The result of the HIV analysis was not detected. ELISA diagnostics: what is the essence, the definition of antibodies, how it is carried out and in what diseases it is effective

Currently, new diagnostic technologies make it possible to identify the etiological and pathogenetic causes of many diseases and radically affect the results of treatment. Perhaps the most impressive results of the introduction of these technologies into clinical practice have been achieved in the field of immunology and diagnostics of infectious diseases.

Test systems based on enzyme-linked immunosorbent and immunochemiluminescent assays make it possible to detect antibodies of various classes, which significantly increases the information content of methods of clinical, analytical sensitivity and specificity for diagnosing infectious diseases. It should be noted that the most significant advances in the diagnosis of infections are associated with the introduction into laboratory practice of the polymerase chain reaction method, which is considered the "gold standard" in the diagnosis and evaluation of the effectiveness of the treatment of a number of infectious diseases.

Various research methods can be used biological material: serum, blood plasma, scraping, biopsy, pleural or cerebrospinal fluid (CSF). First of all, methods laboratory diagnostics infections are aimed at identifying diseases such as viral hepatitis B, C, D, cytomegalovirus infection, sexually transmitted infections (gonorrhea, chlamydia, mycoplasma, ureaplasma), tuberculosis, HIV infection, etc.

HIV infection is a disease caused by the human immunodeficiency virus (HIV), which persists for a long time in lymphocytes, macrophages, and nervous tissue cells, resulting in a slowly progressive damage to the immune and nervous systems organism, manifested by secondary infections, tumors, subacute encephalitis and other pathological changes.

The causative agents of infection - human immunodeficiency viruses of the 1st and 2nd types (HIV-1, HIV-2) - belong to the family of retroviruses, a subfamily of slow viruses. Virions are spherical particles with a diameter of 100-140 nm. The viral particle has an outer phospholipid shell, which includes glycoproteins (structural proteins) with a certain molecular weight, measured in kilodaltons. In HIV-1, these are gpl60, gpl20, gp41. The inner shell of the virus, covering the core, is also represented by proteins with a known molecular weight - p17, p24, p55 (HIV-2 contains gpl40, gpl05, gp36, p16, p25, p55).

The HIV genome contains RNA and the enzyme reverse transcriptase (revertase). In order for the retrovirus genome to connect with the genome of the host cell, DNA is first synthesized on the viral RNA template using reversetase. The provirus DNA is then integrated into the genome of the host cell. HIV has a pronounced antigenic variability, significantly exceeding that of the influenza virus.

In the human body, the main target of HIV is T-lymphocytes, which carry the largest number of CD4 receptors on their surface. After HIV enters the cell with the help of reversetase, the virus synthesizes DNA according to the pattern of its RNA, which is integrated into the genetic apparatus of the host cell (CD4-lymphocytes) and remains there for life in the state of a provirus. In addition to T-lymphocyte helpers, macrophages, B-lymphocytes, neuroglial cells, intestinal mucosa and some other cells are affected. The reason for the decrease in the number of T-lymphocytes (CD4 cells) is not only the direct cytopathic effect of the virus, but also their fusion with uninfected cells. Along with the defeat of T-lymphocytes in patients with HIV infection, polyclonal activation of B-lymphocytes is noted with an increase in the synthesis of immunoglobulins of all classes, especially IgG and IgA, and subsequent depletion of this section of the immune system. Dysregulation of immune processes is also manifested by an increase in the level of α-interferon, β2-microglobulin, and a decrease in the level of IL-2. As a result of dysfunction of the immune system, especially with a decrease in the number of T-lymphocytes (CD4) to 400 cells per 1 μl of blood or less, conditions arise for uncontrolled HIV replication with a significant increase in the number of virions in various body environments. As a result of the defeat of many parts of the immune system, a person infected with HIV becomes defenseless against pathogens of various infections.

Against the background of increasing immunosuppression, severe progressive diseases develop that do not occur in a person with a normally functioning immune system. These are diseases that the World Health Organization (WHO) has defined as AIDS marker or AIDS-defining diseases.

