ELISA blood test for parasites: explanation. What is an ELISA test? What is enzyme immunoassay

In order to determine the level of biologically active substances such as immunological complexes, a special laboratory test is required. If a hematological, infectious or autoimmune disease is suspected, specialists prescribe a blood test for ELISA. As the article progresses, we will look at what it is, how to prepare for it, what it shows and how to decipher it yourself.

What kind of study is this - an ELISA blood test? The abbreviation stands for enzyme immunoassay of blood, during which the quantitative and qualitative content of various biologically active substances, as well as antibodies and antigens in biological material is determined.

What is used in the enzyme immunoassay of blood:

• cerebrospinal fluid;
• mucus of the urethra, cervical canal;
• amniotic fluid;
• contents of the vitreous body.

Enzyme immunoassay is prescribed for the purpose of diagnosing sexually transmitted diseases, infectious and oncological processes, allergic reactions, disorders in the endocrine and reproductive systems, autoimmune and immunodeficiency diseases, as well as to assess the risk of developing certain kidney diseases.

A blood test for ELISA during pregnancy is prescribed for the purpose of early detection, developmental disorders in the initial stages and monitoring their dynamics. In addition, ELISA analysis allows you to control the level of antibodies that can provoke the development of severe neurological disorders in a child, thereby preventing the development of complications not only during pregnancy, but also during childbirth.

It makes it possible to respond to identified problems in advance and significantly improve the health of both the mother and her baby.

The advantages of blood ELISA include:

• high (up to 90%) degree of sensitivity;
• the possibility of early diagnosis, while simultaneously determining the stage of the disease;
• low cost of reagents, and, consequently, availability;
• minimum amount of material for research;
• minimum terms of receipt;
• possibility of long-term storage of reagents;
• the ability to control the dynamics of the development of the pathological process.

Disadvantages of enzyme immunoassay:

• it is impossible to specify the causative agent of the disease; only an immune response to it is obtained in the laboratory
• the possibility of obtaining a false result (in very rare cases, mainly during pregnancy).

Preparation

The principle of this type of blood test is based on the formation of specific antibodies in the human body when antigens enter it, that is, foreign agents that cause the development of a particular disease. As a result of their interaction, “antigen-antibody” complexes are formed, which are studied during enzyme immunoassay.

In this regard, in order not to affect these complexes and, accordingly, the reliability of the results obtained, the analysis of blood and other biological material for ELISA requires certain preparation.

Also, 14 days before the proposed examination, specific treatment is suspended and the use of antibacterial and antiviral drugs is stopped.

Blood for ELISA is donated from a vein. Immediately before blood sampling, you should limit physical activity, and also avoid smoking and drinking alcoholic beverages.

There are several methods and methods for performing enzyme immunoassay: direct and indirect methods, competitive and blocking methods.

The basis for examining blood (any other biological material) using ELISA is an algorithm consisting of two reactions - immune and enzymatic.

During the first, pathological agents bind to the protective elements of the cells of the biological material taken for research. An immune complex “antigen - antibody” is formed. It looks like this. Antigens are present on the surface of absolutely all cells.

When foreign cells enter the body, they are captured by immune cells (on whose surfaces antibodies are located), which compare data on antigens from the surface of newly arrived cells with information stored in their own “memory”. If these data do not coincide, the body’s protective functions are activated, consisting in the formation of an immune complex.

After this, an enzymatic reaction is carried out, which visualizes the results of the previous reaction. Its principle is based on the transformation of a substance from one to another. For each type of ELISA, the material formed during the immune reaction has a specific enzyme, the treatment with which is used to determine the concentration of the foreign agent (based on the intensity of the color of the solution).

Decoding

Thanks to a blood test for ELISA, laboratory technicians determine A, G and M. A positive result when testing blood (other biological raw materials) for ELISA makes it possible not only to make a diagnosis, but also the stage of the disease, as well as the nature of its course (acute or chronic).

