Ifa summary what kind of analysis. Enzyme immunoassay blood test: interpretation, methods. Video: modern enzyme immunoassay

Three variants of solid-phase immunoassay are most often used in practice - indirect immunoassay, direct immunoassay and sandwich-type immunoassay. The differences between these types of immunoassays are as follows. In the indirect version of the immunoassay, at the first stage, the antigen is adsorbed onto the surface of the wells of a polystyrene plate. After removing unbound antigen molecules, a sample containing antibodies specific to that antigen is added. The resulting antigen-antibody complexes are detected using anti-species antibodies conjugated to any label (Fig. 1A). In the direct version of the immunoassay, detection of the sorbed antigen is carried out directly using specific antibodies conjugated to a label (Fig. 1B). In a sandwich-type immunoassay, at the first stage, not the antigen, but antibodies specific to the antigen under study (substrate antibodies) are adsorbed onto the surface of the plate. After removing unbound antibody molecules, a sample containing the antigen is added. To detect the resulting substrate antibody-antigen complex, second antibodies are added that are specific to another, spatially distant, epitope of the antigen, conjugated to any label (Fig. 1B). The use of sandwich-type immunoassays of antibodies specific to two different epitopes of an antigen allows one to achieve high sensitivity and specificity in determining the antigen even in such heterogeneous samples as blood plasma.

Rice. 1. Principle of indirect (A), direct (B) and sandwich-type immunoassay (C)

Indirect enzyme immunoassay (indirect ELISA)

The indirect immunoassay method is characterized by a 3-stage process, at the first stage of which the antigen is adsorbed on specially prepared plastic, at the second stage antibodies specific to it interact with the antigen, and at the third stage anti-species antibodies conjugated with an enzyme are introduced into the system, causing an indicator enzymatic reaction. . In this technique, horseradish peroxidase is used as an enzyme. The reaction is carried out in special 96-well plates.

I. Antigen sorption

The wells of a 96-well plate for immunoassay sorb 0.1-0.5 μg of antigen per well in 100 μl of phosphate-buffered saline (PBS). Incubation is carried out for 30 minutes at room temperature and shaking on a horizontal plate shaker. Washing (2-fold) of unbound antigen molecules is carried out with phosphate-buffered saline containing 0.1% Tween-20 (PBS).

II. Lock

To block nonspecific binding sites, the wells of the plate are filled with PBST and incubated for 10-15 minutes at room temperature.

III. Titration of specific antibodies

Titration can be carried out both horizontal and vertical rows of the tablet. It should be noted that titration of antibodies is carried out if it is necessary to select the optimal concentration of antibodies or determine the titer. If the optimal concentration and/or titer of antibodies is determined, then the recommended dilution for these specific antibodies is used.

When titrating, a ready-made dilution of antibodies is added to the first well of the row - on average 1-10 μg per well, then sequential dilution of the antibodies is carried out in the wells. Incubation with specific antibodies is carried out for 30 minutes at room temperature and shaking on a horizontal plate shaker. Washing is carried out using PBST 3 times.

IV. Addition of anti-species antibodies conjugated to an enzyme tag

Anti-species polyclonal antibodies conjugated to horseradish peroxidase are used as detector (secondary) antibodies. Most often, goat or rabbit antibodies are used, specific to the whole molecule or to the Fc fragments of specific antibodies. The concentration of detector antibodies is usually indicated by the manufacturer as a dilution of the stock solution (for example, 1:1000). Incubation with secondary antibodies is carried out for 30 minutes at room temperature and shaking on a horizontal plate shaker. Washing is carried out using PBST 5-6 times.

Horseradish peroxidase catalyzes the reaction of substrate oxidation with hydrogen peroxide. O-phenylenediamine (OPD) is used as a substrate for horseradish peroxidase. As a result of the reaction, a colored oxidation product of OPD is formed.

Substrate solution: To 10 ml of substrate buffer (0.1 M Na-citrate buffer, pH 4.5), add 0.01 ml of 30% hydrogen peroxide and 0.2 ml of 50x OPD solution (340 mg OPD in 10 ml of ethyl alcohol; store at –20°C).

Incubation is carried out for 10 minutes at room temperature and shaking on a horizontal plate shaker.

V. Stopping the enzymatic reaction

VI. Optical density measurement

Direct enzyme immunoassay (direct ELISA)

The direct immunoassay technique has only slight differences compared to the indirect immunoassay technique. Thus, stages I and II are the same in both types of analysis. The difference is that in the direct version of the immunoassay, stage III uses specific antibodies conjugated to an enzyme tag. If necessary, it is also possible to titrate specific antibodies conjugated to an enzyme tag, similarly to what was previously described for unconjugated antibodies. Stage IV is omitted, and further stages (V-VII) are carried out similarly to those described above for the indirect version of the immunoassay.