AIDS-defining diseases

The first group - diseases inherent only in severe immunodeficiency (CD4 level<200). Клинический диагноз ставится при отсутствии анти-ВИЧ-антител или ВИЧ-антигенов.

The second group - diseases that can develop both against the background of severe immunodeficiency, and in some cases without it.

Therefore, in these cases, laboratory confirmation of the diagnosis is necessary.

First group:

candidiasis of the esophagus, trachea, bronchi;
extrapulmonary cryptococcosis;
cryptosporidiosis with diarrhea for more than 1 month;
cytomegalovirus lesions of various organs other than the liver, spleen or lymph nodes in a patient over the age of 1 month;
an infection caused by the herpes simplex virus, manifested by ulcers on the skin and mucous membranes that persist for more than 1 month, as well as bronchitis, pneumonia or esophagitis of any duration, affecting a patient over the age of 1 month;
generalized Kaposi's sarcoma in patients under the age of 60;
brain lymphoma (primary) in patients under the age of 60;
lymphocytic interstitial pneumonia and/or pulmonary lymphoid dysplasia in children under 12 years of age;
disseminated infection caused by atypical mycobacteria (mycobacteria complex M. avium intracellulare) with extrapulmonary localization or localization (in addition to the lungs) in the skin, cervical lymph nodes, lymph nodes of the roots of the lungs;
pneumocystis pneumonia;
progressive multifocal leukoencephalopathy;
toxoplasmosis of the brain in patients over the age of 1 month.

Second group:

bacterial infections, combined or recurrent, in children under 13 years of age (more than two cases in 2 years of observation): sepsis, pneumonia, meningitis, damage to bones or joints, abscesses caused by Haemophilus influenzae, streptococci;
disseminated coccidioidomycosis (extrapulmonary localization);
HIV encephalopathy (HIV dementia, AIDS dementia);
histoplasmosis with diarrhea persisting for more than 1 month;
isosporiasis with diarrhea persisting for more than 1 month;
Kaposi's sarcoma at any age;
brain lymphoma (primary) in persons of any age;
other B-cell lymphomas (with the exception of Hodgkin's disease) or lymphomas of an unknown immunophenotype: small cell lymphomas (such as Burkitt's lymphoma, etc.); immunoblastic sarcomas (immunoblastic, large cell, diffuse histiocytic, diffuse undifferentiated lymphomas);
disseminated mycobacteriosis (not tuberculosis) with lesions in addition to the lungs of the skin, cervical or basal lymph nodes;
extrapulmonary tuberculosis (with damage to internal organs, in addition to the lungs);
salmonella septicemia, recurrent;
HIV-dystrophy (exhaustion, sudden weight loss).

Table 1 (see reference above) lists AIDS-defining diseases and their etiological agents.

There are many classifications of AIDS.

According to the new classification proposed by the US Centers for Disease Control (Table 2 - see the link to the source above), the diagnosis of AIDS is established for people with a CD4-lymphocyte level of less than 200/μL, even in the absence of AIDS-defining diseases.

Category B includes various syndromes, the most important of which are bacillary angiomatosis, oropharyngeal candidiasis, recurrent vulvovaginal candidiasis, difficult to treat, cervical dysplasia, cervical carcinoma, idiopathic thrombocytopenic purpura, listeriosis, peripheral neuropathy.

Antibodies to HIV-1 and HIV-2 in the blood

Antibodies to HIV-1 and HIV-2 are normally absent in the blood serum.

Determination of antibodies to HIV is the main method of laboratory diagnosis of HIV infection. The method is based on enzyme immunoassay (ELISA) - sensitivity is more than 99.5%, specificity is more than 99.8%. Antibodies to HIV appear in 90-95% of those infected within 1 month after infection, in 5-9% - after 6 months, in 0.5-1% - at a later date. In the AIDS stage, the number of antibodies can decrease until it disappears completely.

The result of the study is expressed qualitatively: positive or negative.

A negative test result indicates the absence of antibodies to HIV-1 and HIV-2 in the blood serum. The laboratory issues a negative result as soon as it is ready. Upon receipt of a positive result - the detection of antibodies to HIV - in order to avoid false positive results in the laboratory, the analysis is repeated 2 more times.

