Ifa the analysis is positive. Ifa blood test

It is characterized by broad spectrum of action, speed and convenience of carrying out, high accuracy of results, acceptable cost. It is in demand, therefore it is available in various clinics and laboratories of the city.

Development modern medicine in the world of diagnostics, it never ceases to amaze with its achievements, and now the doctor does not need to make assumptions about the probable diagnosis, relying only on indirect signs. The creation and introduction into the world of laboratory research of the enzyme-linked immunosorbent assay (ELISA) allows you to quickly and accurately determine not only the presence of the pathogen, but also many other characteristics of the disease.

History of creation and development of IFA

This method of blood testing began to be used in practical medicine in the middle of the last century - somewhere in the 60s. Its original goal was Scientific research in the field of histology, which were limited to the search and study of cellular antigenic structure biological species. Blood test by ELISA method is based on the interaction of related antigens (AG) and specific antibodies (AT), with the formation of an "antigen-antibody" complex, determined by the enzyme.

This phenomenon prompted scientists to decide that the method can be used to recognize protein compounds of various classes formed in blood serum when a pathogen enters the body. Due to their direct involvement in the functioning of the immune system, these compounds are called immunoglobulins (Ig, Ig), and the discovery was the greatest breakthrough in laboratory diagnostics.

At the same time, this highly sensitive method began to be actively used only in the 80s, and it was available only in highly specialized medical institutions. The very first received the opportunity to use enzyme immunoassay analyzers at stations and blood transfusion centers, venereal and infectious medical institutions. This was due to the rapid spread of the "plague of the 20th century" - AIDS, and urgent diagnostic and therapeutic measures were required.

Possibilities of the technique

The scope of the blood test for ELISA is quite wide - for this moment it is impossible to imagine how complicated the search for the cause of many diseases would be. Such research is now used in almost all medical fields, even in oncology. Although it is difficult for the uninformed to understand, but in individual cases after conducting it, they managed to save the lives of patients, finding a tumor on early stages.

Important! ELISA is a blood test that shows markers characteristic of some malignant processes, which makes it possible to detect the disease at that time stage when it is beyond the control of any other method.

In modern diagnostic centers, laboratory examinations are represented not only by tumor markers - they are equipped with an impressive arsenal of panels for carrying out this diagnosis. They can be used to determine the set pathological conditions, such as hormonal abnormalities and infectious processes of various origins.

In addition, conducting and decoding an ELISA blood test will make it possible to track the effect of medications on the body of a sick person and even an animal. The latter is widely used in veterinary clinics helping to maintain the health of our pets or breadwinners, providing a stable supply of meat, dairy products and eggs irreplaceable in the diet.

So, as a result of taking only a few milliliters of venous blood and its diagnosis using the ELISA method, the doctor, having described the research materials, will be able to determine:

• a hormonal condition that includes biologically active substances genital and thyroid glands, as well as adrenal glands;
• the presence of bacterial and viral infection(hepatitis B and C, syphilis, herpes, chlamydia, tuberculosis, myco- and ureaplasmosis, HIV, TORCH) and other diseases of this nature;
• signs of vital activity of pathogens of the pathological process, which ended in recovery and passed into the stage of antibody formation (immune response).

Such complexes are much easier to recognize and eliminate by immune cells. Residual effects in the form of antibodies in multiple cases are contained in the blood for life, which practically reduces the risks of re-infection to zero.

Varieties of immunoglobulins

There are several types of antibodies, and each of them is involved in the process of the immune response at a certain stage. For example, immunoglobulins of class M (IgM) are the first to be formed in response to the ingress of hypertension into the body. Their highest rates are observed in the first days of the disease.

Types of immunoglobulins used in ELISA

Then the immune system launches class G Ig (IgG) into the plasma, which are responsible for the complete destruction of antigens and the patient's recovery. Later, they continue to be in the blood, thereby preparing immunity for the repeated hit of an identical pathogen. This is how vaccination works. With the introduction of weakened antigens of pathological microorganisms, many immunoglobulins appear and remain circulating in the plasma.

The main objects of interest for laboratory diagnostics are Ig classes M, G and A. By the level of their concentration, you can determine the stage of the disease and find out what infectious diseases a person has had in his life. For example, you can check if there was a history of chicken pox or rubella. The doctor, in order to find out whether a certain type of AT or AH is present in the patient's body or the concentration of any hormone, does not need to prescribe multiple laboratory examinations - it is enough to prescribe a referral for ELISA.

The essence of the technique

The research methodology is based on several options (direct and indirect - competitive and non-competitive) for performing the assigned tasks, each of which is intended for specific purposes. This approach allows for a targeted search, and in the shortest possible time to identify the cause of a particular pathology. To detect Ig of different class categories, a 96-well plate (polystyrene panel) is used, and sorbed recombinant proteins are located in its wells. They play the role of antigens, and at the initial stage are in the solid phase.

When it enters the well with blood plasma, antigens or antibodies identify the object in connection with their orientation, and form a complex (AG - AT). This formation is fixed by an enzyme compound (conjugate), which will subsequently manifest itself as an altered coloration of the well. ELISA is produced on specific test systems manufactured in specialized laboratories and equipped with a full set of reagents.

This analysis can be performed on washers - washers and spectrophotometer readouts, but they require manual labor... Of course, it is several times more convenient and faster for a laboratory assistant to carry out all manipulations on fully automated devices. When using them, laboratory staff are freed from a large amount of monotonous activity - washing, instilling and the rest of the routine, but not all medical institutions can afford such expensive equipment.

Therefore, many hospitals and diagnostic institutions continue to carry out ELISA in the old fashioned way - on semiautomatic devices.

Interpretation of research materials is exclusively the competence of a laboratory diagnostics specialist - only he can be told the results about the specifics and subtleties of the course of the disease. In this case, the doctor must take into account the possibility of obtaining false negative or false positive responses.

Decoding of materials

The result of a high-quality enzyme immunoassay should be an unambiguous conclusion - the desired microorganism was found or not in this blood sample. Quantitative analysis will indicate the level of concentration and can be expressed in two ways - by value in numbers or by the number of "+" signs.

