Blood test for Epstein-Barr virus for antibodies and PCR: how to pass, norms

By herpes we are accustomed to understand unsightly painful blistering rashes on the face in the lip area, which subsequently form brown crusts. Alas, this is only one of the faces of the herpes virus, which can occur in humans in 8 forms. What we usually call herpes is type 1 virus, or herpes simplex virus. Type 2 virus causes genital herpes, type 3 - "chickenpox" and shingles, type 4 - infectious mononucleosis and several other quite dangerous pathologies, etc. This list can be continued further, but we will focus on herpes virus type 4, which is otherwise called the Epstein-Barr virus. Let's try to figure out what type 4 herpes virus is, why it is dangerous, when and why an Epstein-Barr virus test is performed, and what the results of laboratory tests indicate.

What is herpes virus type 4?

Herpesvirus type 4, as one of the varieties of herpesvirus infection, was described 53 years ago by the English virologist Michael Epstein. In his work on the project, the professor was assisted by his graduate student Yvonne Barr. It is to these people that the virus owes its name. However, 15 years after the virus was discovered, its scientific name was changed to human herpesvirus 4, and a year ago the virus was called human gammavirus type 4.

But what is the Epstein-Barr virus? Like any other virus, the virion (viral particle) of herpesvirus type 4 consists of genetic material (in this case, double-stranded DNA) and a surrounding protein shell (capsid). In addition, the virus is surrounded by a membrane that helps it easily penetrate into the host's cells.

Any virus is a non-cellular form, which is an infectious factor and cannot develop and reproduce outside the cells of a living organism. The favorite habitat of herpes virus type 4 is considered to be the epithelial cells of the nasopharynx. They do not disdain leukocytes either, giving preference to one of their varieties, called B-lymphocytes. It is B-cells that actively participate in providing the body's immune defense. When in contact with an antigen, which in our case is the herpes virus type 4 (or rather its antigens), B-lymphocytes produce antibodies (immunoglobulin proteins). They can be detected in the patient's blood by conducting an analysis for the Epstein-Barr virus (EBV).

The herpes virus type 4 has 4 antigens that appear in a strictly defined sequence:

• EA is an early antigen that appears at the initial stage of the disease, when viral particles are in the synthesis stage (primary acute infection or reactivation of a latent virus when immunity is reduced),
• VCA is a capsid antigen that is contained in the protein shell and also belongs to the early ones, because clinically the disease may not even manifest itself in this period,
• MA – membrane antigen, appears when the virion has already been formed,
• EBNA – nuclear (polypeptide or nuclear) antigen is one of the late antigens, antibodies to which can be detected even several months after the disease and remain in the blood throughout life.

Herpes virus type 4 is very insidious. Since the virus is inactive outside a living organism, it can only be contracted from a person who is the source of the infection. And it is not at all necessary that he or she exhibit all the symptoms of the disease; the infection can be latent, disguised as ordinary fatigue. For example, chronic fatigue syndrome is in most cases associated with the Epstein-Barr virus.

Individual virions can be found in blood, saliva, sperm, vaginal secretions, and tissues of various organs. Virus particles, along with saliva and blood, can enter objects around us, where they will remain inactive until they somehow enter the human body. In the vast majority of cases, infection occurs by airborne droplets or contact (through kissing). But intrauterine transmission of the virus from mother to fetus, infection during a blood transfusion (if the donor blood contained virus virions), and sexual contact are also possible.

After entering the body and penetrating into cellular structures, it may take from 5 to 50 days before the disease makes itself known. But it may not make itself known, proceeding in a latent form, as is the case in most cases.

Yes, according to research, about 90% of the adult population has had an EBV-associated herpes infection at least once in their lives. Most people don’t even know it because their body is able to fight off the viral attack. But this is not always the case.

How does the Epstein-Barr virus manifest itself?

Most often, doctors have to deal with the following types of herpesvirus infection type 4 in their practice:

• Chronic form (occurs after the acute phase of the disease, has some general symptoms of ill health),
• Latent or hidden form (there are no symptoms, but the virus remains active and is released into the environment),
• Slow form (less common, symptoms occur one at a time over a long period, ending in the death of the patient).

People are first infected with the Epstein-Barr virus, primarily in childhood and adolescence. The peak incidence occurs between the ages of 14 and 18.