Immunoblotting for antibodies to HIV viral proteins in blood serum

Antibodies to HIV viral proteins are normally absent in the blood serum.

The ELISA method for the determination of antibodies to HIV is a screening method. Upon receipt of a positive result, to confirm its specificity, the Western-blot method is used - counter-precipitation in the gel of antibodies in the patient's blood serum with various viral proteins subjected to separation by molecular weight using electrophoresis and applied to nitrocellulose. Antibodies to viral proteins gp41, gpl20, gpl60, p24, pi8, p17, etc. are determined.

According to the recommendations of the Russian Center for the Prevention and Control of AIDS, the detection of antibodies to one of the glycoproteins gp41, gpl20, gpl60 should be considered a positive result. If antibodies to other proteins of the virus are detected, the result is considered doubtful, such a patient should be examined twice - after 3 and 6 months.

The absence of antibodies to specific HIV proteins means that the enzyme immunoassay gave a false positive result. At the same time, in practical work, when evaluating the results of the immunoblotting method, it is necessary to be guided by the instructions supplied by the company to the “Immunoblotting kit” used.

The method of immunoblotting is used for laboratory diagnosis of HIV infection.

p24 antigen in blood serum

The p24 antigen is normally absent in the blood serum.

The p24 antigen is the HIV nucleotide wall protein. The stage of primary manifestations after infection with HIV is a consequence of the beginning of the replicative process. The p24 antigen appears in the blood 2 weeks after infection and can be detected by ELISA in the period from 2 to 8 weeks. After 2 months from the moment of infection, the p24 antigen disappears from the blood. In the future, in the clinical course of HIV infection, a second rise in the content of the p24 protein in the blood is noted. It falls on the period of the formation of AIDS. Existing ELISA test systems for detecting the p24 antigen are used for early detection of HIV in blood donors and children, determining the prognosis of the course of AIDS and monitoring ongoing therapy in AIDS patients. ELISA has a high analytical sensitivity, which makes it possible to detect the p24 antigen of HIV-1 in blood serum at a concentration of 5-10 pg/ml and HIV-2 - less than 0.5 ng/ml, and specificity. However, it should be noted that the level of p24 antigen in the blood is subject to individual variations, which means that only 20-30% of patients can be detected using this study in the early period after infection (Rose N.R. et al., 1997).

Antibodies to the p24 antigen of the IgM and IgG classes appear in the blood from the 2nd week, reach a peak within 2-4 weeks and remain at this level for various times: IgM class antibodies - for several months, disappearing within one year after infection, and IgG antibodies can persist for years.

The algorithm for diagnosing HIV infection depends on the phase of the disease and is characterized by a change in the dynamics of detection antibodies of various classes (Fig. 1, 2 - see the link to the source above).

The result of the study is expressed qualitatively - positive or negative. A negative test result indicates the absence of antibodies to HIV-1 and HIV-2 and the p24 antigen in the blood serum.

The laboratory issues a negative result as soon as it is ready. Upon receipt of a positive result - detection of antibodies to HIV-1 and HIV-2 and / or p24 antigen - in order to avoid false positive results in the laboratory, the analysis is repeated 2 more times.

Regardless of the test results obtained, the patient's blood sample and the results of 3 tests are sent by the laboratory to the regional AIDS center to confirm a positive result or verify an indeterminate result. In such cases, the regional AIDS center issues the final answer for this study.

HIV detection by polymerase chain reaction (qualitatively)

Detection of HIV by polymerase chain reaction - PCR (qualitatively) is carried out in order to:

resolution of questionable immunoblot results;
for early diagnosis of HIV infection;
monitoring the effectiveness of antiviral treatment;
determining the stage of AIDS disease (the transition of infection into a disease).

In case of primary infection with HIV, the PCR method makes it possible to detect HIV RNA in the blood as early as 10-14 days after infection.

The result of the study is expressed qualitatively: positive or negative. A negative test result indicates the absence of HIV RNA in the blood.

A positive result - the detection of HIV RNA - indicates infection of the patient.

HIV detection by polymerase chain reaction (quantitative)

HIV is normally absent in the blood.