These immunoglobulins, in the presence of a pathological process in the patient’s body, are formed during different periods of the disease:

• Immunoglobulins M, IgM– are formed five days after the onset of the disease and remain in the blood for 1.5 months, after which they disappear. Detection of IgM indicates the presence of an acute primary pathological process in the body and the need for enhanced therapeutic intervention.
• Immunoglobulins G, IgG– occur four weeks after infection, their presence in the body can be detected for several months. Detection of IgG indicates a person's history of previous disease. An increase in their level may also indicate re-infection.
• Immunoglobulins A, IgA– appear at the time of infection and are present in the body for four weeks. A decrease in their content indicates the beginning of recovery. Reappearance in tests after a person has undergone treatment and recovered indicates that the disease has become chronic. The simultaneous detection of IgA and IgG in the ELISA results also indicates the presence of a chronic process in the body.

It should also be noted that the simultaneous presence of IgG and IgM in the analyzes indicates that the chronic process has worsened and the patient needs to start treatment. If none of the immunoglobulins were detected during the examination, this means the absence of an immune response.

Content

Modern diagnostics cannot do without highly sensitive laboratory tests. Previously, to establish the cause of the disease and detect the causative agent of the infection, doctors carried out various microscopic multi-stage studies. Today, in order to refute or confirm the initial diagnosis, you need to do a single test - an enzyme-linked immunosorbent assay (ELISA). This laboratory test helps assess a person’s health status and diagnose hematological, oncological, autoimmune, and infectious pathologies.

What is an enzyme immunoassay blood test?

The enzyme immunoassay method is a modern laboratory test of blood for the presence of antigens, antibodies to pathogens and viruses of the disease. The ELISA method helps the doctor identify the etiology of the disease, determine its phase, how old it is, the level of danger to humans, and make the necessary adjustments to treatment. More often than others, enzyme immunoassays examine the presence of antibodies of groups M and G. What are they?

When a pathogenic microorganism enters the bloodstream, the immune system initiates a protective reaction in the form of the release of antibodies (immunoglobulins). These substances bind to the cell and reveal whether it is part of the body or came from outside. If the system has determined that the microorganism is foreign, then the number of antibodies increases in order to combat the pathogenic virus. Immunoglobulins (Ig) come in several types: some appear during infection, others remain throughout life, developing lasting immunity. Antibodies in medicine are designated: A, D, E, M, G.

The ELISA method tests blood, although there are other types of enzyme immunoassays. As a rule, they differ in the type of liquid taken, on the basis of which the composition is further studied and the presence of antigens is determined. In this case, both human blood and other liquids are taken for research:

• vitreous contents;
• mucus from the cervical canal and urethra;
• amniotic fluid;
• strokes;
• cerebrospinal fluid.

Indications for use

• allergic reactions;
• immunodeficiency;
• illnesses of viral origin (hepatitis, herpes, Epstein-Barr virus, cytomegalovirus);
• venereal diseases, sexually transmitted STDs (ureaplasma, syphilis, trichomonas, chlamydia, mycoplasma);
• liver diseases;
• neurosyphilis (infectious lesion of the central nervous system).

A blood test for ELISA is often performed during a preoperative comprehensive examination to determine hormone levels and assess the quality of treatment. The high accuracy of the data obtained helps the doctor to have an idea of ​​the complete picture of the health status. At the same time, results are obtained in a short period of time, which allows you to monitor the dynamics of the development of the disease.

Advantages of the method

The undeniable advantages of the blood ELISA method are its high sensitivity, i.e. the ability to determine the desired substance, even at low concentrations; and specificity, implying error-free diagnosis. In addition, the study of blood serum by ELISA has the following advantages:

Flaws

The main disadvantage of the enzyme-linked immunosorbent test is that when conducting the study, the doctor must have an assumption about the nature of the disease in advance. When diagnosing infectious diseases, it is impossible to accidentally find the pathogen and establish its immunoenzyme properties. The test can only indicate the presence of antibodies in the patient’s blood, which indirectly indicate the presence of a harmful microorganism.

In addition, if the technique is violated or the preparation is incorrect, the analysis may show a false positive or false negative result. Blood serum ELISA testing is an accurate, but expensive method, so it should be used in extreme cases. The interpretation of the results should only be trusted to a qualified specialist.

