Immunoenzyme analysis( EIA) - Causes, symptoms and treatment. MF.
Immunoenzyme assay( ELISA) is a modern laboratory study 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. The results of ELISA 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, venereal);
3) examination of the patient's hormonal status;
4) examination for oncomarkers;
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 monitor the dynamics of the process, that is, the comparison of the number of antibodies in different time intervals.
3) Availability of ELISA diagnostics in any medical institution.
Relative deficiency:
1) Identification of the immune response( antibodies), but not the pathogen itself.
Basic concepts of
Before clarifying the essence of the ELISA method, let us briefly examine some concepts.
Antibodies( or immunoglobulins - Ig) are specific proteins produced by B-
lymphocytes( immune cells) in response to ingestion of any infectious pathogen( viruses, bacteria, fungi, etc.).Immunoglobulins A( IgA), immunoglobulins E( IgE), immunoglobulins M( IgM), immunoglobulins G( IgG), immunoglobulins D( IgD) are isolated. They differ from each other in molecular form and mass, half-life, participation / non-participation in infectious processes, the timing of detection from the moment of infection. If we consider the molecular weight, then most of it in IgM is a pentamer( 950 000 daltons), unlike the rest of Ig( from 150 to 200 000 Da), so that IgM simply can not pass through the placental barrier. Therefore, the detection of IgM in a child 1 year of age is always a sign of infection in the fetus. In the serum, the bulk of immunoglobulins is represented by IgG( 75-85%), and the lowest IgE( 0.003%).In the infectious process, only IgA, M, and G. IgE are directly involved in the infection process, and IgD can be detected only in the tissue of lymph nodes and tonsils, plays a role in the formation of local immunity.
Classes of immunoglobulins
antigens are high molecular substances of organic origin, in particular pathogens of infectious and other diseases, as well as substances of various altered cells formed in a disease( autoimmune diseases, oncology).
The immune complex is an antigen-antibody complex involved in the immune process. What is the IFA method based on?
Several types of ELISA are isolated( direct, indirect, blocking, competitive), but in practice heterogeneous solid phase immunoassay or ELISA( enzyme linked immunosorbent assay)
is used most often. The basis of the enzyme immunoassay is the immune response of the antigen and antibody to form an immune complex: antigen-antibody, which results in a change in the enzymatic activity of specific labels on the surface of antibodies.
The essence of the ELISA method
In simple language, this process can be divided into several stages:
1) The purified antigen of a particular pathogen is located on the surface of the wells of the doctor's plate that conducts the examination. When a biological material( blood serum) is added to the patient, a specific reaction occurs between this antigen and the desired antibody( immunoglobulin).This compound will act as a "special antigen" in the next stage.
2) At this stage, the formation of IR( immune complexes) is a reaction between a "special antigen" and a conjugate( this is an immunoglobulin labeled with a peroxidase enzyme).A special chromogen is added. The result of this enzymatic reaction is the formation of a colored substance in the well of the plate, the intensity of the color 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, comparing the optical density of the test material with the optical density of the control samples, and mathematical processing of the results. The amount of antibodies in a patient depends directly on the height of the optical density of a given well.
Typically, 96 well plates are used in practice.
When measuring the optical density( OP) of the test fluid, the amount( or concentration) of antibodies in a certain unit of volume is counted. Then the result is compared with the control sample.
It should be remembered: for each test system, individual indicators are developed for the recording of results, indicators of norm and pathology( ie "reference values").This should be taken into account when evaluating the results of each particular study. It is incorrect to interpret the results of one laboratory on the "reference values" of another laboratory. It is also incorrect to compare the results of different laboratories with each other.
When stating the reactions of ELISA, the concept of avidity of antibodies also matters.
The avidity of the antibodies is the binding strength of the antibody to the antigen and the amount of antigen in association with the immunoglobulins( antibodies).Avidity is very important in assessing the estimated duration of infection, which is extremely important in the diagnosis of primary infection in pregnant women.
The basis for the antibody test consists of processing the immune complex( antigen-antibody) with a solution of urea to destroy the protein. High-like connections remain intact, and low-visibility bonds are destroyed. The result is given in the form of an index of avidity, expressed as a percentage( %).
What diseases are diagnosed with IFA diagnostics?
1. Infectious diseases
1.1 Viral hepatitis A( IgM, G), antigens -HBsAg, HBeAg, antibodies - IgM HBcorAg, Ig total HBcorAg, IgG HBeAg), C( antibodies Ig M, G HCV), D( IgM,G), E( Ig M, G), and others.
1.2 HIV infection( IgG)
1.3 Cytomegalovirus( IgM, G) infection
1.4 Epstein-Barr infection or mononucleosis( IgM, G antibodies to EA, VCA, NA virus proteins)
1.5 Herpetic infections( IgM, G antibodies)
1.6 Toxoplasmosis( antibodies IgM, G)
1.7 Rubella( IgM, G)
1.8 Measles( IgM, G), Brucellosis( IgM, G), Pseudotuberculosis( IgM, G), Salmonellosis( IgM, G), Shigellosis or dysentery(IgM, G), Aspergillosis( IgM, G), Tick-borne encephalitis, Borreliosis and many others.
1.9 Parasitoses( giardiasis, toxocariasis, trichinosis, opisthorchiasis, echinococcosis, alveococcosis and others), in which IgG is detected.