Direct quantitative determination of HIV RNA using PCR allows more accurate than the determination of the content of CD4 cells to predict the rate of development of AIDS in persons infected with HIV, therefore, to more accurately assess their survival. A high content of viral particles usually correlates with a pronounced violation of the immune status and a low content of CD4 cells. A low viral particle count generally correlates with a better immune status and a higher CD4 cell count. The content of viral RNA in the blood makes it possible to predict the transition of the disease to the clinical stage. With HIV RNA-1 levels >74,100 copies/mL, almost all patients develop clinical picture AIDS (Senior D., Holden E., 1996).

People with blood levels of HIV-1 >10,000 copies/mL are 10.8 times more likely to develop AIDS than people with HIV-1 levels in their blood<10 000 копий/мл. При ВИЧ-инфекции прогноз непосредственно определяется уровнем виремии. Снижение уровня виремии при лечении улучшает прогноз заболевания.

A panel of US experts has developed indications for the treatment of patients with HIV. Treatment is indicated for patients with a CD4 count in the blood<300/мкл или уровнем РНК ВИЧ в сыворотке >20,000 copies/ml (PCR). Assessment of the results of antiretroviral therapy in people infected with HIV is carried out by reducing the level of serum HIV RNA.

With effective treatment, the level of viremia should decrease by 10 times during the first 8 weeks and be below the detection limit of the method (PCR) (<500 копий/мл) через 4-6 месяцев после начала терапии.

Thus, to date, many research methods have been introduced and used in clinical practice for the diagnosis of HIV infection, as for all other viral infections. Among them, the leading role is given to serological studies. The main methods for diagnosing HIV infections are presented in Table 3 (see the link to the source above), where they are divided depending on the importance of each method for detecting viruses into four levels:

A - the test is usually used to confirm the diagnosis;
B - the test is useful in certain circumstances for the diagnosis of certain forms of infection;
C - test is rarely used for diagnostic purposes, but is of great importance for epidemiological surveys;
D - test is not usually used by laboratories for diagnostic purposes.

Since for the diagnosis of viral infections, in addition to choosing the optimal method of analysis, it is equally important to correctly determine and take the biomaterial for research, Table 4 (see the link to the source above) provides recommendations for choosing the optimal biomaterial for the study of HIV infection.

To monitor HIV-infected people, one should use the possibilities of a comprehensive study of the immune status - quantitative and functional determination of all its links: humoral, cellular immunity and nonspecific resistance in general.

In modern laboratory conditions, the multi-stage principle of assessing the immunological status includes the determination of a subpopulation of lymphocytes, blood immunoglobulins. When evaluating indicators, it should be taken into account that HIV infection is characterized by a decrease in the ratio of CD4 / CD8 T cells less than 1. CD4 / CD8 index 1.5-2.5 - indicates a normergic state, more than 2.5 - indicates hyperactivity, less 1.0 - indicates immunodeficiency. Also, the CD4/CD8 ratio may be less than 1 in severe inflammation.

This ratio is of fundamental importance in assessing the immune system in AIDS patients, because HIV selectively infects and destroys CD4 lymphocytes, as a result of which the CD4 / CD8 ratio drops to values significantly less than 1.

Assessment of the immunological status is also based on the identification of general or "gross" defects in the system of cellular and humoral immunity: hypergammaglobulinemia (increased concentration of IgA, IgM, IgG) or hypogammaglobulinemia in the terminal stage; increase in the concentration of circulating immune complexes; decreased production of cytokines; weakening of the response of lymphocytes to antigens and mitogens.

Violation of the ratio of populations in the total pool of B-lymphocytes is characteristic of insufficiency of humoral immunity. However, these changes are not specific to HIV infection and may occur in other diseases. In a comprehensive assessment of a number of other laboratory parameters, it should be taken into account that HIV infection is also characterized by: anemia, lymphopenia and leukopenia, thrombocytopenia, an increase in the level of β2-microglobulin and C-reactive protein, an increase in the activity of transaminases in the blood serum.

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Antibodies produced in the body in response to infection with the human immunodeficiency virus (HIV). Qualitative determination of HIV p24 antigen and antibodies to HIV antigen types 1 and 2.