Analyzed indicators

During the research, a careful study of the main immunoglobulins involved in the immune response is carried out, such as:

• IgM - detection of this class means development acute form infectious disease. A negative result for the search for IgM can be both evidence of the absence of the desired pathogen, and the transition of the disease to chronic course.
• IgA - determination of this class in the absence of IgM in most cases, a signal of a chronic or latent form of development infectious disease.
• IgM and IgA (simultaneous presence) - positive results for both species indicate the very peak of the acute form of pathology.
• IgG - its presence indicates the transformation of the disease into chronic form or for recovery and the formation of immunity to the agent being determined.

The emergence and accumulation of a certain class of IS occurs at different time stages. So, for example, IgM appears first, about 5 days after the pathogen hits. IGs remain in the blood for about 5-6 weeks, and then gradually disappear. At this time, they are available for determination by ELISA. Approximately 3-4 weeks after the onset of the disease, IgG appears, which subsequently can remain for several months. But they are not always found in analysis.

IgA are formed in the blood in the period of 2-4 weeks, while 20% of them are contained in the serum, and 80% - in the secretion of the mucous membranes. As a rule, these immunoglobulins disappear within 2-9 weeks, which indicates the destruction of the pathogen and the patient's recovery. If ELISA nevertheless shows the presence of IgA, then this signals the transition of the process to a chronic form.

Analysis options

Depending on the data obtained, ELISA answers can be issued on a form in the form of a table with a complete list of all AT and AG and an indication of a positive or negative reaction. In certain situations, a quantitative value will be displayed - a sharply positive, positive, weakly positive or negative result. The second option indicates the amount of antibodies contained in the blood sample under study.

ELISA materials interpretation options

In addition to the above values, another quantitative parameter is investigated in the ELISA process - the AT avidity index, calculated as a percentage. It shows how long the disease lasts - that is, the higher the indicator, the more time it takes for the pathology to develop.

Alternative ELISA method

An enzyme-linked immunosorbent assay is a fairly well-known and common diagnosis. Perhaps some have never heard of it, but there is another species that is even less known to a wide range of people. this study in which a non-blood sample is taken. This technique is called the analysis of feces for occult blood, and in many cases it allows you to avoid additional exhausting procedures, which are also accompanied by unpleasant sensations.

An ELISA test for occult blood (hemoglobin molecules) makes it possible to detect bleeding of the digestive system, even insignificant, the signs of which in the patient's stool, as they say, cannot be found with the naked eye. An enzyme-linked immunosorbent assay in human feces in a short time is able to show peptic ulcer disease, polyposis, diverticulosis, tumors that are not accompanied by certain symptoms in the early stages.

To date, thousands of varieties of enzyme immunoassay test systems have been created, which provide the ability to find antibodies and antigens of a huge list of pathologies. Therefore, this analysis is used in almost all branches of medicine, for any age category. And the absolute harmlessness allows you to resort to it both during pregnancy and for the diagnosis of debilitated patients.

Content

Modern diagnostics is not complete without highly sensitive laboratory tests. Previously, to establish the cause of the disease and detect the causative agent of the infection, doctors performed various microscopic multistage 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 research helps to assess the state of human health and diagnose hematological, oncological, autoimmune, infectious pathologies.

What is an enzyme immunoassay

The enzyme-linked immunosorbent assay 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 to identify the etiology of the disease, determine its phase, the age of origin, the level of danger to humans and make the necessary adjustments to the treatment. More often than others, enzyme immunoassay examines the presence of antibodies of groups M and G. What are they?

On hit pathogenic microorganism into the bloodstream, the immune system turns on a protective response 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 has come from outside. If the system has established that the microorganism is foreign, then the number of antibodies increases in order to fight the pathogenic virus. Immunoglobulins (Ig) are of several types: some appear during the period of infection, others remain throughout life, developing strong immunity. Antibodies in medicine are designated: A, D, E, M, G.

Blood is examined by ELISA, although there are other types of enzyme-linked immunosorbent assay. As a rule, they differ in the type of fluid 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:

• the contents of the vitreous body;
• mucus from the cervical canal and urethra;
• amniotic fluid;
• smears;
• cerebrospinal fluid.

Indications for appointment

• allergic reactions;
• immunodeficiency;
• ailments of viral origin (hepatitis, herpes, Epstein-Barr virus, cytomegalovirus);
• sexually transmitted diseases STDs (ureaplasma, syphilis, Trichomonas, chlamydia, mycoplasma);
• liver disease;
• neurosyphilis (infection of the central nervous system (central nervous system)).

An ELISA blood test is often done during preoperative comprehensive survey to determine the level of hormones and assess the quality of the treatment. The high accuracy of the data obtained helps the doctor to have an idea of ​​the detailed picture of the state of health. At the same time, the results are obtained in a short time period, which makes it possible to track the dynamics of the development of the disease.

Method advantages

The indisputable advantages of the blood ELISA method is its high sensitivity, i.e. the ability to determine the desired substance, even at its low concentration; and specificity, implying that the diagnosis is accurate. In addition, the study of blood serum by the ELISA method has the following advantages:

Flaws

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

In addition, if the technique is violated or improper preparation, the analysis may show a false positive or false negative result. The study of serum ELISA is an accurate, but at the same time expensive method, so it should be consulted in extreme cases. The interpretation of the results should be trusted only by a qualified specialist.

Training

An enzyme-linked immunosorbent assay must be carried out following the doctor's recommendations. research results are often influenced by various external factors. Basic rules for preparing for IFA:

• venous blood must be donated strictly on an empty stomach (normally, the last meal should be 12 hours before the study);
• it is necessary on the eve of the analysis to exclude the reception of any drugs(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 canceled);
• you must not smoke or drink alcoholic beverages before the examination, because this will negatively affect the result;
• you should definitely get some sleep before analysis;
• any physical activity, stress factors must be excluded;
• diagnosis of most female hormones reproductive system will require a blood draw on certain days of the menstrual cycle.

How is

For the enzyme immunoassay, the patient takes blood from the cubital vein strictly on an empty stomach. The patient must inform the doctor in advance about the presence of diseases and the drugs taken so that the results of the study are not distorted. As a rule, all medications should be stopped 16 days before the ELISA. The sensations during the procedure are similar to taking blood during a biochemical test.

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 receiving the reaction, all residual serum is discarded. Using special indicators, specialists determine the amount of antibodies. The ELISA lead time depends on the laboratory. As a rule, the results of the study can be provided in a period from two days to a week..