Preparation

Enzyme immunoassay must be carried out in accordance with the doctor’s recommendations, because Research results are often influenced by various external factors. Basic rules for preparing for ELISA:

• venous blood must be donated strictly on an empty stomach (normally, the last meal should be 12 hours before the test);
• on the eve of the test, it is necessary to exclude taking any medications (if the patient is taking antihistamines (antiallergic) drugs, you should consult with the doctor about how long before the start of the ELISA they should be discontinued);
• You are not allowed to smoke or drink alcohol before the examination, because this will negatively affect the result;
• you should definitely get enough sleep before the analysis;
• it is necessary to exclude any physical activity and stress factors;
• Diagnosis of most hormones in the female reproductive system will require blood sampling on certain days of the menstrual cycle.

How is it carried out?

To conduct an enzyme immunoassay, blood is taken from the patient's antecubital vein on an empty stomach. The patient must inform the doctor in advance about the presence of diseases and medications taken so that the results of the study are not distorted. As a rule, all medications should be stopped 16 days before ELISA. The sensations during the procedure are similar to taking blood during a biochemical analysis.

The material is sent to a laboratory, where serum containing antibodies is isolated from the blood. The resulting composition is placed in a test tube with antigens. These can be a variety of allergens (milk, wool, pollen, citrus fruits), pathogens of viral and infectious diseases, and others. After obtaining the reaction, all remaining whey is discarded. Using special indicators, specialists determine the amount of antibodies. The turnaround time for ELISA depends on the laboratory. As a rule, research results can be provided within two days to a week..

Decoding ELISA

An enzyme immunoassay blood test helps determine the presence of antibodies in the body. There are several classes of immunoglobulins:

1. IgM. The very first ones appear after infection. The presence of these antibodies indicates the occurrence of the disease in any case, because a healthy person does not have this class. Typically, IgM immunoglobulins are present in the blood for approximately 6 weeks.
2. IgA. Antibodies are found in large quantities in the mucous membranes, protecting the body from the penetration of pathogenic microbes. If a patient has this class, it is necessary to fight the disease more intensively. After all, immunoglobulins A arise only in chronic diseases. The disappearance of IgA indicates the destruction of the infection.
3. IgG. Immunoglobulins of this class indicate that a person is either a carrier of the infection or has already suffered from the disease. These antibodies are produced after IgM one month after infection. Class G immunoglobulins can be present in the body for 5-6 years, protecting it from relapse of the disease, and with syphilis such antibodies are present for life.

When performing an ELISA test in children (up to 1.5 years), it should be taken into account that the child’s blood contains mother’s IgG antibodies to infections. Although this does not mean that the baby is sick, rather this fact is the norm. The presence of class M indicates an intrauterine or acquired infection after birth, because Mother's IgM antibodies are not able to penetrate the child's body through the placenta. A breakdown of possible combinations of the presence or absence of antibodies of 3 classes is presented in the table:

The ELISA results must be interpreted by a qualified physician.. As a rule, (+) indicates a positive outcome of the analysis, and (-) indicates a negative outcome. A result that shows the absence or presence of a substance is called qualitative. Sometimes it is supplemented by quantitative, which displays the number of different substances in the body. Often the test system has its own reference (correlated) values. Exceeding such indicators means the presence of pathologies in the patient.

Contraindications

No cardinal contraindications for enzyme immunoassay were identified. Sometimes during pregnancy, when the patient experiences continuous changes in hormones in the blood, it may be necessary to conduct the analysis more than once for a more reliable result. The diagnostic method is not recommended for use after:

• surgical interventions;
• hemolysis (destruction of red blood cells);
• blood transfusions;
• taking a puncture or biopsy of biological material.

Price of enzyme immunoassay blood test

The cost of an ELISA test depends on the policy of the medical institution, the type of analysis and the antigen being determined, because Based on these factors, the price of reagent kits is calculated and the complexity of the study is established. As a rule, an enzyme immunoassay blood test is a publicly available procedure; the average cost of the method varies from 300 to 2000 rubles. Approximate prices for enzyme immunoassay blood tests in Moscow are presented in the table:

Video

ELISA (enzyme-linked immunosorbent assay, ELISA) entered the life of practical medicine somewhere in the 60s of the last century. Its initial task was histological research 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 types of malignant neoplasms underlies the early detection of a tumor, when it is not yet determined by any other method 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 monitor pharmaceutical drugs in order to identify their effect on the patient’s body and, by the way, not only human. 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 (thyroid and adrenal hormones, sex hormones);
• The presence of viral and bacterial infections (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 machines (“washers”) and reading spectrophotometers, which mostly involve manual labor. 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.