Primary viral infection has 3 different forms:

• asymptomatic (no clinical manifestations),
• respiratory (symptoms of respiratory infection: fever, nasal discharge, general weakness, etc.),
• Infectious mononucleosis with a triad of main symptoms: high temperature, signs of sore throat with yellowish crusts on the tonsils, enlargement of organs such as the liver and spleen; there is an increase in the level of leukocytes and enlargement of the lymph nodes.

There are several options for getting out of the acute phase of the disease:

• full recovery,
• the symptoms of the disease disappear, but the virus remains in the body and develops, although it no longer leads to noticeable changes in the cells (carrier state),
• there are no symptoms of the disease, the virus does not leave the body, but also shows little activity (latent form),
• reactivation of the virus from a latent form,
• chronic course of infection (with relapses of the disease, chronic active form, generalized with damage to organs and body systems).

The result of a long stay of the virus in the body can be:

• Chronic form of infectious mononucleosis.
• Hematophagocytic syndrome: stable fever, decreased blood components (increased coagulability), enlarged liver and spleen, bleeding of mucous membranes, jaundice (due to liver dysfunction), enlarged lymph nodes, neurological symptoms.
• Latent form with the development of secondary immunodeficiency: hyperthermia over a long period of time, general weakness, enlargement and soreness of the lymph nodes, muscle and joint pain, frequent infectious diseases.
• Development of autoimmune pathologies in the form of lupus erythematosus, rheumatoid arthritis, etc.
• Manifestations of chronic fatigue syndrome with deterioration of general well-being and performance.
• Generalized form of chronic infection with damage to the central nervous system, cardiac myocardium, kidneys, liver, and lungs.
• Development of oncological diseases (lymphocytic leukemia and lymphomas), in which a pathological increase in the number of lymphatic system cells is observed. The herpes virus type 4 does not destroy carrier cells, but forces them to actively multiply, as a result of which neoplasms from lymphoid tissue are detected.

As we can see, the Epstein-Barr virus is not as harmless as it seems at first glance, which means that it should not be treated carelessly. Moreover, herpes virus type 4 is characterized by frequent episodes of virus carriage and latent form, not to mention various forms of chronic infection, when a person remains a source of infection without even suspecting it.

In this case, the presence of an infectious agent in the body can only be determined using a special analysis for the Epstein-Barr virus, the biomaterial for which is usually blood.

Indications for the procedure Epstein-Barr virus test.

Since herpesvirus infection type 4 is sometimes not so easy to detect, it is not always suspected. But there are certain signs by which a doctor may suspect the presence of the virus in the body:

• severely weakened immune system (patients with HIV infection and AIDS, patients after organ transplantation or chemotherapy are at risk),
• enlargement of regional lymph nodes in the chin and occipital region of the head and their soreness, especially if this is observed after a blood transfusion or organ transplant from a donor.
• acute respiratory viral infection (ARVI), occurring against the background of very high temperatures (38-40 degrees),
• the appearance of signs of infectious mononucleosis, which most often occurs under the influence of the Epstein-Barr virus.

Even if a person does not have the above-mentioned symptoms, a specialist may be suspicious based on some results of routine tests (general blood analysis and biochemistry), as well as immune status studies.

A complete blood count for Epstein-Barr virus may show:

• increase in the number of lymphocytes,
• low hemoglobin, which indicates a decrease in the level of red blood cells,
• increased blood clotting due to a large number of platelets,
• the appearance of virocytes (atypical lymphocytes similar in structure to monocytes).

A biochemical blood test, which provides information about the state of internal organs, will show changes in the functioning of the liver and spleen.

An immunoassay for EBV may show a change in the number of specific lymphocytes, a discrepancy in the number of immunoglobulins of different classes (dysimmunoglobulinemia), and a deficiency of immunoglobulin G, which indicates a weak immune system and its inability to contain the onslaught of the virus.

Such results of non-specific analyses may alert doctors, but it is still impossible to say exactly what they are dealing with. Everything will remain at the stage of assumptions and preliminary diagnosis. In most cases, doctors suspect a latent form of infectious mononucleosis, although other viral pathologies (flu, hepatitis, etc.) can manifest themselves in the same way.