1.10 STDs( sexually transmitted diseases) - Mycoplasmosis( IgM, G), Ureaplasmosis( IgM, G), Chlamydia( IgG, M, G), Syphilis( IgG)
1.11 Helicobacter pylori infection( IgG)
2. Markers of autoimmune diseases and human immunodeficiency indices ( total IgE, total IgG, total IgA, total IgM, total IgD, secretory IgA, IgG 2, IgG4, CIC-circulating immune complexes, IgA and IgG to gliadin and others)
3. Oncologic markers ( FNO-tumor necrosis factor, REA-cancer-embryonic antigen, PSA-prostate-specific antigen, CH-chorionic gonadotropin, CA 125, alveomucine and many others)
4. Reproductive disorders i( estradiol, progesterone, prolactin, testosterone, AFP-alfa-proteprotein, FSH - follicle-stimulating hormone and others)
5. Thyroid gland diseases ( free and bound T3, T4, thyreoglobulin, thyreperoxidase - TPO, thyroid-stimulating hormone - TSH).
This list is not presented by all diseases, which are diagnosed by 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 serve as cerebrospinal fluid, amniotic fluid, vitreous contents, mucus of the cervical canal and urethra, strokes.
Preparation of patients for delivery of material for IFA
Blood is taken up on an empty stomach. Before donating blood, you do not need to take any medications. Specific treatment( antibacterial, antiviral, antiparasitic and others) should be discontinued no less than 2 weeks before the study.
Timing of the preparation of the ELISA
The enzyme immunoassay is carried out quickly, during the day. Delays can be in different laboratories due to the accumulation of a certain number of sera.
Possible results of the ELISA diagnostics
When assessing the results for specific infections, the class of antibodies detected and their number are important. It depends not only on 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 active infection( acute or exacerbation of the chronic) at the time of the examination.
What are the approximate terms for the appearance of antibodies( immunoglobulins-Ig)?
The earliest antibodies are IgM .Identify them can be 1-3 weeks after possible infection, which characterizes the acute phase of the infectious process. The second situation with the appearance of IgM antibodies is the activation( or exacerbation) of the chronic process. IgM antibodies circulate on average about 3 months, then their number gradually disappears. However, in some patients, trace amounts of IgM can be detected within 1-2 years from the time of infection.
Modern test systems are highly sensitive, resulting in non-specific false-positive results( often in pregnant women).Therefore, in this group of patients, positive IgM should be rechecked!
IgA antibodies appear 2-4 weeks after infection, but in an amount sufficient for detection - in a month. Serum IgA is synthesized by plasmatic cells of the spleen, lymph nodes and mucous membranes, secretory IgA concentrate on the mucous membranes to perform its protective function - participate in local immunity.
From the 4th week after infection antibodies IgG begin to appear. In 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. With some diseases( mycoplasmosis, chlamydia, trichomoniasis), the IgG level is not high, it decreases substantially because of the lack of immunity in these infections.
Variants of detection of antibodies of different classes:
- Isolated detection of IgM antibodies presupposes the presence of primary
infection.
- Simultaneous detection of IgM and IgG in blood is characteristic for primary infection of
in the previous 2-3 months, as well as in exacerbation of a chronic disease. Therefore, in pregnancy, the presence of IgM is not always a sign of primary infection.
- Detection of isolated IgG may indicate both immunity to the disease,
and chronic infection. In the second situation, the number of antibodies( titer) is also important, and the change in this titer is in dynamics. Usually, studies are conducted at intervals of 2-4-6 weeks.
- Detection of IgA isolated or with IgM indicate a primary infection. With
, the appearance of IgA together with IgG is expected to activate chronic infection( an average of 2 weeks from the time of exacerbation).
The definition of IgG antibodies IgG is an excellent complementary stage in the diagnosis of primary infection from long-term infection, which has its clinical significance, especially in assessing the risk of intrauterine fetal infection. Detection of low-grade IgG indicates a primary infection and is detected on average 4-6 months after infection, less often longer. Low-dose IgG requires other laboratory evidence of primary infection( IgM).High-antibodies are either a sign of a chronic disease and its aggravation, or of a formed immunity.
Features in infants: in children under one year, and sometimes 1.5 years in the blood, maternal IgG is circulating to different infections( that is, their penetration through the placenta from mother to fetus during the period of fetal development).It is not in itself a sign of infection in the present. If at the same age IgM are found( recall that maternal IgM can not penetrate the placenta), then this is a sign of intrauterine infection or acquired infection after birth.
Quantitative ELISA method
The result of the ELISA diagnostics( with the help of an enzyme immunoassay) is given in certain units:
- Optical density( OP) of the sample is the concentration of specific antibodies per unit volume. The higher the OP sample, the higher the concentration of antibodies. Some results refer to the coefficient of positivity( KP) - this 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 titres( in which the disease is diagnosed, and not the fact of infection) for different diseases are different.
- In the form of symbols - "+", "-", "?"( +, ++, +++, ++++).
- In the form of a qualitative assessment based on a given criterion( positive or negative).
It is right to estimate the number of antibodies, the variant of class detection of immunoglobulins, and, consequently, to expose the stage of the disease and the need for treatment can only be done by a doctor.
It should not be forgotten that for any test system, their "reference values"( variants of the norm) are developed, when exceeding which one or another disease is diagnosed( pathology variants).For different test systems, the "reference values" are different.
A correct comparison of the ELISA results taken in dynamics is possible only if they are manufactured in the same laboratory.
Physician infektsionist Bykova N.I.