ELISA decoding

An enzyme-linked immunosorbent assay (ELISA) 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 onset of the disease in any case, because a healthy person does not have this class. Typically, IgM immunoglobulins are present in the blood for about 6 weeks.
2. IgA. Antibodies are found in large quantities in the mucous membranes, protecting the body from the penetration of pathogenic microbes. If the patient has this class, it is necessary to fight the disease more intensively. After all, immunoglobulins A appear only in chronic disease. 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 a 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 recurrence of the disease, and in syphilis, such antibodies are found for life.

When analyzing ELISA in childhood(up to 1.5 years), it should be borne in mind that the child's blood contains IgG antibodies mothers to infections. Although this does not mean that the baby is sick, rather this fact is the norm. The presence of class M indicates intrauterine or acquired after birth infection, because the mother's IgM antibodies are not able to enter the child's body through the placenta. The decoding of possible combinations of the presence or absence of antibodies of 3 classes is presented in the table:

Interpretation of ELISA results should be done by a qualified doctor.... As a rule, (+) indicates a positive outcome of the analysis, and (-) - a negative one. The result, which shows the absence or presence of a substance, is called qualitative. Sometimes it is supplemented by a quantitative one, which displays the number of various 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 the enzyme immunoassay have been identified. Sometimes during pregnancy, when the patient has a continuous change in hormones in the blood, it may be necessary to perform the analysis several times for a more reliable result. The diagnostic method is not recommended for use after:

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

The price of an enzyme-linked immunosorbent assay

The cost of the ELISA study depends on the policy of the medical institution, the type of analysis and the antigen being determined. based on these factors, the price of reagent kits is calculated and the complexity of the study is established. Typically, an enzyme-linked immunosorbent assay (ELISA) is a generally available procedure. average cost method varies from 300 to 2000 rubles. Approximate prices for an enzyme immunoassay in Moscow are presented in the table:

Video

Samoilikov Pavel Vladimirovich intern of the Department of "Clinical laboratory diagnostics"

Russian State Medical University

Immunoassay methods are widely used in medical practice. In all areas of modern medicine, immune analysis is used, mainly for diagnostic and analytical purposes. It is especially important that they make it possible to identify biological components (hormones, enzymes, neuropeptides, products of the immune system, antigens, etc.) in low and very low concentrations. All products against which antibodies can be obtained are detected by these methods.

Immunoassay is based on the interaction of antigen (AG) and antibody (AB) using different labeling options for one of the components (enzyme, radionuclide, fluorescent dye, and others). The assessment of the reaction is carried out automatically on special equipment, which makes it possible to standardize these methods.

Depending on the type of label used and the conditions for setting the test, the immunoassay is designated as enzyme immunoassay (ELISA), radioimmunoassay (RIA), immunofluorescent and others. When formulating reactions in one or more stages, they are designated as direct or indirect. The environment in which the reaction is carried out matters. If the reaction is carried out with reagents fixed on the surface, then the test is designated as a solid phase, for example, ELISA (enzyme linked immunosorbent assay).

In this work, only enzyme immunoassay will be considered - a method widely used in biology and medicine, both practical and fundamental.

ELISA appeared in the mid-60s and was originally developed as a method for identifying antigen in a histological specimen, as well as for visualizing precipitation lines in immunodifusion and immunoelectrophoresis tests, and then began to be used for the quantitative determination of antigens and antibodies in biological fluids... E. Engvall and R. Palman took part in the development of the method, as well as, independently of them, V. Van Veeman and R. Schurs.

Figure 1. Basic principle of ELISA.

1) To detect antigens. 2) To detect antibodies.

The method is based on the specific binding of an antibody to an antigen, while one of the components is conjugated to an enzyme; as a result of the reaction with the corresponding chromogenic substrate, a colored product is formed, the amount of which can be determined spectrophotometrically (Fig. 1).

The discovery of the possibility of antigen and antibody immobilization on various carriers with the preservation of their binding activity made it possible to expand the use of ELISA in various fields of biology and medicine.

The emergence monoclonal antibodies served the further development of ELISA, which made it possible to increase its sensitivity, specificity and reproducibility of results.

Theoretically, ELISA is based on the data of modern immunochemistry and chemical enzymology, knowledge of the physicochemical laws of the antigen-antibody reaction, as well as on the main principles of analytical chemistry. The sensitivity of ELISA and the time of its conduct is determined by several main factors: kinetic, thermodynamic characteristics of the antigen-antibody reaction, the ratio of reagents, the activity of the enzyme and the resolution of methods for its detection. In general, the antigen-antibody reaction can be described by a simple scheme:

+ [AG] ↔ [ATAG]

A variety of research objects from low molecular weight compounds to viruses and bacteria, as well as an unusually wide range of tasks associated with the variety of conditions for using ELISA, determine the development of an extremely a large number variants of this method.

Any ELISA version contains 3 mandatory stages:

1. stage of recognition of the test compound by a specific antibody to it, which leads to the formation of an immune complex;

2. the step of forming a bond of the conjugate with the immune complex or with free binding sites;

3. stage of conversion of the enzyme label into a recorded signal.

The classification of ELISA methods is based on several approaches:

1. According to the type of reagents present at the first stage of ELISA, competitive and non-competitive methods are distinguished.

A) In competitive ELISA at the first stage, the system contains both the analyzed compound and its analogue, labeled with an enzyme and competing for specific binding sites with it.

B) Non-competitive methods are characterized by the presence in the system at the first stage of only the analyzed compound and binding sites specific to it.

2. All ELISA methods are divided into homogeneous and heterogeneous.

If all three stages of ELISA take place in solution and between the main stages there are no additional stages of separation of the formed immune complexes from unreacted components, the method belongs to the group of homogeneous ones.

Homogeneous ELISA, which is usually used to determine low molecular weight substances, is based on the inhibition of enzyme activity when it is combined with an antigen or antibody. The enzyme activity is restored as a result of the antigen-antibody reaction.

When an antibody binds to an antigen containing an enzyme label, the enzyme activity is inhibited by 95% in relation to the high molecular weight substrate, which is due to steric exclusion of the substrate from the active center of the enzyme. As the concentration of antigen increases, more and more antibodies are bound and more and more free antigen-enzyme conjugates are retained, capable of hydrolyzing a high molecular weight substrate. The analysis is carried out very quickly, it takes 1 minute for one determination. The sensitivity of the method is quite high. With its help, you can determine the substance at the level of picomoles.