Sandwich-type immunoassay

Rice. 2. Schematic representation of a sandwich-type immunoassay. ATP - substrate antibody, ATD - detector antibody, AG - antigen, M - label covalently bound to the detector antibody, P - substrate on which the substrate antibody is sorbed.

This version of the immunoassay (Fig. 2) uses a pair of antibodies specific to spatially distant epitopes of the antigen being studied.

I. Sorption of substrate antibodies

The substrate antibodies, 1-2 μg per well, are absorbed into the wells of a 96-well plate for immunoassay in 100 μl of phosphate-buffered saline (PBS). Incubation is carried out for 30 minutes at room temperature and shaking on a horizontal plate shaker. Washing (2-fold) of unbound antigen molecules is carried out with phosphate-buffered saline containing 0.1% Tween-20 (PBS).

II. Lock

To block nonspecific binding sites, the wells of the plate are filled with PBST and incubated for 10-15 minutes at room temperature.

III. Incubation with antigen

50 µl of the test solution or standard dilutions of the antigen are added to the wells of the plate with pre-adsorbed antibodies. Antigen dilutions should be prepared using PBST because Tween-20 reduces the nonspecific binding of protein molecules to each other and to the plate surface. Both the test solution and standard antigen dilutions are added in pairs (or in 3 replicates), using two (three) wells for each protein dilution. Incubation is carried out at room temperature for 30 minutes with constant stirring. Washing is carried out with PBST solution 3 times.

IV. Incubation with antibodies conjugated to an enzyme tag

100 μl of a solution of specific antibodies conjugated to an enzyme label is added to the wells of the plate. The optimal concentration of conjugated antibodies is usually indicated by the manufacturer (usually a concentration of 2-4 μg/ml is used). Incubation with antibodies containing an enzyme tag is carried out for 30 minutes at room temperature and shaking on a horizontal plate shaker. Washing is carried out using PBST 5-6 times.

V. Carrying out an enzymatic reaction accompanied by the appearance of a colored product

100 μl of substrate solution is added to the wells and incubated for 10 minutes at room temperature and constant stirring.

VI. Stopping an enzymatic reaction

Before measuring the optical density, the color reaction is stopped using 0.5 M H2SO4. After incubation, 50 μl of a solution of 0.5 M sulfuric acid is added to the wells with the OPD working solution. After this, you can immediately begin measuring optical density.

VII. Optical density measurement

The optical density of the colored product solution is measured at λ=490 nm using a plate spectrophotometer.

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Immunoassay assay (ELISA)

Immunoassay assay, or method (ELISA) — detection of antigens using their corresponding antibodies conjugated to a tag enzyme (horseradish peroxidase, beta-galactosidase and or alkaline phosphatase). After combining the antigen with the enzyme-labeled immune serum, the substrate/chromogen is added to the mixture. The substrate is cleaved by the enzyme and the color of the reaction product changes - the intensity of the color is directly proportional to the number of bound antigen and antibody molecules.

The components are detected by adding labeled antibodies or antigens. If the result is positive, the color of the chromogen changes.

Solid-phase indirect enzyme-linked immunosorbent assay

Each time after adding another component, unbound reagents are removed from the wells by washing.
I. When determining antibodies, the patient’s blood serum, antiglobulin serum labeled with an enzyme, and a substrate/chromogen for the enzyme are successively added to the wells of plates with sorbed antigen.

II. When determining an antigen, an antigen is added to the wells with sorbed antibodies (for example, blood serum with the desired antigen), a diagnostic serum against it and secondary antibodies (against the diagnostic serum), labeled with an enzyme, are added, and then a substrate/chromogen for the enzyme.

Competitive ELISA for antibody detection: the desired antibodies and enzyme-labeled antibodies compete with each other for antigens adsorbed on the solid phase.

Determination of peroxidase label using enhanced chemiluminescence. Development of rapid enzyme immunoassay methods performed using portable devices. Synergy and degree of enhancement. Intensity and duration of light emission.

Enzyme-linked immunosorbent assay with enhanced chemiluminescence

INTRODUCTION

The enzyme label can be quantified using various methods, for example, using colorimetry, fluorimetry, radiometry and potentiometry.

Recently, chemiluminescence or bioluminescence has also been used for this purpose. Enzyme tags that can be determined using reactions accompanied by light emission are presented in table. 1. In general, the sensitivity of chemi- and bioluminescent detection methods is very high, and if amplification of the signal from an enzyme label is also used, then the sensitivity of the corresponding immunoassay methods should be extremely high.

The capabilities of the bioluminescent method were recently demonstrated by the example of the determination of D-galactosidase. Using the coupled reaction of galactose dehydrogenase AOH:PMN oxidoreductase-bacterial luciferase, it was possible to detect 2 * 10″22 mol of /3-galactosidase.

Horseradish peroxidase is widely used as a label in enzyme-linked immunosorbent assays; methods for preparing and stabilizing conjugates with which have been well studied.

Horseradish peroxidase is most often determined colorimetrically, but various chemi- and bioluminescent reactions can also be used for this purpose.