Due to the high prevalence of hepatitis type 4 and the possibility of its transmission from mother to child, the Epstein-Barr virus test will also be useful when planning a pregnancy. If the mother has previously suffered from the infection, her body has developed antibodies to it. Reinfection is usually excluded due to the formation of strong immunity, and if it does occur, it will no longer have the consequences that are possible with the first encounter with the virus. Immunity will restrain the activity of the virus throughout life, although the virus itself will remain inside the body, like any of the herpes viruses.

If the expectant mother contracts the herpes virus during pregnancy, this can lead to miscarriages and premature births, or the virus will negatively affect the intrauterine development of the baby.

An oncologist may order an EBV test if Burkett's lymphoma is suspected or to diagnose tumor processes in people with HIV. A therapist may use this test to diagnose herpes infections (differential diagnostics to determine the type of virus). Sometimes the test is also used to assess the effectiveness of treatment.

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Preparation

Depending on the type of research, the biomaterial for it can be blood, saliva, urine, sputum, amniotic fluid sample, scraping taken from the cervical canal or urethra, cerebrospinal fluid (CSF). Most often, doctors resort to blood testing, which is considered the most informative.

It is clear that some factors may negatively affect the quality and quantity of biomaterial, so it is worth following certain rules the day before:

• Any tests (especially blood tests) are recommended to be taken in the morning on an empty stomach. The last meal should be no later than 12 hours before blood sampling, so it is better to drink water for dinner.
• The most acceptable material for analysis of the Epstein-Barr virus is considered to be venous blood, and before donating blood from a vein, a 15-minute rest is always recommended if a person has just arrived at the laboratory,
• To ensure that blood sampling takes place without consequences and the test results are reliable, it is not recommended to perform active physical work or play sports, drink alcohol, or smoke within 12 hours before the procedure.
• The results of the tests may also be affected by the medications you are taking. You should stop taking medications at least 2 days before the test. If this is not possible, you must inform the lab nurse about the medications you are taking.
• During pregnancy, before testing for EBV, a test for toxoplasmosis is performed to exclude a false positive reaction.
• If a blood test for the Epstein-Barr virus is done on a child under 5 years old, half an hour before the procedure the child should be given a lot of boiled water to drink in relatively small portions.

If another biomaterial is taken for analysis, you need to clarify with your doctor in advance all the nuances of preparation for the analysis depending on the material used.

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Technique Epstein-Barr virus test.

Doctors assign an important role in the diagnosis of herpes type 4 and infectious mononucleosis to specific tests that help identify the DNA of the virus or unique antibodies in the patient's biomaterial. The main types of laboratory tests used to detect the Epstein-Barr virus in the human body include enzyme-linked immunosorbent assay (ELISA) and PCR diagnostics. Let's take a closer look at the essence of both methods and the features of their implementation.

EBV enzyme immunoassay

ELISA is a study (analysis) of venous blood of patients for antibodies to the Epstein-Barr virus. As a result of diagnostics, immunoglobulins of the IgG or IgM type (there are 5 types in total) to one of the 3 antigens of the virus (early, capsid or nuclear) are detected in the blood of patients with EBV.

The analysis is carried out in an immunological laboratory, where about 10 ml of blood is taken from the patient's vein. The biomaterial is then left at room temperature for a quarter of an hour, during which time the blood coagulates. The clot is carefully separated from the liquid part. The liquid is centrifuged and pure blood serum is obtained. It is this that is subjected to further examination.

The idea of the method arose on the basis of data that our body produces specific antibodies to each type of virus and bacteria that penetrate the body from the outside. The body recognizes them as a stranger and destroys them with the help of unique antibodies that firmly adhere to the antigen.

The essence of the ELISA analysis is based on this reaction. Antibodies with tags attached to them combine with antigens. A substance is applied to the tags, which changes the color of the sample when reacting with a special enzyme. The more such "chains", the more intense the color of the biomaterial.

Enzyme immunoassay can be performed using three methods:

• Direct ELISA. The test fluid is placed in the wells and left for about half an hour so that the antigens can attach to the well walls. Liquid with labeled antibodies is added to the sorbed antigens. After the required time (from half an hour to 5 hours), when the antibodies have detected and bound to the antigens, the liquid is drained, the wells are carefully washed and the enzyme is added to them. The concentration of the virus in a unit of blood is determined using the colorization method.
• Indirect ELISA. In this method, the blood serum being tested and labeled antibodies are added to the antigens sorbed on the surface of the wells. As a result, 2 types of ligaments are obtained, some of which are labeled. The result depends on the concentration of antigens in the sample being tested. The more unlabeled antibodies, the fewer compounds labeled by the enzyme.