The heterogeneous method is characterized by an analysis in a two-phase system with the participation of a solid phase - a carrier, and an obligatory stage of separation of immune complexes from unreacted components (washing), which are in different phases (the formed immune complexes are on a solid phase, and unreacted complexes are in solution) ... Heterogeneous methods, in which the formation of immune complexes in the first stage occurs on a solid phase, are called solid phase methods.

Methods are homogeneous-heterogeneous, if stage 1 - the formation of specific complexes occurs in solution, and then a solid phase with an immobilized reagent is used to separate the components.

3. Based on the principle of determining the test substance:

A) Direct determination of the concentration of a substance (antigen or antibody) by the number of binding sites interacting with it. In this case, the enzyme label will be in the formed specific AG-AT complex. The concentration of the analyte will be directly proportional to the recorded signal.

B) Determination of the concentration of a substance by difference the total binding sites and remaining free binding sites. In this case, the concentration of the analyte will increase, and the recorded signal will decrease, therefore, in this case, an inverse dependence on the magnitude of the recorded signal can be traced.

Enzymes.

Enzyme labels have an extremely powerful catalytic action, one enzyme molecule can react with large quantity substrate molecules. Thus, an enzyme present in trace amounts can be identified and quantified by the formation of products of the reaction it catalyzes. Another advantage of using enzymes as labels is due to the presence in the molecule of numerous functional groups (sulfhydryl, carboxyl, tyrazine residues, etc.) through which the ligand molecules can be covalently attached.

Enzyme markers used in ELISA must have following properties:

- high activity and stability of the enzyme under the conditions of the analysis, with modification and in conjugate with antibodies or other proteins;

- the presence of sensitive substrates and the simplicity of the method for determining the products or substrates of an enzymatic reaction;

- the ability to adapt substrate systems for further enhancement;

- the absence of the enzyme and its inhibitors in the investigated biological fluid.

ELISA can use at least 15 different enzymes. Horseradish peroxidase (HRP), alkaline phosphatase (ALP) and β-D-galactosidase have found the greatest application, in accordance with the above requirements (Table 1). All three are stable and catalyze highly sensitive reactions. In addition, the products obtained as a result of reactions catalyzed by these enzymes, depending on the substrate used, can be detected not only by colorimetric methods, but also by fluorescence methods. Other enzymes are used much less frequently. This is due to their lower specific activity in comparison with HRP and AP.

Substrates.

The choice of substrate is primarily determined by the enzyme used as a label, since the enzyme-substrate reaction is highly specific.

Basic requirements for the substrate:

- ensuring high sensitivity of the method when detecting the enzyme in the conjugate;

- the formation of well-taken into account (for example, colored) products of the enzyme-substrate reaction;

- the substrate should be safe, cheap, affordable and convenient to use.

Table 1.

Enzymes and their substrates are the most widely used in ELISA.

Chromogenic substrates are more often used, which, breaking down, form a colored substance. The use of high-energy substrates - fluorescent, chemipuminescent - is promising. The use of such substrates makes it possible to theoretically increase the ELISA sensitivity by two orders of magnitude.

Antigens and antibodies.

AG and AT used in ELISA should be highly purified and highly active. In addition, antigens should have high antigenicity, optimal density and number of antigenic determinants, foreignness and homogeneity. Many synthetic and recombinant AGs of viruses and bacteria have proven themselves well when used in ELISA. This significantly increased the specificity and reproducibility of the method by minimizing cross-reactions.

Antibodies are one of the most important reagents in ELISA. The ELISA sensitivity depends on the concentration, activity and specificity of the antibodies used. The antibodies used can be poly- or monoclinal, of various classes (IgG or IgM) and subclasses (IgGl, IgG2), anti-allotypic or anti-idiotypic. At low affinity AT, the disintegration of the AG-AT complex leads to the removal of bound AG from the system. The sensitivity and specificity of the method increases with the use of monoclonal antibodies. In this case, it becomes possible to detect low concentrations of AG (AT) in the test samples.

Conjugate formation

A conjugate is an antigen or antibody labeled with an enzyme tag. Conjugate formation is one of important milestones carrying out ELISA.

When forming a conjugate, an optimal method for introducing an enzyme label is selected so that both components of the conjugate retain their biological activity: the enzyme is the ability to interact with the substrate, and the antigen or antibody is antigenic and antigen-binding activity, respectively. The presence of a labeled, highly purified antigen allows competitive methods to be used. In this case, at the final stage, it is possible to measure the activity of the conjugate that is not bound to the immobilized antibodies, which avoids the washing procedure and makes the analysis more convenient. However, antigens are diverse in their physical and chemical properties and structure, which means that it is impossible to develop universal methods for obtaining a conjugate with an antigen. In this case, the preparation of an antigen-enzyme conjugate is a separate challenge. The preparation of labeled antibodies for ELISA is methodologically more accessible.

Conjugation of the enzyme with immunochemically active proteins is performed different methods: chemical cross-linking, covalent binding of an enzyme molecule with AG or AT and the formation of connections through non-covalent bonds, for example, when the bond between the enzyme and AG or AT is carried out immunologically, through the interaction of antigen-antibody.

The most widespread are covalent methods for the preparation of conjugates. The choice of the binding reaction is determined by the type of functional groups available in the given protein molecules. Glutaraldehyde, sodium periodate, etc. are used as reagents used to introduce the enzyme into antigen and antibody molecules.

There are one-step and two-step methods for obtaining conjugates using glutaraldehyde. Conjugates of various sizes with reduced enzymatic activity (15-60% of free enzyme) can be formed. The resulting conjugate large sizes can sterically hinder the determination of the test substance. Relatively low molecular weight conjugates consist of a Fab fragment and one enzyme molecule.

As a result of a two-step synthesis, which consists in the step-by-step production of an enzyme modified with a cross-linking agent, its isolation, and then its subsequent interaction with an antigen (antibody), molecules of a homogeneous composition are formed, containing 1-2 enzyme molecules per immunoglobulin molecule and maintaining a high enzymatic and immunological activity. However, the amount of such formed conjugates is small (for horseradish peroxidase it is 5 - 10%).