The determination of peroxidase using a chemiluminescent reaction in the luminol-hydrogen peroxide system enhanced with phenolic compounds was studied in detail.

This approach to determining enzyme activity has a number of advantages compared to other determination methods; its main characteristics are listed in table. 2.

Amplifiers. Various compounds, including 6-hydroxybenzothiazoles, such as luciferin, phenols, naphthols and amines, have the ability to enhance light emission in the luminal oxidation reaction with hydrogen peroxide, catalyzed by peroxidase.

In Fig. Figure 1 shows the structures of representatives of substances of each class. The degree of enhancement of light emission depends on the concentration of the reagents. A typical dependence of the luminescence intensity on the concentration of the amplifier in the reaction of luminol with hydrogen peroxide, catalyzed by peroxidase and enhanced by 6-hydroxy-benzothiazole, is shown in Fig.

Synergy and degree of enhancement. In the system peroxidase - luminal - hydrogen peroxide, a blue-

rgism, as evidenced by the data given in table. 3. The degree of enhancement is influenced by many factors; Nevertheless, an increase in light emission of more than 500 times has been achieved.

In the presence of certain amplifiers, the background light emission in the luminol-hydrogen peroxide system decreases. When determining peroxidase activity, this makes it possible to increase the signal-to-noise ratio by several orders of magnitude only through the use of such amplifiers.

Increased luminescence is characteristic of various cyclic diacylhydrazides when exposed to oxidizing agents in the presence of peroxindase. The enhancement is not observed for some substances with peroxidase activity, such as hemoglobin, which catalyzes only colorimetric reactions.

This fact indicates a higher specificity of enhanced chemiluminescent analysis compared to colorimetric methods. The spectra of emitted light are almost the same in amplified and non-amplified reactions. This “suggests that the amplifier itself does not emit light.

In the absence of amplifiers, chemiluminescence in the peroxidase-luminol-peroxide system is apparently limited by the relatively slow reaction of peroxindase, in the form of the so-called compound II, with luminol, as a result of which luminol radicals are formed and peroxidase is regenerated.

Amplifiers capable of reacting rapidly with such a compound can increase light emission, accelerating the conversion of Compound II into active peroxidase.

The conversion products of the amplifier can then react rapidly with the luminol to form additional luminol radicals and thereby further enhance the light emission. Boosters can also target inactive peroxidase intermediates™, such as Compound III.

Reagents

An important factor in determining peroxidase activity using enhanced chemiluminescence is the chemical purity of the cyclic diacylhydrazides.

A study of various luminol samples showed that impurities negatively affect light emission. As a result of photochemical and thermal processes, luminol is partially decomposed, and the resulting impurities affect the kinetics and intensity of light emission.

In table 4 presents the results of a comparison of the purity and properties of some commercially available luminol preparations, as well as the sodium salt of luminol, purified by recrystallization from a sodium hydroxide solution.

2. ENHANCED CHEMILUMINESCENT IMMUNOASSAY

Enhanced chemiluminescence immunoassay with peroxidase as an enzyme tag has been used for the determination of many compounds. A number of examples are given in table. 5. These immunoassays used competitive and immune complex extraction methods as well as most commonly used solid supports.

a Signal/background ratio for chemiluminescent determination of horseradish peroxidase enhanced with l-iodophenol; 6 TLC - thin layer chromatography.

rice. Figure 3 shows a diagram for determining follicle-stimulating hormone. In this method, the detection limit of FSH on a microtiter plate is about 0.01 ppm. The method is characterized by excellent reproducibility both within one assay and between assays.

The enhanced chemiluminescence method has also been successfully used to detect peroxidase labels in DNA hybridization studies.

FLUORESCING ANTIBODY METHOD (FFA).

a method of quantitative determination using a plate photometer and qualitative analysis using photographic detection with a sensitivity of several picograms.

Devices

The industry currently produces several types of luminometers suitable for measuring the light emitted in enhanced chemiluminescence reactions.

Some of these luminometers have been modified specifically for enhanced chemiluminescence analysis. The intensity and duration of light emission in luminescence-enhancing reactions has facilitated the development of simple luminometers that use silicon photodiodes or high-speed photographic film as detectors.

Such luminometers are relatively inexpensive, portable, and therefore can be used outside laboratories. Currently, MAST Immunosystems produces special cameras for chemiluminescent detection in the ELISA method of allergen-specific immunoglobulin E. It can be assumed that in the future kits will be created with photographic recording of the results of ELISA and other compounds, the determination of which must be carried out in clinics, hospitals or at home.

Manufactured instruments and reagent kits.

Enhanced chemiluminescence enzyme immunoassay kits and associated instruments are now marketed under the name Amerlite by Amersham International pic.

The analysis is performed on white microtiter plate strips, and luminescence measurements are quickly and automatically performed by a special microplate luminometer.