The interpretation of ELISA results is within the competence of the laboratory diagnostics physician, and the inherent property of almost all immunochemical reactions to give false-positive or false-negative answers must also be 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, the patient's venous blood taken on an empty stomach is used. 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 confidence that many social problems have disappeared (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 immunoassay method used to diagnose HIV infection is one of the most important standard studies, which, however, requires special conditions, since the topic is very delicate.

It makes sense to carry out HIV ELISA after sexual contact, blood transfusion, other medical procedures that suggest 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 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 at the earliest 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 (+) involves a new blood draw 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 individually using 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 to a month and a half, that is, the 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 of the onset of a 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 the immune status of a particular 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 later “neutralize” it (AT avidity). At the initial stage of the disease, IgG binds very poorly to viral antigens (low avidity) and only then begins 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

Modern diagnostic techniques make it possible to identify a particular disease in a laboratory using special tests. One of these is an enzyme-linked immunosorbent blood test, which can confirm a previously made diagnosis.

Enzyme immunoassay ELISA is one of the most effective and modern methods for identifying disorders associated with immune and hormonal imbalances, as well as oncological processes. During the test, antibodies produced when there is an infection in the body can be detected in the blood. Taking this nuance into account, the disease can be detected even at the earliest stage of its development.

What is the basis of the technique?

The results of ELISA analysis are based on obtaining chemical reactions to enzymes, which serve as special identification marks for recognizing antibodies. Consequently, during immunochemical reactions, antibodies begin to interact with certain antigens. All this gives grounds to assert that false results when donating blood to ELISA are minimal.

The study allows you to determine the number of immune cells, their properties, as well as the presence of the necessary antibodies

The result is considered positive when coloration of the solution is detected. The color indicates that the antigens are interacting with the antibody. If nothing like this happens, the result is negative.

– a modern laboratory test that searches for specific antibodies in the blood or antigens to specific diseases in order to identify not only the etiology, but also the stage of the disease. ELISA results can be given qualitatively and quantitatively.

Currently, ELISA is used in the following situations:

1) Search for specific antibodies to any infectious disease;
2) search for antigens of any diseases (infectious, venereological);
3) study of the patient’s hormonal status;
4) examination for tumor markers;
5) examination for the presence of autoimmune diseases.

Advantages of the ELISA method:

1) High specificity and sensitivity of the ELISA method (more than 90%).
2) The ability to determine the disease and track the dynamics of the process, that is, comparing the number of antibodies in different time periods.
3) Availability of ELISA diagnostics in any medical institution.

Relative disadvantage:

1) Detection of the immune response (antibodies), but not the pathogen itself.

Basic Concepts

Before we clarify the essence of the ELISA method, let us briefly understand some concepts.
Antibodies (or immunoglobulins - Ig) – specific proteins produced by B -
lymphocytes (immune cells) in response to any infectious pathogen (viruses, bacteria, fungi, etc.) entering the body. There are immunoglobulins A (IgA), immunoglobulins E (IgE), immunoglobulins M (IgM), immunoglobulins G (IgG), immunoglobulins D (IgD). They differ from each other in molecular shape and weight, half-life, participation/non-participation in infectious processes, and timing of detection from the moment of infection. If we consider the molecular weight, then IgM has the largest weight - it is a pentamer (950,000 daltons) in contrast to other Igs (from 150 to 200,000 Da), due to which IgM simply cannot pass through the placental barrier. Therefore, the detection of IgM in a child of 1 year of age is always a sign of the presence of infection in the fetus. In blood serum, the bulk of immunoglobulins is represented by IgG (75-85%), and the lowest is IgE (0.003%). Only IgA, M, G are directly involved in the infectious process. IgE is a sign of allergic reactions and diseases, and IgD can only be found in the tissue of the lymph nodes and tonsils and plays a role in the formation of local immunity.