The greatest practical use found a method for obtaining immunoperoxidase conjugates based on the oxidation of the carbohydrate component of the enzyme with sodium periodate (the binding of peroxidase to the conjugate reaches 70-90% of the initial amount of the enzyme).

A reliable conjugate should have the following properties:

High antibody tiger and high affinity for the antigen, so that it can be used in a large dilution, and thus reduce non-specific binding;

Sufficient specificity in working breeding;

The predominance of monomeric forms over polymeric ones, because polymer forms have a tendency to adhere nonspecifically to plastic, resulting in a high background level of reaction;

The optimal molar ratio between enzyme and antibodies (the optimal ratio is about 1: 1);

Sufficient enzymatic activity of the conjugate. This property is mainly determined by the conjugation conditions and the ratio of enzyme and antibody molecules in the conjugate.

Solid phase

As a solid phase for ELISA, you can use various materials: polystyrene, polyvinyl chloride, polypropylene and other substances. The walls of a test tube, 96-well and other plates, beads, beads, as well as nitrocellulose and other membranes that actively sorb proteins can serve as a solid phase.

Immobilization of antigen or antibodies on the solid phase is possible in three ways:

- passive adsorption based on strong hydrophobic interactions between proteins and a synthetic surface;

- covalent attachment to the solid phase;

- immunochemical, etc. (non-covalent and non-adsorption).

Passive adsorption of proteins is widely used for ELISA on titration plates and on nitrocellulose membranes. Passive adsorption follows the saturation principle and correlates with the molecular weight of the adsorbed substance. The adsorption surface of various types of membranes (nitrocellulose, nylon, etc.) is 100-1000 times higher than that of plastic.

Polysaccharides and highly glycosylated proteins often have low affinity for polystyrene. Other methods are required for their immobilization, for example, covalent attachment using glutaraldehyde. Covalent attachment is effective if hydrophilic beads (agarose) and polystyrene beads are used as the solid phase.

Immunochemical methods are based on the use of pre-adsorbed "trap" antibodies to immobilize antigen or antibodies. An antigen immobilized immunochemically is 10 times more active than a passively adsorbed antigen. Can be used lectins or immunoglobulin-binding proteins of bacteria that are easily adsorbed on plastic or other hydrophobic surfaces, such as concanavalin A (Con A) or staffed protein A. Con A is able to immobilize gp 120, a protein of the HIV virus.

Free sites on the surface of the solid phase that are not bound to the sorbed agent can fix other molecules, including conjugates, during the test, which leads to an increase in the background signal. To prevent nonspecific binding, after immobilization on the solid phase of the base material, treatment with substances neutral for the test is carried out. The most popular blocking agents are bovine serum albumin (BSA), casein, etc. The choice of a blocking agent and the conditions for this stage depend on the type of solid phase and the sensitivity of the system.

Currently, a huge number of all kinds and modifications of ELISA are used. Widespread different variants enzyme-linked immunosorbent assay (ELISA).

Solid phase ELISA was proposed in 1971. The basic principles of solid-phase ELISA, regardless of modification, are as follows:

1. At the 1st stage of the reaction, antigens or antibodies are adsorbed on the solid phase. In this case, the reagents not bound to the solid phase are easily removed by washing.

2. Incubate the test sample in the sensitized wells. Positive control wells contain standard reagents. In this case, immune complexes are formed on the surface of the solid phase. Unbound components are removed by washing.

3. When adding conjugate antibody-enzyme or antigen-enzyme and binding it to the immobilized immune complex active center the enzyme remains available for subsequent interaction with the substrate. Incubation of the substrate in the wells with the immobilized conjugate leads to the development of a color reaction. This reaction can be stopped at the desired stage, the severity of the staining can be assessed visually or by optical density.

An important step in any variant of solid-phase analysis is the procedure for removing unbound reagents. It is important not only to rinse the components fixed on the solid phase, but to remove the reagents from the entire depth of the layer. These are the longest and most laborious stages of the analysis. Sample washing can be carried out automatically using special device- washer or manually, with a multichannel pipette. To carry out ELISA, you need:

- polystyrene plate or other solid phase options used;

- washing solution;

- conjugate (enzyme-labeled antigens or antibodies);

- a mixture of substrates used;

- stopping solution (Stop reagent - solution for stopping the reaction);

- samples used for positive and / or negative control;

- standard antigen (to build a calibration curve);

- single and multichannel pipettes;

- washer (washer);

– optical instrument to determine the optical density of the test solution (ELISA reader, reader, which sequentially photometric all wells);

- 5-100 μl of the biological material under study.

Direct ELISA

1. Antigens or antibodies (test material) are adsorbed in the wells of the panels. It was noted above that antigens differ significantly in their ability to adsorb on different types plastic depending on which class of substances (proteins, carbohydrates or lipoproteins) they belong to. Often in direct ELISA, the antigen immobilized on the solid phase is cells and other corpuscular antigens.

Control. As a control, use wells with an adsorbed positive control sample, which necessarily contains the desired antigen, and a negative control sample that obviously does not contain the test antigen. If a purified standard antigen is available, the reaction is carried out in several dilutions so that a calibration curve can be generated.

2. “They block free binding sites remaining on the solid phase using BSA casein and others (to prevent nonspecific sorption of the conjugate on the solid phase).

3. Enzyme-labeled antibodies or antigens (conjugate) are added to the wells, incubated. The binding of the conjugate to the solid phase will occur only if both components of the system are complementary. After incubation with the conjugate, the wells are washed, thus removing the unbound part of the conjugate.

4. Subsequently, a substrate specific for the enzyme used is added to the wells and incubated. Upon reaching the optimum level of staining in the positive control wells, the enzymatic reaction is stopped.

5. Taking into account the reaction. First, the reaction results are taken into account visually. For a more accurate account of the results, the intensity of staining is assessed using an ELISA reader with an appropriate light filter. Based on the results of the analysis, a graph of the dependence of the optical density on the concentration is plotted (Fig. 2).

Figure 2. Direct ELISA.

a) to detect antigen; b) to detect antibodies.