Functions. The human immunodeficiency virus (HIV) belongs to the family of retroviruses. The virus mainly infects cells of the immune system - T-lymphocytes. In a host (human) cell, the virus forms a piece of DNA and integrates it into the host's genome. A cell affected by a virus produces materials for building viral particles, and virus antigens appear on its surface. When dividing, daughter cells receive viral DNA. To the antigens of the virus located on the surface of the cells, antibodies are produced, which are used to diagnose the infection. Antibodies to HIV begin to be detected in the blood of an infected person, usually after 3-6 weeks, they are almost always detected after 12 weeks, in rare cases they appear only a few months or more after the virus enters the bloodstream. Also, their number can decrease markedly in the terminal period of the disease . In rare cases of HIV infection, antibodies may disappear for a long time.

features of the infection. Infection.

The condition of the mucous membranes of the genital organs / mouth / rectum during sexual transmission, the number of viral particles that enter the body, the state of the immune system, and the general condition of the body affect the likelihood of HIV infection. With a massive intake of viral particles, clinical signs of infection appear earlier. When infected with HIV I, the first symptoms of the disease occur faster than with HIV II.

Training

Special preparation for the study is not required.

The timing of the analysis is determined by the possible transmission of the virus: the more likely the infection (see table), the earlier the study becomes informative. It is advisable to conduct a test for the detection of antibodies to HIV not earlier than 3-4 weeks after a possible infection with a repeat after 3 and 6 months in case of a negative result.

Indications

Enlargement of lymph nodes in 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.
Planning for pregnancy.
Preoperative preparation, hospitalization.
Identification of the following infections or their combinations: tuberculosis, manifest toxoplasmosis, often recurrent herpesvirus infection, candidiasis of internal organs, repeated herpes-zoster neuralgia, pneumonia caused by mycoplasmas, pneumocystis or legionella.
Kaposi's sarcoma at a young age.
Casual sex.

Interpretation of results

Units of measurement in the INVITRO laboratory: the HIV 1/2 antibody test is qualitative. In the absence of antibodies, the answer is “negative”. In case of detection of antibodies to HIV, the study is repeated in another series. When a positive result is repeated in the enzyme immunoassay, the sample is sent for examination by a confirmatory immunoblot method, which is the "gold standard" in the diagnosis of HIV.

Reference values: negative.

Positive result:

HIV infection;
false positive result (antibodies to the Epstein-Barr virus, rheumatoid factor, HLA major histocompatibility complex);
the study is not informative (in children born from HIV-infected mothers up to 18 months)

Negative result:

not infected (diagnostic terms of the analysis were observed);
seronegative variant of the course of infection (antibodies are produced late);
terminal stage of AIDS (impaired formation of antibodies to HIV);
the study is not informative (diagnostic terms are not observed).

Note! Information on positive results of serological tests for markers of parenteral viral hepatitis and detection of antibodies to HIV.

Dear patients, in accordance with the regulations in force on the territory of the Russian Federation:

- information on positive results of serological tests for markers of parenteral viral hepatitis is transmitted by the INVITRO laboratory to the Department for Registration and Registration of Infectious Diseases of the Center for Sanitary and Epidemiological Surveillance in Moscow. ORUIB TsGSEN in Moscow, in turn, brings the information to the medical institution providing outpatient medical care at the place of registration of the patient;

- information on positive results of the determination of antibodies to HIV - to the AIDS Center.

Registration of applications for research in LLC "Independent Laboratory INVITRO" is carried out according to a passport or a document replacing it. In the absence of a passport (a document replacing it), the patient has the right to issue an anonymous application for the delivery of biomaterial. With an anonymous examination, the application and the biomaterial sample received from the client are assigned a number 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 with the ORUIB.

To determine HIV infection, the following specific indicators are used: antibodies to HIV, HIV antigens, HIV RNA and provirus DNA. Antibodies to HIV are determined by enzyme immunoassay (ELISA) or immunoblotting, which is essentially a type of ELISA. Antigens (proteins) of HIV are determined by ELISA. With the help of molecular genetic methods of polymerase chain reaction (PCR), it is possible to determine HIV RNA and provirus DNA.