Antigens – high-molecular substances of organic origin, in particular pathogens of infectious and other diseases, as well as substances of various altered cells formed during a particular disease (autoimmune diseases, oncology).

Immune complex – an antigen-antibody complex involved in the immune process.

What is the ELISA method based on?

There are several types of ELISA (direct, indirect, blocking method, competitive), but in practice the heterogeneous solid-phase immunoassay or ELISA (enzyme linked immunosorbent assay) is most often used.

The basis of enzyme immunoassay is the immune reaction of antigen and antibody with the formation of an immune complex: antigen-antibody, which results in a change in the enzymatic activity of specific marks on the surface of the antibodies.

In simple terms, this process can be divided into several stages:

1) On the surface of the wells of the tablet of the doctor conducting the examination, there is a purified antigen of a certain pathogen. When biological material (blood serum) from the patient is added, a specific reaction occurs between this antigen and the desired antibody (immunoglobulin). This compound will act as a “special antigen” in the next step.

2) At this stage, the formation of IC (immune complexes) occurs - a reaction between a “special antigen” and a conjugate (this is an immunoglobulin labeled with the enzyme peroxidase). A special chromogen is added. The result of this enzymatic reaction is the formation of a colored substance in the well of the tablet, the color intensity of which depends on the amount of immunoglobulins (antibodies) contained in the patient's material.

3) Next, the result is evaluated: photometry using a multichannel spectrophotometer, comparison of the optical density of the material under study with the optical density of control samples, mathematical processing of the results. The amount of antibodies in a patient directly depends on the height of the optical density of a given well.

Typically, 96-well plates are used in practice.

When measuring the optical density (OD) of the test liquid, the amount (or concentration) of antibodies in a certain unit of volume is calculated. The result is then compared with a control sample.

Need to remember:For each test system, individual indicators are developed to record the results, indicators of normality and pathology (that is, “reference values”). This must be taken into account when assessing the results of each specific study. It is incorrect to interpret the results of one laboratory based on the “reference values” of another laboratory. It is also incorrect to compare the results of different laboratories with each other.

When performing ELISA reactions, the concept of antibody avidity is also important.
Antibody avidity - this is the strength of the connection between the antibody and the antigen and the amount of antigen that is in relationship with immunoglobulins (antibodies). Avidity is of great importance in assessing the expected duration of infection, which is extremely important when diagnosing primary infection in pregnant women.

The basis of the antibody avidity test consists of treating the immune complex (antigen-antibody) with a urea solution to destroy the protein. High-avidity bonds remain intact, while low-avidity bonds are destroyed. The result is given as an avidity index expressed as a percentage (%).

What diseases are detected using ELISA diagnostics?

2. Markers of autoimmune diseases and indicators of human immunity(total IgE, total IgG, total IgA, total IgM, total IgD, secretory IgA, IgG 2, IgG4, CEC-circulating immune complexes, IgA and IgG to gliadin and others)

3. Oncological markers(TNF - tumor necrosis factor, CEA - carcinoembryonic antigen, PSA - prostate-specific antigen, hCG - human chorionic gonadotropin, CA 125, alveomucin and many others)

4. Reproductive disorders I (estradiol, progesterone, prolactin, testosterone, AFP-alphafetoprotein, FSH - follicle-stimulating hormone and others)

5. Thyroid diseases(free and bound T3, T4, thyroglobulin, thyroid peroxidase - TPO, thyroid-stimulating hormone - TSH).

This list does not represent all diseases that are diagnosed using enzyme immunoassay.

Material for ELISA analysis and rules for its collection

The most common material for the ELISA reaction is the patient's blood serum taken on an empty stomach. The material can also be cerebrospinal fluid, amniotic fluid, vitreous contents, mucus of the cervical canal and urethra, smears.

Preparing patients to submit material for ELISA

Production time for ELISA

Enzyme immunoassay of the material is carried out quickly, within 24 hours. Delays may occur in different laboratories due to the accumulation of a certain amount of serum.

Possible results of ELISA diagnostics

When assessing the results for specific infections, the class of antibodies detected and their quantity are important. Not only the question of the etiology of the infection (whether it exists or not), but also the expected stage of the disease (acute, chronic), as well as the presence of an active infection (acute or exacerbation of chronic) at the time of examination depends on this.

