks), in the later stages of AIDS with severe suppression of the immune system and with improper blood preparation;
• false positive result: can be observed in some diseases and in case of improper blood preparation;
• positive result: indicates HIV infection, requires carrying out an IB and the patient contacting a specialist at the AIDS center.

Why can an ELISA test give false positive results?

False-positive HIV ELISA test results can occur due to improper blood processing or in patients with the following conditions and diseases:

• multiple myeloma;
• infectious diseases caused by the Epstein-Barr virus;
• state after ;
• autoimmune diseases;
• against the background of pregnancy;
• condition after vaccination.

For the reasons described above, nonspecific cross-reacting antibodies may be present in the blood, the production of which was not provoked by HIV infection.

In recent years, the frequency of false-positive results has decreased significantly due to the use of generation III and IV test systems, which contain more sensitive peptide and recombinant proteins (they are synthesized using genetic engineering in vitro). After the introduction of such ELISA tests, the frequency of false positive results decreased significantly and is about 0.02-0.5%.

A false positive result does not mean that the person is infected with HIV. In such cases, WHO recommends conducting another ELISA test (necessarily IV generation).

The patient’s blood is sent to a reference or arbitration laboratory with the mark “repeat” and tested using a IV generation ELISA test system. If the result of the new analysis is negative, then the first result is considered erroneous (false positive) and IS is not carried out. If the result is positive or questionable during the second test, the patient must undergo IB after 4-6 weeks to confirm or refute HIV infection.

Immune blotting

A definitive diagnosis of HIV infection can only be made after a positive immunoblotting (IB) result is obtained. To carry it out, a nitrocellulose strip is used, on which viral proteins are applied.

Blood sampling for IB is performed from a vein. Next, it undergoes special treatment and the proteins contained in its serum are separated in a special gel according to their charge and molecular weight (manipulation is carried out on special equipment under the influence of electric field). A nitrocellulose strip is applied to the blood serum gel and blotting (“blotting”) is carried out in a special chamber. The strip is processed and if the materials used contain antibodies to HIV, they bind to the antigenic bands on the IB and appear as lines.

IB is considered positive if:

• according to American CDC criteria - there are two or three lines gp41, p24, gp120/gp160 on the strip;
• according to the American FDA criteria, the strip has two lines p24, p31 and a line gp41 or gp120/gp160.

In 99.9% of cases, a positive IB result indicates HIV infection.

If there are no lines, the IB is negative.

When identifying lines with gr160, gr120 and gr41, IB is doubtful. This result may occur when:

• oncological diseases;
• pregnancy;
• frequent blood transfusions.

In such cases, it is recommended to repeat the study using a kit from another company. If after additional IB the result remains doubtful, then observation is necessary for six months (IB is carried out every 3 months).

Polymerase chain reaction

A PCR test can detect the RNA of the virus. Its sensitivity is quite high and it allows detecting HIV infection within 10 days after infection. In some cases, PCR may give false-positive results, since its high sensitivity may also respond to antibodies to other infections.

This diagnostic technique is expensive and requires special equipment and highly qualified specialists. These reasons do not make it possible to carry out mass testing of the population.

PCR is used in the following cases:

• to detect HIV in newborns born from HIV-infected mothers;
• to detect HIV in the “window period” or in case of doubtful IB;
• to control the concentration of HIV in the blood;
• for the study of donor blood.

The PCR test alone does not make a diagnosis of HIV, but is carried out as an additional diagnostic method to resolve controversial situations.

Express methods

One of the innovations in HIV diagnostics is rapid tests, the results of which can be assessed within 10-15 minutes. The most effective and accurate results are obtained using immunochromatographic tests based on the principle of capillary flow. They are special strips on which blood or other test fluids (saliva, urine) are applied. If antibodies to HIV are present, after 10-15 minutes a colored and control strip appears on the test - a positive result. If the result is negative, only the control strip appears.

As with ELISA tests, the results of rapid tests must be confirmed by IB analysis. Only after this can a diagnosis of HIV infection be made.

There are rapid home testing kits available. The OraSure Technologies1 test (USA) is FDA approved, available over the counter and can be used to detect HIV. After the test, if the result is positive, the patient is recommended to undergo examination at a specialized center to confirm the diagnosis.

Other tests for home use have not yet been approved by the FDA and their results may be very questionable.

Despite the fact that rapid tests are inferior in accuracy to IV generation ELISA tests, they are widely used for additional testing of the population.

You can take tests to detect HIV infection at any clinic, central district hospital or specialized AIDS centers. On the territory of Russia they are carried out absolutely confidentially, or anonymously. Each patient can expect to receive medical or psychological consultation before or after the test. You will only have to pay for HIV tests in commercial medical institutions, and in state clinics and hospitals they are performed free of charge.