During primary infection, the following dynamics of HIV markers in the blood of those infected is observed. In the first month, as a result of activation of the replicative process, there is a sharp increase in viral load (HIV RNA content in plasma), then, due to dissemination of the virus and massive infection of target cells in the blood and lymph nodes, it becomes possible to determine proviral DNA. Of paramount diagnostic value is the fact of detection of provirus DNA integrated into the genome of the target cell.

Viral load reflects the intensity of the replication process in infected cells. During the period of primary infection, the level of viral load is different when infected with different HIV subtypes, but the dynamics of its changes is approximately the same. So, when infected with subtype B, for example, if in the first month after infection the value of the viral load is 700 copies / ml, then in the 2nd month there is a decrease to 600, in the 3rd - to 100, in the 4th - to 50 copies/ml Such dynamics is observed against the background of an increase in the content of specific antibodies to HIV in the blood. The content of proviral DNA in the blood mononuclear cells of HIV-infected patients is characterized by relative constancy during the first 6 months with slight fluctuations in some subtypes. Thus, RNA and DNA loads are not identical.

During the incubation stage, for some time, there is no formation of specific antibodies to HIV in an amount sufficient to be determined by existing laboratory methods. Before the detection of antibodies, the appearance in the blood of the Nef protein, which represses the replicative process, and the structural protein p24 are observed for a very short time. The p24 antigen can be detected in the blood by enzyme-linked immunosorbent assay already 1-2 weeks after infection and can be determined until the 8th week, then its content decreases sharply. Further, in the clinical course of HIV infection, a second rise in the content of the p24 protein in the blood is noted. It falls on the period of the formation of AIDS. The disappearance of free (not bound by antibodies) p24 core proteins in the blood and the appearance of specific antibodies to HIV proteins mark the onset of seroconversion

Viremia and antigenemia cause the formation of specific antibodies of the IgM class (anti-p24, anti-gp41, anti-gp120, anti-gp160). Free antibodies of the IgM and IgG classes to the p24 protein may appear starting from the 2nd week, their content increases within 2-4 weeks, reaching a certain level, which remains for months (IgM) and years (IgG).

The appearance of complete seroconversion, when a high level of specific antibodies of the IgG class to the structural proteins of HIV p24, gp41, gp120, gp160, is recorded in the peripheral blood, greatly facilitates the diagnosis of HIV infection. Antibodies to HIV appear in 90-95% of those infected within 3 months after infection, in 5-9% - in the period from 3 to 6 months from the moment of infection, and in 0.5-1% - at a later date.

Despite the fact that antibodies to HIV appear last, the main laboratory diagnostic indicator so far is the detection of specific antibodies by ELISA and immunoblotting.

ELISA (enzyme-linked immunosorbent assay, ELISA - English) entered the life of practical medicine somewhere else in the 60s of the last century. His initial task was histological research for scientific purposes, which was limited to the search and identification of the antigenic structure of the 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 led scientists to the idea 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 ELISA analyzers were supplied to blood transfusion centers and stations, infectious and venereal hospitals, since the formidable AIDS, born on the African continent, appeared on the horizon with us and immediately joined the “old” infections, required immediate measures for diagnosing and searching for therapeutic drugs that affect him.

Scope of the ELISA method

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

Modern clinical laboratory diagnostics (CDL), in addition to tumor markers, has a significant arsenal of panels for ELISA and uses them to diagnose various pathological conditions (infectious processes, hormonal disorders) and monitor pharmaceutical drugs in order to identify their effect on the patient's body and, by the way, does not only human. Currently, enzyme immunoassay is widely used in the veterinary service, because "our smaller brothers" are also susceptible to many diseases, from which, sometimes, they suffer very much.

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

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

What is the essence of IF?

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

A viral or bacterial dose significantly affects 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-linked immunosorbent assays as an 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. The fact is that the 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 detect immunoglobulins of one or another class, a traditional 96-well polystyrene panel (tablet) is used, in the wells of which adsorbed recombinant proteins are concentrated in the solid phase. The antibodies or antigens that got into the well with blood serum find a “familiar” object and form a complex with it (AG - AT), which, fixed by the enzyme conjugate, will manifest itself as a change in the color of the well when reading the results.