Currently, kits are produced for the determination of thyroid stimulating hormone, human choriogonadotropin, carcinoembryonic antigen, alpha-fetoprotein, thyroxine, triiodothyronine and for determining the free thyroxine index. These assays have high sensitivity and excellent reproducibility.

CONCLUSIONS

Determination of the peroxidase tag using enhanced chemiluminescence is highly sensitive, specific, and simple and can be used as a basis for the development of convenient and rapid enzyme-linked immunosorbent assay methods performed using inexpensive portable devices.

Principles of enzyme immunoassay, main types of ELISA, application in diagnostics

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Types of immunoassay depending on the type of label and test conditions. Characteristics of components used in enzyme immunoassay.

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Luminescent tags

The concept of luminescence, its physical nature and causes of occurrence.

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Enzyme-linked immunosorbent assay (ELISA)– 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.

Immunoglobulin classes

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.

The essence of the ELISA method

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?

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)

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

5. Diseases of the thyroid gland (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

Blood is drawn on an empty stomach. You do not need to take any medications before donating blood.

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.

Enzyme-linked immunosorbent assay (ELISA)

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).

Determining the avidity of IgG antibodies is an excellent complementary step in diagnosing a primary infection from a 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

ELISA analysis (enzyme-linked immunosorbent assay) is a modern diagnostic procedure aimed at identifying specific antibodies in the blood to pathogens of a number of diseases, determining the stage of development of the pathology and its etiology. It is used as a method of monitoring the treatment process and its effectiveness. During the research process, qualitative and quantitative indicators are analyzed.

The ELISA method is highly informative; the accuracy of the results is more than 96%. Diagnostics are available in almost any laboratory and medical institution.

Main indications

An enzyme immunoassay is prescribed to confirm the presence of sexually transmitted infectious diseases:

• trichomoniasis;
• chlamydia;
• ureaplasmosis;
• syphilis, etc.

ELISA is also used to diagnose viral diseases:

• herpes;
• herpes type 4 (Epstein-Barr virus);
• viral hepatitis;
• cytomegalovirus infection.

How to prepare for research

The biological material for research is blood. A few days before the test, you should not take medications. Antibiotics, anthelmintics and antivirals are stopped within 14 days.

The material is collected in the morning on an empty stomach. An hour before the procedure, you are allowed to drink a glass of still water; food intake is excluded.

Features of the analysis

To conduct the study, venous blood is taken in a volume of at least 5 ml. In some cases, cerebrospinal fluid, amniotic fluid or the contents of the vitreous body are used as biomaterial.

Blood sampling is carried out using an injection needle, strictly on an empty stomach, in order to avoid false positive results. You should stop smoking and drinking alcoholic beverages 12 hours before. When using narcotic substances, the analysis results are distorted.

If the conclusion indicates negative values ​​of immunoglobulins G, A, M, this indicates the initial phase of the development of the pathological process or its absence. This result is recorded upon complete recovery following therapy.

A negative IgM and IgA result and a positive IgG result indicate the formation of immunity after an infectious disease or vaccination.

A positive IgM result and a negative or positive IgG, IgA result indicate the presence of an acute infectious pathology.

ELISA analysis is carried out quite quickly, the results are ready within a day after collecting the material.

Content

To assess the body's ability to resist infectious diseases or to determine the phase of pathology, a blood test is used. The ELISA method occupies an important place among laboratory tests; it helps to comprehensively study the activity of the protective function of the blood, determine immunodeficiency in infectious diseases, blood ailments, hormonal, and autoimmune processes.

What is an enzyme immunoassay blood test?

This method refers to laboratory research that determines the presence of protective blood factors of a protein nature (antibodies) to certain pathogenic agents (antigens). An enzyme-linked immunosorbent test detects immunoglobulins, which can be found in the form of immunocomplexes. They appear when complex neurohumoral reactions of the human immune defense occur, which become a response to the introduction of foreign antigens.

The body produces specific antibodies against each type of pathogen. Next, the pathological microorganism or antigen binds and a complex “antigen-antibody” compound is formed. Then it is neutralized, enzymatic lysis occurs, a phagocytosis reaction occurs, and the process ends with removal from the body. The presence of specific complexes, determined by ELISA, indicates the type of pathogen or harmful substance in the patient.

Immunoglobulin classes

Scientists have discovered and studied 5 types of immunoglobulins: IgE, IgD, IgG, IgM, IgA. There are other classes, but they are still at the research stage, and their role is not fully understood. In practical medicine, A, M, G are important. The information content and accuracy of determination is based on the time intervals during which they appear, reach a maximum and disappear.

Indications for blood testing using ELISA

Using this analysis, you can evaluate the effectiveness of treatment, conduct a comprehensive study before transplant operations, determine the state of immunodeficiency and antibodies to more than 600 types of allergens. Blood testing using ELISA is used as an additional way to detect cancer cells. An analysis is prescribed if it is necessary to detect antibodies to microbes that provoke sexually transmitted pathologies:

• trichomoniasis;
• syphilis;
• toxoplasmosis;
• mycoplasmosis;
• ureaplasmosis.