Read about the ways in which you can become infected with HIV and what myths exist about the possibilities of becoming infected.

The most widespread virus in the world today is AIDS, a virus from the subfamily of retroviruses; they are also called lentiviruses, or “slow”. Indeed, in the human body they begin to show activity after a relatively long time, approximately 10 years.

There are immunodeficiencies of the first and second types. But we can safely talk about the expansion of the first type of virus. Once in the blood and joining the cell that is responsible for immunity, the virus begins to multiply rapidly. While the CD4 molecule, responsible for its recognition, identifies the insidious enemy, it manages to infect the entire body. Therefore, the earlier it is diagnosed, the greater the patient’s chances of receiving timely treatment and living longer.

If a person has a reason to test himself for AIDS, this should be done immediately. Most often, the basis of such diagnosis is the determination of antibodies to immunodeficiency in the blood - proteins that arise as a reaction to the presence of the virus, and they begin to form in the period from 1.5 to 6 months from the time of possible infection. Accordingly, it is more advisable to undergo diagnostics after this period.

The amount of antibodies is determined using an analysis called enzyme-linked immunosorbent assay (ELISA). This method is trusted, it is relatively accurate and highly sensitive, these figures are over 99.5%. The pharmaceutical market offers a number of diagnostic test kits for ELISA: CombiBest HIV, Anti-HIV 1 2, CombiBest anti-HIV, CombiBest anti-HIV 1 2, CombiBest anti-HIV 1 2 d 0172. These test systems belong to the third generation. The disadvantage of these reagents: they do not always detect the disease in early dates. The person is already a virus carrier, he is potentially dangerous to partners, but antibodies are not yet present in the blood.

The kits are mainly equipped with a number of tests to conduct 12*8 or 192 anti-HIV tests. It is not recommended to use them on your own. The description of the drugs indicates that their main purpose is to detect the amount of antibodies to both types of AIDS. And also determine antibodies of all classes to AIDS. If there are no antibodies, then the disease has not been diagnosed.

The fourth generation of tests “sees”, in addition to antibodies, AIDS antigens; they are able to detect infection earlier than third-generation methods. For comparison: HIV antigen is in the blood starting from the 5th day, and the first antibodies appear no earlier than a month later. CombiBest HIV 1, 2 AG/AT (set 1) and CombiBest HIV 1, 2 AG/AT (set 2) belong to the 4th generation tests. Using a set of these reagents, antibodies to type 1, 2 and p24 antigen are detected by examining human blood serum or plasma. Many countries use ELISA testing of the 4th generation, which is also called Antigen/Antibody, abbreviated as AGAT.

Testing is carried out anonymously.

Description

Preparation

Indications

Interpretation of results

Description

Determination method Enzyme-linked immunosorbent assay (ELISA).

Material under study Blood serum

Home visit available

Combined detection of antibodies to HIV types 1 and 2 and HIV p24 antigen, qualitative test.

Attention. In case of positive and questionable reactions, the period for issuing results can be extended to 10 working days. HIV (human immunodeficiency virus), which causes AIDS (acquired immunodeficiency syndrome), belongs to the family of retroviruses. Transmitted from person to person through the use of contaminated needles and syringes. intravenous administration drugs or therapeutic procedures, during sexual contacts, both heterosexual and homosexual. Transmission of the virus can occur through transfusion of infected blood and its products, donation of organs or seminal fluid, and among medical workers - through injury from contaminated needles or instruments. HIV infection is possible through transmission from an infected mother to a child (vertical route), although modern methods of prevention using antiretroviral therapy, if all recommendations are followed, can reduce this risk to a minimum.

The process of interaction of a virus with a cell includes a number of stages: binding of the virus to the cell, releasing it from the envelope, penetration into the cytoplasm, synthesis of DNA using viral RNA, integration of viral DNA into the genome of the host cell. After this, the latent stage of infection begins. In this state, proviral DNA can exist for some time without showing activity and without affecting the life of the host cell. While there is no expression of viral proteins, there is no immune response to the virus. Antibodies to HIV, which characterize the body’s immune response, appear after the activation of viral DNA and the beginning of active reproduction of the virus. The duration of the latent period depends on a number of factors, including the individual genetic characteristics of the organism.

Antibodies to HIV may appear starting from the second week after infection; their content increases within 2-4 weeks and remains for many years. In 90-95% of infected people they appear in the first three months after infection, in 5-9% - in the period from three to six months, in 0.5-1% - at a later date.