Next, a special reagent is added to the washed composition, which is used to determine the enzymatic activity of the antigen-antibody complexes.

• "Sandwich". Differs from the indirect method in that initially, not antigens, but antibodies are sorbed on the surface. A solution containing the antigens being studied is added to them. After washing the carrier, antibodies with enzymatic labels are added. Excess antibodies are removed again and, using hydrogen peroxide, a colored substance is obtained, which is studied using a spectrometric method.

This type of analysis allows not only to identify specific antibodies and determine the concentration of antigens, but also to clarify the stage of the disease. The fact is that different antigens of the Epstein-Barr virus appear at different stages of the herpes infection, which means that antibodies to them are produced at a certain period of the disease.

Thus, IgG antibodies to the early antigen (IgG EA) appear in the blood 1-2 weeks after infection, when the disease is in the acute stage or the stage of virus reactivation. Immunoglobulins of this type disappear after 3-6 months. In the chronic course of a viral infection, such antibodies are especially numerous, and in the atypical form, they are absent altogether.

IgG antibodies to the capsid antigen (IgG VCA) also appear early, during the first 4 weeks of the disease, but their greatest number is determined by the second month of infection. In the acute phase, they are found in most patients, but they may not appear in children. In the chronic course of the disease, especially during periods of virus reactivation, the amount of IgG VCA is especially high. These antibodies remain in the human blood forever, like the virus itself, which indicates the formed immunity to the infectious agent.

IgM antibodies to capsid antigen (IgM VCA) may appear even before the first signs of the disease. Their concentration (titers) is especially high in the first 6 weeks of the disease. This type of antibody is characteristic of acute infection and reactivation of chronic infection. IgM VCA disappear after 1-6 months.

IgG antibodies to the nuclear gene (IgG EBNA) may indicate that a person has previously directly encountered a herpes infection. In the acute phase of the disease, they are detected extremely rarely, usually appearing during the recovery period (in the 3rd-10th month). They can be detected in the blood several years after the infection.

Detection of individual antigens does not provide a complete picture of the disease, so tests for different antibodies should be performed together. For example, if only IgM VCA is present, and IgG EBNA is not detected, this is a primary infection.

Unfortunately, enzyme immunoassay is often insufficient to detect primary herpes infection or congenital pathology. In the latter case, antibodies may not be detected at all. As a confirmatory test for primary disease, a molecular study of blood or other biological material for the Epstein-Barr virus is used.

PRC analysis for Epstein-Barr virus

This analysis is carried out at the stage of acute primary infection, otherwise its result will be incorrect.

The essence of the PCR (polydimensional chain reaction) method is that each infectious agent has its own set of genes contained in a DNA molecule. The DNA of the pathogen is contained in the biomaterial taken for research in small quantities (the viruses themselves are microscopic in size), so it is very difficult to assess the situation. But if a specific reaction is carried out, the amount of genetic material will increase significantly, which will make it possible to name the pathogen.

Using disposable instruments, material is taken for molecular research, which is placed in a special device for analysis. The device is a thermostat with a special program - a thermal cycler or amplifier. The device runs a full PRC cycle several dozen times (about 2-3 minutes), which has 3 stages:

• Denaturation (at a temperature of 95 degrees, DNA strands are separated).
• Annealing (at a temperature of 75 degrees, specially prepared “primers” for EBV are introduced into the material being studied, which attach to the DNA of the virus).
• Extension or multiplication of genetic material (a special enzyme is added to the seed at a temperature of 72 degrees, which recreates a new DNA chain, thereby doubling the amount of genetic material).

If the full cycle of the polydimensional reaction is run 50 times, the amount of material will increase 100 times. This means that it will be much easier to identify the pathogen.

Epstein-Barr virus test for a child

As we already know, the Epstein-Barr virus can cause the development of many diseases. Once it enters the human body, it remains its permanent resident, and only the well-coordinated work of the immune system does not allow it to actively parasitize inside cells.