Enzyme immunoassay is carried out on test systems of a certain specificity, made in special laboratories and equipped with all the necessary reacting components. Studies can be carried out using washers ("washers") and reading spectrophotometers, where most manual labor is involved. On full automatic machines, freeing 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 a luxury and continue to work in the old fashioned way - on semi-automatic devices.

Interpretation of the results of ELISA is within the competence of the doctor of laboratory diagnostics, while the property inherent in almost all immunochemical reactions to give false positive or false negative answers is necessarily taken into account.

Video: modern enzyme immunoassay

ELISA results on the example of syphilis

ELISA is suitable for detection of all forms, and, in addition, it is used in screening studies. For analysis, venous blood of the patient taken on an empty stomach is used. In the work, plates with a certain specificity (AT classes A, M, G) or total antibodies are used.

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 decoding of the results obtained can be presented in the following form:

IgM indicate the duration of the infectious process (may appear during exacerbation of chronic inflammatory diseases);
IgA state that the infection happened more than a month ago;
IgG indicate that the infection is in full swing or recent treatment, which is easily found out when collecting anamnesis.

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

The most exciting of enzyme immunoassays - ELISA for HIV

Analysis on, perhaps more than others, is of interest to a wide range of the population, because it is not yet possible to say with certainty that many social problems (prostitution, drug addiction, etc.) have disappeared. Unfortunately, HIV affects not only these sections of human society, you can get infected under various circumstances that are not related to sexual promiscuity or drug use. But if there is a need for an HIV test, then you should not be afraid that everyone around will find out about visiting 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 immunoassay used to diagnose HIV infection is one of the primary standard studies, which, however, require special conditions, since the topic is very sensitive.

It makes sense to carry out ELISA for HIV after sexual contact, blood transfusion, other medical procedures that involve 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. Blood is donated for HIV 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 laboratories provide a response on the same day or the next.

What can be expected from HIV results?

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

The task of HIV ELISA is to search for class G antibodies that are detected on all test systems, but in a later period, and class A and M antibodies detected on new generation recombinant test kits, which make it possible to detect antibodies at the earliest stages (the incubation period is seronegative window). The following answers can be expected from the ELISA:

Primary positive result: blood is subject to rechecking on a test system of the same type, but, if possible, of a different series and by another person (laboratory assistant);
Repeated (+) involves a new blood sampling from a patient with a study of it similar to the primary analysis;
The next positive result is subject to a reference analysis, which uses highly specific test kits (2-3 pcs.);
A positive result in both (or three) systems is sent for immunoblotting (the same ELISA, but performed individually on test kits of especially high specificity).

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

Analyzes for chlamydia and cytomegalovirus by enzyme immunoassay have also gained particular popularity, due to the fact that they allow you to determine the time of infection, the stage of the disease and the effectiveness of therapeutic measures.

During the introduction, it is also possible to observe the appearance of antibodies of various classes. in different phases of a pathological condition caused by an infectious agent:

IgM can be detected as early as seven days after infection;
IgA indicate that the infection has been living in the body for more than a month;
IgG confirms the diagnosis of chlamydia, helps to 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, for the correct interpretation of the analysis, reference values (norms) must be taken into account, 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 norm values are entered into the form next to the ELISA result.

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

The presence of class G antibodies is characteristic for the onset of a primary acute infection or reinfection. The analysis states that the virus is present, but does not provide information on the stage of the infectious process. Meanwhile, determining the IgG titer norm also causes difficulties, since it entirely depends on the immune status of a particular person, which, however, is established by the detection of class G immunoglobulins. Given this behavior of antibodies, in the diagnosis of CMVI, it becomes necessary to assess the ability of class G antibodies to interact with CMV, in order to “neutralize” it later (AT avidity). At the initial stage of the disease, IgG binds very poorly to the antigens of the virus (low avidity) and only then they begin to show activity, therefore, we can talk about an increase in the avidity of antibodies.

We can talk about the advantages of enzyme immunoassay for a long time, 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 taking material for research. In addition, test systems for ELISA continue to improve and the day when the test will give a 100% reliability of the result is not far off.