In case of helminthic infestations, the ELISA analysis will indicate an increase in the amount of immunoglobulins. Studies are carried out to confirm whether the patient has:

• Epstein-Barr virus;
• herpetic infections;
• cytomegalovirus;
• group of viral hepatitis.

Blood test using ELISA method

Enzyme immunoassay blood testing is not the only option for determining immunoglobulins. Sometimes for this study, cerebrospinal fluid, vitreous tissue, and amniotic fluid are collected. When using blood, it is collected from the antecubital vein using an injection needle. The test must be taken on an empty stomach; before ELISA, it is not recommended to take medications that could affect the result. You should give up alcohol, smoking, and drug use before donating biomaterial. Test result options:

1. If the immunoglobulins IgG, IgM, IgA are negative, doctors say there is no pathology or initial stage. The same result (negative) will be after complete recovery after a long period.
2. If IgG is positive, but IgM and IgA are not detected, this indicates the formation of immunity after vaccination or an infectious disease.
3. With high titers of IgM and negative IgA, IgG, a diagnosis of an acute infectious disease is made.
4. If IgG, IgM, IgA are positive, doctors talk about the acute phase of a relapse of an existing chronic disease.
5. For a chronic infection that is at the stage of abating (remission), the ELISA test shows negative IgM titers, while IgA and IgG will be positive.

Advantages and disadvantages of ELISA analysis

The main negative aspect of this study is the possibility of obtaining false positive or false negative results. The reason for unreliability is the use of medications and technical defects in the laboratory. The process of metabolic disorders in the body can falsify the analysis. The main advantages of the ELISA analysis are:

• accuracy, diagnostic specificity;
• low cost of analysis;
• speed of obtaining results;
• the possibility of dynamic monitoring of the stage of pathology and the effectiveness of treatment;
• ease of research;
• the ability to perform mass examinations of foci of infection;
• painlessness, safety for the patient;
• application in information technology processing.

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Linked immunosorbent assay(or ELISA for short) is a laboratory test that helps make an accurate diagnosis, identify hidden diseases, determine predisposition to certain diseases, and also monitor the effectiveness of prescribed therapy. During the study, antigens characteristic of specific pathogens and antibodies to them are detected in the patient’s blood serum.

What's the point

In order to understand the principle of ELISA, it is necessary to remember how the body’s immune system works, what “antigen” and “antibodies” are, and what functions they perform.

An antigen is a molecule that carries certain information about a cell. If a foreign antigen enters the body, antibodies (or immunoglobulins (Ig), in response to the appearance of a foreign microorganism in the body, bind to it and recognize whether it is one’s own or a foreign one. When receiving a “foreign” signal, the antibodies begin to destroy the potentially dangerous object. This “antigen” interaction -antibody" is called immune complex. The ELISA method is based on it.

Indications

The analysis is widely used to diagnose various types of diseases. And what is especially valuable is that this study quite accurately diagnoses diseases that occur in the body latently, without symptoms.

With its help you can identify:

• sexually transmitted infectious diseases (chlamydia, ureaplasmosis, mycoplasmosis, syphilis, herpes, HIV, etc.);
• toxoplasmosis, tuberculosis, hepatitis, measles, etc.;
• autoimmune problems;
• oncology;
• sex hormones;
• thyroid hormones;
• allergies.

ELISA can even detect markers of heart disease. In addition, it is prescribed to evaluate the effectiveness of the course of treatment, as well as before certain types of surgical interventions.

Preparation

Blood for research is taken from a vein on an empty stomach, preferably in the morning.

The day before you should refrain from alcohol, sugary drinks, coffee and a large dinner. In addition, the results of the indicators may be affected by the use of certain medications, so consultation with your doctor is necessary.

Smoking should be avoided 4 hours before the procedure.

Enzyme immunoassay blood test at ON CLINIC

The international medical center ON CLINIC is equipped with its own laboratory, which has an international quality control certificate. Here, experienced and qualified specialists conduct various types of analyzes (more than 1000 items).

Among the advantages of this method, one can highlight the fact that it detects it already at the earliest stages of the development of the disease. The sensitivity of the test is 90%. The study accurately shows the dynamics of the infectious process, which allows the specialist to monitor the effectiveness of therapy. The entire process is fully automated, which eliminates the influence of the so-called “human factor”.

In addition, high-precision equipment allows you to obtain reliable research results in a minimally short period of time.

You can decipher the results on the same day with your doctor. Based on them, the doctor will choose a treatment tactic that is right for you.

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GENERAL PHARMACOPOEIAN ARTICLE

Introduced for the first time

This general pharmacopoeial monograph applies to the enzyme-linked immunosorbent assay (ELISA) method. The ELISA method is a highly sensitive and highly specific immunodiagnostic method, which is used to carry out the qualitative and quantitative determination of various substances that have the properties of an antigen, hapten (incomplete antigen) or antibody. The ELISA method is widely used for the diagnosis of infectious and non-infectious diseases of humans and animals and can also be used to confirm the quality of immunobiological medicinal products (IBPs).