Almost 95% of the world's adult population lives with EBV inside, and most of them became acquainted with the virus in early childhood. Some inherited it from their mother, while others got the virus from parents and relatives rushing to the child with kisses, or by airborne droplets in kindergarten or school (infectious diseases there usually acquire "universal" proportions).

Small children generally tend to put everything in their mouths, and the largest number of virions are found in saliva. And if several children lick the same toy in kindergarten while the teachers are busy with their own affairs, then it is not surprising that the virus spreads so actively in large groups of children.

EBV can easily be called a disease of children and young people, because by adolescence, half of children already have the virus in their body (and by the age of 30, about 90% of adults). Children at different ages get sick in their own way. Up to a year, while the child does not actively communicate with people, the probability of him getting sick is small. A child over a year old, even if he has not gone to kindergarten, becomes more sociable, plays with peers on the street, goes on active shopping trips with his mother, etc., and the probability of catching the virus becomes much higher.

But this is not a reason to lock the child within 4 walls. At the age of 1-3 years, the disease in the vast majority of cases proceeds without any symptoms, except perhaps with a slight rise in temperature and a slight runny nose, reminiscent of a cold. It turns out that the earlier the child gets acquainted with the virus, the easier such acquaintance proceeds.

It is not good if a child gets sick without the appearance of IgG VCA antibodies in the blood, which may indicate that immunity to the virus has not been formed, and reactivation of the virus is possible as soon as the immune system weakens. The reason is most likely the imperfection of the immune system of small children, which is in the process of formation for several years.

School life provides even more prerequisites for the disease, especially in adolescence, when young people actively practice kissing. But in children over 3 years old, the disease is less often asymptomatic. In most cases, doctors encounter infectious mononucleosis with its characteristic symptoms.

Despite the fact that the pathology can have a long course (about 2 months), it is not so dangerous and does not require the use of serious drugs. Doctors prescribe anti-inflammatory and antiviral drugs, if a bacterial infection joins, they seek help from antibiotics. By the way, penicillins are not recommended in this case due to the fact that they can provoke the appearance of a skin rash.

Don't think that if a child or teenager gets infectious mononucleosis, it means that the Epstein-Barr virus has taken up residence in their body. The disease has other less common pathogens, such as cytomegalovirus (herpes virus type 5). To understand what they are dealing with, doctors prescribe an Epstein-Barr virus test and, if necessary, other laboratory tests.

It is also true that infectious mononucleosis is not the only manifestation of EBV in childhood. There are other diseases associated with this pathogen, but in our region they are rare.

Thus, Burkitt's lymphoma (which is what EBV owes its detection to) is found mainly in children in African countries, very rarely in America, and even more rarely in Europe (and then only against the background of AIDS). A jaw tumor with damage to the lymph nodes, kidneys, and other organs is found in children aged 3-8 years.

Nasopharyngeal cancer, a significant proportion of other lymphomas, hairy leukoplakia of the mouth - all these are manifestations of EBV against the background of greatly reduced immunity, which occurs with HIV infection and its later stage of AIDS.

Congenital immunodeficiency and the addition of the Epstein-Barr virus is a dangerous mixture that can lead to the development of proliferative syndrome in a child. In this case, an increase in the number of B-lymphocytes leads to the appearance of granules in many organs, which prevents them from functioning normally. This is a disease with a high mortality rate, but it does not develop against the background of normal immunity.

It can be said that in childhood, the Epstein-Barr virus is dangerous mainly in cases of immunodeficiency due to the development of various complications. In most cases, everything is limited to infectious mononucleosis. And although it does not require special treatment, doctors still prefer to establish the nature of the pathogen, for which the child is prescribed a general blood test, enzyme immunoassay and PCR.

Since in childhood, primary infection mainly occurs, it is quite possible to limit oneself to only a complete blood count and a PCR, which is quite informative when the disease is first detected.

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Normal performance

The results of the PCR analysis are processed by electrophoresis or using labeled "primers". In the latter case, it is sufficient to simply add a reagent (chromogen) and determine by color whether there are virions in the sample. A positive result in electrophoresis is indicated when DNA strands of different lengths are detected in the sample being studied.