This ELISA variant is usually used to detect specific antibodies. In the wells of the panels, a standard antigen is adsorbed and incubated with samples of serum or other biological material obtained from the patient (cerebrospinal fluid, saliva, etc.). Specific antibodies bound to the antigen on the solid phase are detected using an antiglobulin conjugate. Depending on the purpose of the analysis, different antiglobulin reagents are used that detect antibodies of all isotypes, or specific to individual classes and subclasses of immunoglobulins. The main advantage of the method is the versatility of the conjugate. The same conjugate can be used to detect human antibodies to a wide variety of antigens in any sample. The reaction is methodically simple.

The main stages of an indirect ELISA for the determination of antibodies:

1. The antigen is adsorbed on the solid phase, then washed from unbound components.

2. Block free meshes of binding. They are washed.

3. The test material is introduced into the wells, incubated and then the washing procedure is carried out. Samples with positive and negative controls are put in parallel.

4. Add antiglobulin conjugate in working dilution, incubate, wash from unbound components.

5. Add the substrate, incubate. When the optimum staining level is reached in the positive control wells, the reaction is stopped by adding stop solution.

6. Measure the amount of the reaction product on an ELISA reader (Fig. 3).

Under optimal conditions for the analysis, the method is highly specific and sensitive. It allows you to detect nanogram amounts of antibodies in the sera of the studied patients. To obtain satisfactory results, it is necessary to standardize reagents and methodologies. This ELISA variant can also be used to test monoclonal antibodies.

Antigens determined using this ELISA variant must have several epitopes capable of binding antibodies, or have repeated, spatially separated epitopes of the same specificity.

When carrying out this variant of ELISA, highly specific poly- or monoclonal antibodies adsorbed on the solid phase are incubated with the test sample. After the washing procedure, enzyme-labeled antibodies (conjugate) to the same antigen are introduced into the wells, and then all other stages of the reaction are carried out. The efficiency of the formation of a specific complex at each stage of the analysis depends on the binding constant of the antigen-antibody reaction.

The main stages of the analysis:

1. Monoclonal antibodies or affinity-purified polyclonal antibodies are immobilized on the solid phase.

2. The test sample is introduced into the wells of the panels, a positive control sample and a negative control sample in different dilutions are placed in parallel. Incubate and wash.

3. Enzyme-labeled monoclonal or polyclonal antibodies - conjugate are introduced into the wells. After incubation, wash is carried out.

4. Add the substrate, incubate. The reaction is stopped when optimal staining is achieved in the positive control wells.

5. Accounting for the results on the ELISA reader.

The main advantage of the method is its high sensitivity, which surpasses the capabilities of other ELISA schemes (Fig. 4).

Figure 3. Indirect ELISA for antibody detection.

This version of the analysis is based on the competition of labeled (conjugate) and unlabeled (investigated) antibodies for binding to the antigen adsorbed on the solid phase. The amount of the enzyme attached to the solid phase will decrease in proportion to the content of free antibodies in the mixture. To determine the antigen, the same variant is used, but in this case the desired antigen competes with the labeled, standard antigen for binding to antibodies immobilized on the surface of the solid phase.

The competitive method requires a minimum number of operations, low consumption of reagents, and can be easily automated. When conducting competitive ELISA to detect antibodies, it is better to use labeled monoclinal antibodies, then the competition of the conjugate with the test sample occurs for a single epitope of the antigen adsorbed on the solid phase. This ELISA version is used to determine various compounds, such as human immunoglobulins, cancer-embryonic antigen, insulin, etc. It allows the detection of antibodies to diagnostically significant epitopes of infectious agents.

The main stages of the analysis for the detection of antigen (Fig. 5):

1. Monoclonal antibodies specific for the antigen to be detected are immobilized on the solid phase.

2. In the wells of the panels, the antigen labeled with the enzyme and the test sample are introduced at a known concentration. Incubation and washing are carried out. Positive and negative controls are placed in parallel in adjacent wells. To construct the calibration, a standard unlabeled antigen in various dilutions is used.

3. Add substrate, incubate, stop reaction when optimal staining develops in positive control wells.

4. Taking into account the reaction on the ELISA reader.

In this case, the amount of antigen in the test sample is inversely proportional to the enzymatic activity on the solid phase.

In this variant of ELISA, the antigen present in the test sample binds to monoclonal antibodies labeled with an enzyme label and inhibits their interaction with a standard antigen immobilized on a solid phase. The presence of even trace amounts of a conjugate-specific antigen in a sample will inhibit the binding of labeled antibodies to the immobilized antigen. The degree of inhibition is directly proportional to the antigen content in the solution. For quantitative analysis, a calibration curve is constructed using serial dilutions of a standard antigen. The main stages of inhibitory ELISA for antigen detection (Fig. 6).

1. In the wells of the panels, a standard antigen is adsorbed. A working dilution of labeled antibodies is selected by titration.

Figure 4. "Sandwich" - ELISA variant.

2. Conduct a preliminary incubation of the conjugate in a dilution preceding the working one, with dilutions of the test sample, standard antigen and positive control samples.

3. Transfer the mixture to the wells of the panels. To control 100% binding, only labeled antibodies are added to several wells, without inhibitory antigen. The panels are incubated and then washed.

4. Add the substrate.

5. Record the results.

The concentration of the determined antigen in the test sample is inversely proportional to the enzymatic activity on the solid phase.

ELISA can be used not only to determine a soluble antigen or antibody, but also cells that produce various proteins.

In 1983, the solid-phase ELISA technology was adapted to determine lymphoid cells secreting antibodies or antigens (for example, cytokines) in vitro. The method is called ELISPOT (method of immunoassay zones or spots). The basic principle of the method:

1. On the surface of a polystyrene well (using 24-well panels for cell cultivation), antigens or antibodies are sorbed, which serve as "trap" reagents.

2. Add the studied lymphoid cells, cultured for several hours at 37 ° C, giving them the opportunity to take a certain place and perform a secretory function. Antibodies or antigens secreted by such cells are captured by the reagents adsorbed on the solid phase.

3. The cells are removed using a wash solution with a cell lysing detergent.

4. Areas of accumulation of secretory products are developed by adding enzyme-linked antibodies (antiglobulin reagent).

5. Add a mixture of substrate with agarose (the substrates used must dissolve in agarose and form insoluble reaction products), brown or blue spots are formed on the surface of the solid phase (depending on the enzymes and substrates used), revealing the areas where the cells were located.