The principle of the method is the specific interaction of an antigen with an antibody with the formation of an immune complex and subsequent detection of the resulting complex using spectrophotometry, chemiluminescence and other adequate techniques. Detection can be either direct (when the test substance itself has enzymatic activity, or it is labeled with an enzyme label), or indirect or indirect (when the test substance, bound to antibodies immobilized on the solid phase, is incubated with enzyme-labeled antibodies). Qualitative analysis allows you to obtain information about the content of antigen or antibody in the material under study according to the “yes/”no” principle. When carrying out a quantitative analysis, the concentration of antigen or antibody in the test material is determined using a calibration graph.

GENERAL PROVISIONS

The ELISA method includes 3 main stages: 1) formation of an immune complex “antigen (test substance) – antibody specific to it” or vice versa; 2) the formation of a bond between the conjugate and the immune complex formed at the previous stage or with free binding sites (determinants); 3) transformation of the substrate under the action of an enzyme tag into a recorded signal as a result of a biochemical reaction.

All methods for performing enzyme immunoassays are classified as homogeneous or heterogeneous.

Methods in which all 3 stages of ELISA take place in solution, and between the main stages there are no additional stages of separating the formed immune complexes from unreacted components, belong to the group of homogeneous ELISA methods. The basis of homogeneous ELISA, used, as a rule, for the determination of low-molecular substances, is the process of inhibiting the activity of the enzyme when it combines with an antigen or antibody. As a result of the antigen-antibody reaction, enzyme activity is restored. When an antigen–antibody immune complex containing an enzyme tag is formed, the enzyme activity is inhibited by 95% in relation to the high molecular weight substrate, which is due to the steric exclusion of the substrate from the active center of the enzyme. As the concentration of the antigen increases, more and more antibodies bind, and more and more free antigen-enzyme conjugates remain, capable of hydrolyzing the high molecular weight substrate. The homogeneous ELISA method is very fast. It takes 1 minute to analyze one definition. The sensitivity of the method is quite high. It can be used to determine a substance at the picomole level.

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

Methods are classified as 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.

The heterogeneous ELISA method consists of 3 main steps:

• immobilization of an antigen or antibody on a solid phase, the resulting complex is called an immunosorbent;
• removal of unbound reagent and blocking of binding sites on a solid support using blocking proteins, such as albumin, casein; incubation of the analyzed drug with an immunosorbent so that their binding occurs;

3) detection due to the enzymatic activity of the test substance itself or due to an enzymatic label associated with the analyzed drug (direct version). In some cases, additional incubation of the “immunosorbent–test substance” complex with secondary antibodies conjugated to an enzymatic label is performed (indirect option).

Quantitative determination of the test substance is carried out by adding a substrate suitable for the detector used and comparing the signal of the test substance with a standard sample.

The heterogeneous ELISA method is divided into non-competitive ELISA and competitive ELISA. Assay schemes can be modified during the drug development process in accordance with the necessary requirements. Changes must be indicated in the pharmacopoeial monograph or regulatory documentation. The choice of ELISA method depends on the nature of the test substance and its quantity, since different types of ELISA have different sensitivities. To assess the quality of substances containing antibodies, it is possible to use specific anti-idiotypic antibodies.

Non-competitive ELISA method

The non-competitive ELISA method is divided into several types according to the type of detection (direct competitive, indirect (indirect) competitive) and according to the type of substance immobilized on the solid phase (antigen or antibody).

Direct ELISA option

Can be done in 2 ways. In the first case, the test substance (antigen) is directly immobilized on the solid phase; then the labeled antibody bound to the antigen is the detector. When performing the test in a different way, antibodies immobilized on the solid phase are used. In this case, the detector is the test substance labeled with an enzyme.

Indirect (indirect) version of ELISA

When performing the indirect version of ELISA, the antigen is immobilized on the solid phase. After blocking, a solution of antibodies specific to it is added to the antigen. After incubation, the resulting antigen-antibody complex is washed from unbound antibodies and enzyme-labeled anti-immunoglobulin (anti-Ig) is added, which acts as a detector. Anti-Ig detectors are commercially available for specific Ig classes and subclasses, making this assay format convenient for antibody isotyping. In addition, the use of labeled anti-Ig enhances the signal compared to the direct enzyme-linked immunosorbent assay method, thereby increasing the sensitivity of the assay.

The “sandwich” method as an option for performing ELISA

The most common non-competitive method is the sandwich method. When it is performed, primary antibodies are immobilized on the solid phase and subsequently blocked. Then the test substance containing the antigen is added to them and incubated. After incubation, the antigen-antibody complex is washed away from unbound antigen and secondary antibodies labeled with an enzyme are added and detection is carried out.