During the incubation period of the disease and asymptomatic virus carriage, the PCR will give a negative result, as well as in the case of the absolute absence of the virus in the body. At the beginning of the development of the primary infection and in its early stages, the PCR in real time can give both positive and negative results, which does not clarify the situation in any way.

However, in the midst of the disease (acute phase), in its chronic course or reactivation of the virus (exacerbation) and in the case of atypical forms, the analysis will be positive. If a person has been ill for a long time and the virus in his body is in an inactive state, the PCR analysis will give a negative result, i.e. conducting this analysis during this period is also inappropriate, as well as at too early stages of the disease.

It must be said that accurate results of this type of laboratory research are possible only in the case of primary infection and the absence of virions of other viruses.

Now, regarding the enzyme immunoassay for the Epstein-Barr virus. It has the same requirements. The presence of herpes virus types 5 or 6, toxoplasmosis, and HIV infection in the body can distort the result no less than a careless attitude to the analysis or poor quality of the reagents used. In this case, additional studies may be required, taking into account possible pathogens.

Normal test results, which indicate the absence of the virus in the body, are considered to be a negative result for all 4 tests: IgG EA, IgM VCA, IgG VCA and IgG EBNA. Yes, each test is carried out separately, because antigens appear at different periods of the disease. Sometimes only individual tests may be prescribed, but in most cases all 4 tests have to be done, but at different periods of the disease.

For example, during the incubation period of the disease, as well as in the absence of infection, none of the 4 types of antibodies are detected in the blood. Such a result cannot be considered sufficient, because it does not allow differentiating a person who has fallen ill for the first time from a healthy person.

At the beginning of the development of the primary disease, only IgM VCA antibodies appear in the blood. In the early stages of the disease, IgG VCA join them.

The acute stage of primary infection proceeds with the formation of three types of antibodies: IgG VCA, IgM VCA and IgG EA, with the greatest number of IgG antibodies to the capsid antigen detected. The same composition of antibodies is maintained for six months after the acute phase of the disease, but the amount of IgM VCA gradually decreases to zero.

Six months after the illness, IgG EBNA antibodies appear in the blood, while IgG EA immunoglobulins become less and less, and IgM VCA are absent altogether.

In chronic cases of the disease or reactivation of the virus, there may be different indicators. Most often, all 4 types of antibodies are found in the blood. But it may well be that immunoglobulins IgM VCA and IgG EBNA are not detected.

Complication of viral infection by tumor processes occurs with the absence of IgM VCA antibodies, and IgG EBNA immunoglobulins are not detected in all cases.

But enzyme immunoassay determines not only the presence of certain antibodies, but also their concentration, which allows us to judge the stage of the pathology and its possible consequences with greater accuracy. There is no need to talk about specific figures here. After all, each laboratory conducts the analysis in one of the possible ways, using various reagents, so the results of the analysis of different laboratories may differ in digital format.

The patient is required to be given a form indicating the threshold (reference) values. If the result is below the threshold, it is considered a normal (negative) indicator. If the determined number is above the reference value, everything indicates a positive result, which means the virus is living in the body. The value of the determined value indicates the stage of the disease and the colonization of the body with EBV virions, i.e. the severity of the pathology.

If the ELISA test is negative, it only means that the person has not had contact with EBV in the past. But it is impossible to say with certainty whether the virus is currently present in the body. A negative result can be caused by incubation of the pathogen in the body and asymptomatic virus carriage. Sometimes, to make sure that the body is not inhabited by the virus, it is necessary to conduct a second series of tests after some time.

If the Epstein-Barr virus ELISA result only slightly exceeds the reference values, the result is considered questionable. The cause is most often the early stage of the disease or the presence of virions of another virus in the body. In this case, after 2 weeks, it is recommended to do a repeat test for EBV and possibly for other pathogens.

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As for the question of how long it takes to do an Epstein-Barr virus test and when you can expect the results, there are no particular difficulties to be expected. In a serious, well-equipped laboratory, you will have to wait no more than 2 days after submitting the biomaterial. In situations requiring urgent analysis, the answer can be obtained even after a couple of hours.

The Epstein-Barr virus test should be performed in a trusted laboratory, where there are both high-quality reagents and qualified specialists. After all, the test costs money (and not a small amount, the test for one type of antibody costs about 150-170 UAH), and I would not like to get a false result, and then possibly contact again, but to a different laboratory, for a repeat test.

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