In the first weeks of infection, even before the appearance of antibodies to the virus (i.e., before seroconversion), the presence of HIV antigens, including its p24 capsid protein, can be detected in serum or plasma samples. Later, after seroconversion, it usually becomes undetectable.

4th generation combined test systems, which include the HIV Ag/Ab Combo test (Architect, Abbott), detect both antibodies to HIV types 1 and 2 and HIV p24 antigen, which allows for early detection of infection. The special characteristics of the screening test used in the INVITRO laboratory to detect HIV infection include the high specificity of the study (> 99.5%); The assay is 100% sensitive to antibodies characteristic of the period of seroconversion, and the sensitivity of the test to the p24 antigen is about 18 pg/ml.

The procedure for conducting a laboratory examination for HIV is strictly regulated by orders of the Ministry of Health of the Russian Federation and includes the stage of a screening (selection) study of the presence of antibodies to HIV using enzyme-linked immunosorbent assay (ELISA) methods approved for use, and the stage of a verification (confirmation) more detailed study in the laboratory of the city AIDS center. It should be noted that even the best screening ELISA systems do not guarantee 100% specificity, that is, there is some probability of obtaining nonspecific, false-positive results associated with the characteristics of the patient’s blood serum. Therefore, a positive result of a screening ELISA examination may not be confirmed in confirmatory tests, after which the patient will be given a negative or indeterminate result. If the result of the confirmatory study is uncertain, testing should be repeated over time after 2-3 weeks.

Laboratory diagnosis of HIV infection in children born to HIV-infected mothers has its own characteristics. Maternal antibodies to HIV (IgG class) can circulate in their blood for up to 18 months from the moment of birth. The absence of antibodies to HIV in newborns does not mean that the virus has not penetrated the placental barrier. Children of HIV-infected mothers are subject to laboratory diagnostic examination within 36 months after birth.

Preparation

No special preparation required. It is recommended that blood be taken no earlier than 4 hours after the last meal. WITH general recommendations To prepare for research, you can read. It is advisable to conduct a test to detect antigen and antibodies to HIV no earlier than two weeks after possible infection, repeating it after three and six weeks in case of negative result. Applications for research at INVITRO LLC are filled out using a passport or a document replacing it (migration card, temporary registration at the place of residence, military personnel ID, certificate from the passport office in case of loss of a passport, registration card from a hotel). The presented document must necessarily contain information about temporary or permanent registration in the Russian Federation and a photograph. In the absence of a passport (a document replacing it), the patient has the right to fill out an anonymous application for the donation of biomaterial. During an anonymous examination, an application and a sample of biomaterial received from the client, a number is assigned that is known only to the patient and the medical staff who placed the order. ! The results of studies performed anonymously cannot be submitted for hospitalization, professional examinations and are not subject to registration in the ORUIB.

Indications for use

• Increase lymph nodes more than two areas.
• Leukopenia with lymphopenia.
• Night sweats.
• Sudden weight loss of unknown cause.
• Diarrhea for more than three weeks of unknown cause.
• Fever of unknown cause.
• Pregnancy planning.
• Preoperative preparation, hospitalization.
• Detection of the following infections or combinations thereof: tuberculosis, manifest toxoplasmosis, often recurrent herpes virus infection, candidiasis internal organs, repeated neuralgia herpes zoster caused by mycoplasmas, pneumocystis or legionella pneumonia.
• Kaposi's sarcoma at a young age.
• Casual sexual contacts.

Interpretation of results

Interpretation of research 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. The doctor makes an accurate diagnosis using the results this survey, as well as the necessary information from other sources: medical history, results of other examinations, etc.

Units of measurement in the Independent Laboratory INVITRO: qualitative test. Form of presentation of results: in the absence of antibodies to HIV 1 and 2 and p24 antigen, the answer is “negative”. If antibodies to HIV or an antigen are detected in a screening enzyme-linked immunosorbent test, a serum sample is sent for confirmation by immunoblotting to the city AIDS center, which verifies positive and indeterminate results.

Positive result:

1. HIV infection;
2. false positive result requiring repeated or additional studies *);
3. the study is not informative in children under 18 months born from HIV-infected mothers.

*The specificity of the screening test system Antibodies to HIV 1 and 2 and HIV antigen 1 and 2 (HIV Ag/Ab Combo, Abbott), according to estimates provided by the reagent manufacturer, is about 99.6% in both the general population and the group patients with potential interferences (infections HBV, HCV, Rubella, HAV, EBV, HNLV-I, HTLV-II, E.coli, Chl.trach., etc., autoimmune pathologies(including rheumatoid arthritis, presence of antinuclear antibodies), pregnancy, increased level IgG, IgM, monoclonal gammopathies, hemodialysis, multiple blood transfusions).