Competitive ELISA method

The competitive ELISA method is divided into several types: by the type of detection (direct competitive, indirect (indirect) competitive) and by the type of substance immobilized on the solid phase (antigen or antibody).

Direct competitive ELISA

To detect or quantify soluble antigens, a direct competitive version of the ELISA with an antigen immobilized on the solid phase is used. To do this, use antigen-specific antibodies conjugated to an appropriate detector (for example, horseradish peroxidase, alkaline phosphatase, ruthenium or fluorescein). A standard antigen is immobilized onto the solid phase, followed by blocking. The antibody conjugated to an enzymatic tag is incubated with the test substance (soluble antigen). This mixture is then added to the immobilized antigen, incubated, and then washed to remove the unbound antigen-antibody complex. The next step is to add a suitable substrate for the enzyme being used as a tag. The inhibition of the reaction due to the presence of 2 antigens in the system, compared to a control sample without a competitive soluble antigen, is inversely proportional to the amount of the test substance.

The direct competitive solid-phase antibody ELISA is similar to the direct competitive solid-antigen ELISA, but is used to detect or quantify antibodies.

Indirect (indirect) competitive ELISA variant

This ELISA method is similar to the direct competitive version, but instead of a labeled antibody or antigen, a labeled anti-Ig reagent or labeled secondary antibodies are used for detection, respectively.

General conditions for carrying out the methodELISA

Various materials are used as a solid phase for enzyme immunoassay: silicone, nitrocellulose, polyamides, polystyrene, polyvinyl chloride, polypropylene, acrylic and others. The solid phase can be the walls of a test tube, 96-well and other plates, balls, beads, as well as nitrocellulose and other membranes that actively absorb proteins. The principle of immobilization (hydrophobic, hydrophilic, covalent interaction) depends on the choice of solid phase. Most often, 96-well plastic microtiter plates are used as the solid phase. The number of wells in the plate may vary. The tablet can be transparent (colorimetric detection) or matte (chemiluminescent detection, fluorimetry).

Immobilization must be carried out without air bubbles in the well, since their presence changes the optical density reading. It is possible to use biotinylated immobilized reagents. In this case, the reaction uses streptavidin and a biotinylated enzyme tag. This method is used to amplify the signal. The time and temperature of immobilization, depending on the kinetic nature, stability and concentration of the reagent, must be indicated in the pharmacopoeial monograph and regulatory documentation.

All stages of the enzyme immunoassay, washing and blocking solutions, time intervals and temperature conditions for each stage, the number of revolutions per minute for incubation on a shaker, detection conditions must also be specified in the pharmacopoeial monograph and regulatory documentation.

Examples of methods for some types of ELISA

Indirect non-competitive ELISA method

1. Antigen sorption. Add 0.1-0.5 μg of antigen and 100 μl of 0.05 M carbonate-bicarbonate buffer solution (pH 9.6) to each well of a 96-well plate, unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation, and then carry out sorption at a temperature of 4 ° C for 16 hours. It is possible to use other buffer solutions with high pH values. Incubation is carried out by shaking on a horizontal plate shaker.

Washing (twice) of unbound antigen molecules is carried out with a phosphate-buffered saline solution (pH 9.0) containing 0.1% Tween-20 (300 μl per well), unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation.

1. Blocking. To block sites of nonspecific binding of antigens or antibodies, the wells of the tablet are filled with a phosphate-buffered saline solution (pH 9.0) or another buffer solution specified in the pharmacopoeial monograph or in regulatory documentation containing a 1% solution of bovine serum albumin or other proteins (casein, gelatin , milk powder, etc.), and incubate for 10–15 minutes at room temperature (unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation).

III. Titration of specific antibodies. If quantitative assessment is necessary, the test substance (antigen or antibody) is titrated in serial dilutions in parallel with the standard sample (RM).

Titration can be carried out in both horizontal and vertical rows of the plate. It should be noted that titration of antibodies is carried out if it is necessary to select the optimal concentration of antibodies or determine their titer. If the optimal concentration and/or titer of antibodies is determined, then the recommended dilution for these antibodies (serum) is used.

When titrating, a ready-made dilution of antibodies is added to the first well of the series - an average of 1-10 μg per well, then sequential dilution of the antibodies is carried out in the wells. Incubation with specific antibodies is carried out for 30 minutes at room temperature with shaking on a horizontal plate shaker.

Washing is carried out at least 3-4 times using a phosphate-buffered saline solution pH 9.0 containing 0.1% Tween-20.

1. Addition of anti-species (antiglobulin) antibodies conjugated to an enzyme tag. Anti-species polyclonal antibodies conjugated to an enzymatic tag are used as detector (secondary) antibodies. Most often, goat or rabbit antibodies are used, specific to the whole molecule or to the Fc fragments of specific antibodies. The concentration of detector antibodies is usually specified by the manufacturer as a dilution of the stock solution (for example, 1:1000).