The resulting spots are counted under a microscope, this will be the number of secreting cells.

A nitrocellulose membrane can be used as a solid phase. In this case, there are a number of advantages: due to the high adsorption capacity of the NCM, a much smaller amount of antigen is required to be used as a "trap" reagent; in addition, there is no need to include agarose into the substrate.

With parallel determination of the number of secreting cells and the total of the secreted antigen or antibody in the well, which is possible when using a different substrate, it is possible to reveal the amount of the secreted substance by a single cell.

This method found wide application to assess the number of cells secreting antigen captured by adsorbed antibodies, it is used to determine the number of cells secreting cytokines (IL-1, IL-2, IL-4, IL-6, IFN-y, TNF-a).

When using high-affinity antibodies, the sensitivity of individual ELISA variants is very high and theoretically makes it possible to detect single antigen molecules, but in practice the sensitivity is limited by a number of factors: enzyme activity, signal intensity, and signal accounting methods. Signal amplification systems make it possible to increase the sensitivity of various ELISA options. Consider some of these systems:

Based on the Avidin-Biotin Interaction.

Molecules of the coenzyme biotin (mw 244 Da) are conjugated to antibodies using biotinyl-N-hydroxysuccimide. A small molecule of biotin is easier to attach to an immunoglobulin or other protein without affecting its immune or enzymatic properties. The enzyme in this case is bound to a glycoprotein. egg white avidin. The binding affinity of avidin with biotin is very high (the dissociation constant of the complex is 10-15 mol), the avidin-enzyme conjugate is firmly fixed on the antigen-antibody-biotin complex. After adding an appropriate substrate, the reaction product is determined spectrophotometrically or by luminescence intensity.

One avidin molecule consists of four identical subunits, capable of interacting with four molecules of biotin, which allows it to be used as a linking molecule between two biotin-containing compounds. In this case, the enzyme is also biotinylated, and avidin acts as a bridge, connecting two molecules containing biotin residues. Free avidin and then biotinylated enzyme are added to the resulting antigen-antibody-biotin complex. The reaction is recorded.

The avidin protein can be sorbed nonspecifically on other molecules; therefore, another biotin-binding protein, streptavidin, found in the bacteria Streptomyces avidinii, is increasingly being used. Streptavidin also forms a strong complex with biotin and is composed of four identical subunits.

The use of the avidin-biotin complex can significantly increase the sensitivity of ELISA, since during the synthesis of the conjugate with one AT molecule, dozens of biotin molecules can be bound. The production of conjugates (antibodies and enzymes with biotin) is carried out quite easily and is accompanied by minimal changes in their immunological and enzymatic activity. Biotin-conjugated enzymes can be used as versatile reagents.

Use of chemiluminescent reactions.

Chemiluminescent reactions can be used to obtain a signal in ELISA, thereby increasing the sensitivity of the method and shortening the analysis time. Horseradish peroxidase is widely used as a label in ELISA; various chemiluminescent reactions can also be used to detect it. Chemiluminescent reactions are based on the ability of luminol to glow when oxidized with hydrogen peroxide. In direct analysis, during an enzymatic reaction, hydrogen peroxide is formed and oxidizes luminol, horseradish peroxidase acts as a catalyst for this reaction. To amplify the signal, various compounds are used, for example, luciferin, phenols, in this case the luminescence intensity is increased by 10-100 times, in some cases by 500 times (enhanced chemiluminescence analysis). The luminescent signal is very stable, its level reaches a maximum in 30 s (for comparison: the color reaction with OPD as an indicator fully develops in only 30 min).

In indirect analysis, the antibody is labeled with luminol or its derivatives. Such a label in a free state is capable of being oxidized by hydrogen peroxide with the release of light. If it has formed a complex, then it loses the ability to oxidize.

Based on cascade systems.

Enzyme cascade systems can be used to increase the sensitivity of ELISA. In this case, the first enzyme associated with antibodies leads to the formation of a reducible substrate for the second enzyme system. The second enzyme system can be substrate cyclic or redoxycyclic. In this case, phospho-glucoisomerase, aldolase, alkaline phosphatase can serve as enzyme labels. The final product of the reaction is determined visually or spectrophotometrically.

ELISA amplification systems provide high sensitivity. Such ELISA systems are used to determine the level of hormones (thyreostimulating, progesterone, etc.).

ELISA has found wide application in various fields of medicine and biology due to the relative simplicity and high sensitivity of the method. ELISA is successfully used for:

Mass diagnostics of infectious diseases (detection of various specific antigens or antibodies to them);

Detection and determination of the level of hormones and drugs in biological samples;

Determination of isotypes (IgG, IgM and others) of antibodies against a specific antigen;

Revealing immune complexes;

Identification of tumor markers;

Determination of serum proteins (ferritin, fibronectin, etc.);

Determination of total IgE and specific IgE antibodies;

Screening myoclonal antibodies;

Determination of cytokines in biological fluids.

Method sensitivity

ELISA has replaced the methods of agglutination, precipitation and RIA that were widely used earlier in clinical practice. Compared with the above methods, ELISA is less laborious and less time-consuming, convenient to perform a large number of the same type of analyzes.

ELISA combines the unique specificity of an immunochemical assay with a high sensitivity for the determination of an enzyme label. The sensitivity of the method (sensitivity means the minimum detectable amount of antibodies or antigen) is determined by the following factors: the affinity of antibodies, preferably the use of monoclonal antibodies; specific enzyme activity; signal intensity; signal metering sensitivity. Various options EIAs differ in their sensitivity. Certain versions of the solid-phase ELISA allow the detection of single molecules in the sample. The average ELISA sensitivity is 10-9 - 10-12 mol.

Galaktionov V.G. Immunology. Moscow University Publishing House, 1998

A.A. Kishkun Immunological studies and methods for the diagnosis of infectious diseases in clinical practice. Medical News Agency, 2009

Kondratyeva I.A. Workshop on immunology. Textbook for universities. Academy, 2004

Lefkovits I., Pernis B. Immunological research methods. World, 1988

Royt A., Brostoff D., Mail D. Immunology. World, 2000

E. I. Sokolov Clinical immunology. Medicine, 1998

Frimel G. Immunological methods. Medicine, 1987

Khaitov R.M. Immunology. Medicine, 2000

Yu.V. Shigina Immunology: Textbook. RIOR Publishing House, 2007

Yarilin A.A. Fundamentals of Immunology. Medicine, 1999

An enzyme-linked immunosorbent assay is a common method for detecting various pathologies. The result of a blood test, the ELISA decoding of which was interpreted by a laboratory diagnostician, is reliable in most cases.