What is the approximate time frame for the appearance of antibodies (immunoglobulins - Ig)?

The earliest antibodies are IgM. They can be detected 1-3 weeks after possible infection, which characterizes the acute phase of the infectious process. The second situation for the appearance of IgM antibodies is the activation (or exacerbation) of a chronic process. IgM antibodies circulate on average for about 3 months, then their number gradually disappears. However, in some patients, trace amounts of IgM can be detected within 1-2 years after infection.

Modern test systems are highly sensitive, resulting in nonspecific false-positive results (often in pregnant women). Therefore, in this group of patients, positive IgM must be rechecked!

IgA antibodies appear 2-4 weeks after infection, but in quantities sufficient for detection within a month. Serum IgA is synthesized by plasma cells of the spleen, lymph nodes and mucous membranes. Secretory IgA is concentrated on the mucous membranes to perform its protective function - they participate in local immunity.

From the 4th week after infection, IgG antibodies begin to appear. With most infections, their titer gradually increases with a maximum at different times (on average after 1.5-2 months), then the titer remains at a low level and indicates immunity. In some diseases (mycoplasmosis, chlamydia, trichomoniasis), the IgG level is not high and decreases significantly due to the lack of immunity in these infections.

Options for detecting antibodies of different classes:

Isolated detection of IgM antibodies suggests the presence of a primary
infection.
- Simultaneous detection of IgM and IgG in the blood is characteristic of primary infection
in the previous 2-3 months, as well as during exacerbation of a chronic disease. Therefore, during pregnancy, the presence of IgM is not always a sign of primary infection.
- Detection of isolated IgG may indicate immunity to this disease,
as well as for chronic infection. In the second situation, both the amount of antibodies (titer) and the change in this titer over time are important. Typically, studies are carried out at intervals of 2-4-6 weeks.
- Detection of IgA isolated or with IgM indicates a primary infection. At
The appearance of IgA along with IgG suggests activation of a chronic infection (on average 2 weeks from the moment of exacerbation).

Definition IgG antibody avidity is an excellent complementary stage in the diagnosis of primary infection from long-standing infection, which has its clinical significance, first of all, when assessing the risk of intrauterine infection of the fetus. The detection of low-avidity IgG indicates a primary infection and is detected on average 4-6 months after infection, rarely longer. Low-avidity IgG requires other laboratory confirmation of primary infection (IgM). High-avidity antibodies are either a sign of a chronic disease and its exacerbation, or of formed immunity.

Features in infants:in children up to one year old, and sometimes even 1.5 years old, maternal IgG to various infections circulates in the blood (that is, they penetrated through the placenta from mother to fetus during intrauterine development). They are not in themselves a sign of the presence of infection in the present. If IgM is detected at this age (remember that maternal IgM cannot penetrate the placenta), then this is a sign of intrauterine infection or an infection acquired after birth.

Quantitative ELISA method

The result of ELISA diagnostics (using an enzyme immunoassay analyzer) is given in certain units of measurement:
- Optical density (OD) of a sample – the concentration of specific antibodies per unit volume. The higher the OD of the sample, the higher the concentration of antibodies. Some results refer to the positivity coefficient (CP), which is also the optical density of the sample.
- Units of antibody concentration (nanogram/milliliter or ng/ml).
- In the form of serum titers: 1:20, 1:40, 1:100, 1:200, 1:400, 1:800, 1:1200 and so on. Diagnostic titers (at which the diagnosis of the disease is made, and not the fact of infection) are different for different diseases.
- In the form of symbols – “+”, “-”, “?” (+, ++, +++, ++++).
- In the form of a qualitative assessment according to a given criterion (positive or negative).

Only a doctor can correctly assess the number of antibodies, the option of class detection of immunoglobulins, and, therefore, set the stage of the disease and the need for treatment.

We must not forget that for any test system its own “reference values” (variants of the norm) are developed, when exceeded, a particular disease is diagnosed (variants of pathology). For different test systems, the “reference values” are different.

Correct comparison of ELISA results taken over time is only possible if they were produced in the same laboratory.

Infectious disease doctor N.I. Bykova