Incubation with labeled secondary antibodies is carried out for 30 minutes at room temperature with shaking on a horizontal plate shaker.

Washing is carried out at least 3-4 times using a phosphate-buffered saline solution (pH 9.0) containing 0.1% Tween-20.

Incubation is carried out for 10 minutes at room temperature and shaking on a horizontal plate shaker.

1. 100 μl of substrate solution is added to the wells and incubated for 10 minutes at room temperature and constant stirring. To stop the enzymatic reaction, a “stop reagent” is used, which is added to all test and control samples in equal quantities. Sulfuric acid is most often used as a “stop reagent”.

Direct non-competitive ELISA method

The direct ELISA method has only slight differences from the indirect non-competitive ELISA method. Thus, stages I and II are the same in both types of analysis. The difference is that in the direct version of ELISA, at stage III, antibodies specific to the test antigen are used, conjugated to an enzyme label, and they directly interact with the test substance. If necessary, titration of conjugates can also be carried out similar to the principle described previously for unconjugated antibodies. Stage IV of the direct non-competitive ELISA is not performed.

"Sandwich" method ELISA

This version of ELISA uses a pair of antibodies (primary and secondary) specific to spatially distant epitopes of the antigen being studied.

1. Sorption of antibodies on the solid phase. The antigen sorption technique is similar to the antibody sorption technique in the section “Indirect non-competitive ELISA method”.
2. Blocking. The technique for blocking nonspecific binding sites on the substrate (solid phase) is similar to the blocking technique described in the section “Indirect non-competitive ELISA method”.

III. Incubation with antigen. 50 μl of the test substance and standard dilutions of the antigen are added to the wells of the plate with pre-adsorbed antibodies, unless otherwise indicated in the pharmacopoeial monograph or regulatory documentation. Antigen dilutions should be prepared in phosphate-buffered saline (pH 9.0) containing 0.1% Tween-20, since Tween-20 reduces the nonspecific binding of protein molecules to each other and to the plate surface. Both the test substance and standard dilutions of the antigen are added in pairs to adjacent wells in a horizontal row (or in 3 replicates), using 2 (3) wells for each protein dilution.

Incubation is carried out at room temperature for 30 minutes with constant stirring. Washing is carried out at least 3-4 times with a phosphate-buffered saline solution (pH 9.0) containing 0.1% Tween-20, or another buffer solution specified in the pharmacopoeial monograph or regulatory documentation.

1. Incubation with antibodies conjugated to an enzyme tag. 100 μl of a solution of specific antibodies conjugated to an enzyme label is added to the wells of the plate. The optimal concentration of conjugated antibodies is usually indicated in the pharmacopoeial monograph or regulatory documentation (usually a concentration of 2–4 μg/ml is used).

Incubation with antibodies containing an enzyme tag is carried out for 30 minutes at room temperature with shaking on a horizontal plate shaker.

Washing is carried out at least 3-4 times using a phosphate-buffered saline solution (pH 9.0) containing 0.1% Tween-20, or another buffer solution specified in the pharmacopoeial monograph or regulatory documentation.

1. Carrying out an enzymatic reaction accompanied by the appearance of a colored product. The technique for carrying out the enzymatic reaction is similar to the technique described in the section: “Indirect non-competitive ELISA method.”

Detection

As stated above, antibodies labeled with an enzyme tag or other reagent are used for detection. The enzyme tag can be, for example, horseradish peroxidase, alkaline phosphatase or galactosidase. Antibodies or antigens with other labels can be used as a detector. The choice of detector reagent depends on the type of label conjugated to the antibody or antigen and the detection method.

Spectrophotometry, chemiluminescence, fluorimetry and other methods can be used as detection methods, based on the choice of label.

Results of the quantitative ELISA method

Quantitative ELISA results are calculated from a linear calibration curve with inverse regression or by a complex method using a nonlinear calibration curve with inverse regression. The method for interpreting the results depends on the ELISA method used. For example, the test results can be used to estimate the concentration of an unknown sample, the half-maximal inhibition concentration, or the effective concentration using a calibration curve. This allows the amount of the test substance or its activity to be determined in comparison with a reference/calibration standard (RM). Typically, the type of calibration curve when performing a quantitative ELISA method, characterizing the concentration of the analyzed drug, depends nonlinearly on the calculated average value. In this regard, it is recommended to use various mathematical models to analyze the resulting curve. In other cases, the ELISA method is used as a qualitative method that allows one to assess the presence of a particular test substance in a sample within the sensitivity limits of the technique.

Note.

Preparation of carbonate-bicarbonate buffer solution (pH 9,6). Add 1.59 g of sodium carbonate (anhydrous) or 4.29 g of sodium carbonate 10-aqueous and 2.93 g of sodium bicarbonate into a measuring cylinder with a capacity of 1000 ml, dissolve in 800 ml of purified water, adjust the pH to 9.6, mix, then bring the volume of the solution to the mark with purified water and mix again.