What is ELISA

ELISA is a modern diagnostic method that is used to diagnose infections, hormonal and immune disorders, and oncological diseases in laboratory conditions. The method allows you to detect antibodies to infection already on early dates diseases. This method refers to indirect diagnostic methods, as it reveals the body's immune response. Among the advantages of ELISA in comparison with other diagnostic methods, one can single out a high level of manufacturability, which reduces the likelihood of errors. The method is highly sensitive and is used to diagnose diseases in both children and adults. There are a wide variety of different ELISA modifications.

The ELISA method is based on the specificity of immunochemical reactions, as well as the physicochemical laws of the reactions of antigen-antibody complexes. Reactions are carried out with the participation of specific enzymes, which are labels for the detection of antibodies. As a result of an immunochemical reaction, strictly defined antibodies bind to the corresponding antigens. A blood test by enzyme immunoassay almost eliminates the possibility of obtaining a false positive result. Experts in laboratory diagnostics judge whether the solution is stained during the enzymatic indication of the antigen-antibody complex about a positive or negative result. If the solution becomes colored, then the antigen interacts with the antibody, the ELISA result is positive.

What diseases can be detected by ELISA

The study by the method of enzyme immunoassay allows:

• identify a number of infectious diseases;
• diagnose autoimmune diseases;
• identify the presence of oncology;
• identify hormonal disorders;
• make other research.

An ELISA blood test allows you to determine the presence of the following infections:

The method is used to detect antigens of pathogens of a number of infections, as well as to detect antibodies of various classes. The ELISA method has become very popular for the detection of syphilis, HIV, viral hepatitis. It is not recommended to determine the presence and level of antibodies in the blood serum for the primary diagnosis of STIs. In this case, the presence of antibodies in the blood can only be a sign that the patient's body has been in contact with the infectious agent in the past.

Diagnosis of various autoimmune diseases by the ELISA method is carried out through research:

• antinuclear bodies;
• antibodies to double stranded DNA;
• antibodies to soluble nuclear antigens (ENA screen);
• anticardiolipin antibodies;
• IgG to citrulline peptide;
• rheumatoid factor;
• C-reactive protein;
• autoantibodies to cytoplasmic antigens of neutrophils (ANCA screen).

Specific immune complexes are characteristic of certain autoimmune diseases. For example, double-stranded DNA antibodies are characteristic of diseases such as systemic lupus erythematosus.

Determination of oncological diseases is carried out by enzyme-linked immunosorbent assay of blood serum for specific tumor markers, such as PSA, CA-125.

PSA indicates the presence of prostate adenoma and prostate cancer. CA-125 is a tumor marker malignant tumor ovary. The value also increases with cancerous tumors uterus, mammary glands, endometrium.

Preparation for analysis

To ensure the most accurate results, you need to follow certain rules preparation for the study. Analysis for laboratory diagnosis by ELISA, as a rule, is taken in the morning from the cubital vein. It is necessary to donate blood strictly on an empty stomach. In addition to this simple prescription, the following preparation recommendations should be followed:

• 24 hours before the study, it is necessary to exclude alcohol and smoking;
• avoid heavy physical exertion;
• be in a calm state;
• avoid nervous tension;
• donate blood for ELISA no earlier than 10 days after drug withdrawal;
• notify the doctor about taking the necessary medications.

In addition, it is recommended to stick to a diet a few days before the test. At the same time, exclude fatty foods, fried foods from the diet. Before the analysis for viral hepatitis, exclude from the diet not only fatty and fried, but also citrus fruits and orange vegetables.

It should be noted that for the results of a certain spectrum hormonal research influenced by such a factor as the phase of the menstrual cycle. The need to take an analysis at one or another phase of the menstrual cycle should be discussed in advance with your doctor. Otherwise, you may get unexpected indicators. For example, normal level luteinizing sex hormone in women varies depending on the day of the cycle:

• 1-12 days - 2-14 mU / l;
• 12-14 days - 24-150 mU / l;
• from the 15th day before the start of a new cycle - 2-17 mU / l.

Interpretation of ELISA results

The analysis allows you to establish the presence of antibodies of various classes in the body. There are 3 classes of antibodies:

The production of these antibodies occurs on different stages diseases. The very first after infection in the body begins to produce IgM antibodies. Their presence is an indicator of the disease in any case. In the body of a healthy person, this class of antibodies is absent.

These immunoglobulins are present in the blood serum for about 5-6 weeks.

Class G immunoglobulins present in the blood indicate that a person has either already had a disease or is a carrier of infection. These antibodies begin to develop after antibodies of class M, in most diseases 3-4 weeks after infection. Their presence in the body is possible for several years. And in some diseases (for example, syphilis) IgG are present in the blood for life.

If IgA is present in the body, it is necessary to fight the infection as intensively as possible. Antibodies of this class appear only in the case of a chronic infectious disease. The disappearance of IgA indicates the destruction of the infection.

If the ELISA analysis is performed in children under 1.5 years of age, the following feature should be taken into account: the child's blood contains mother's IgG to various infections. This does not mean at all that the child is sick. In this case it is rather the norm... The presence of IgM is evidence of intrauterine infection or an infection acquired after birth. Mother's IgM cannot enter the baby's body through the placenta.

The table shows possible combinations of the presence or absence of antibodies of 3 classes in the body and their interpretation.

When decoding analyzes, (+) indicates a positive result, and (-) indicates the opposite, that is, negative. The result, showing the presence or absence of any substance in the body, is called qualitative. It can be supplemented with a quantitative one. The quantitative result reflects the quantitative content of various substances in the body.

It should be noted that the test systems have their own manufacturer-defined reference values ​​that characterize the indicators. Exceeding the reference values, as a rule, means the presence of certain pathologies in the subject's body.

After receiving the ELISA results, the attending physician should decipher the obtained values. Only he will be able to make a correct assessment of the results obtained and determine the stage of the disease.

In contact with