Enzyme immunoassay (EIA ) - Causes, Symptoms and Treatment .MF .
immunosorbent assay (ELISA) - modern laboratory study in which the search is conducted of specific antibodies or antigens in the blood to specific diseases in order to identify not only the cause, but the stage of the disease.ELISA results may be issued both qualitatively and quantitatively.
Nowadays ELISA is used in the following situations:
1) Search for specific antibodies to any infectious disease;
2) search for antigens of any diseases (infectious diseases, venereal diseases);
3) study the hormonal status of the patient;
4) testing for tumor markers;
5) examination for the presence of autoimmune diseases.
Advantages of ELISA:
1) The high specificity and sensitivity of the ELISA method (90%).
2) Ability to define and track the disease process dynamics, that is, comparing the amount of antibody in different time intervals.
3) IFA-diagnostics available in any health care facility.
1) Identification of the immune response (antibodies), but not of the pathogen.
Before clarify the essence of the ELISA method, briefly's understand some concepts.
Antibodies (or immunoglobulins - Ig) - specific proteins produced by B -
lymphocytes (immune cells) in response to the entry into the body of an infectious pathogen (virus, bacteria, fungi, etc.).Secrete immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin M (IgM), immunoglobulin G (IgG), immunoglobulin D (IgD).They differ from each other and form the molecular weight, half-life, participation / non-participation in infectious processes, detection timing from the moment of infection.If we consider the molecular weight, the most he's IgM - is a pentamer (950,000 Da), in contrast to the rest of Ig (from 150 to 200 000 Da), so IgM simply can not pass through the placental barrier.Therefore, the detection of IgM in a child 1 year of life is always a sign of infection in the fetus.The bulk of serum immunoglobulins is presented IgG (75-85%), and the lowest - IgE (0,003%).The infectious process take direct part only IgA, M, G. IgE allergic reactions are a sign and disease, and IgD - can be detected only in the lymph node and tonsil tissue, plays a role in the formation of local immunity.
classes of immunoglobulins
Antigens - macromolecular substances of organic origin, in particular of infectious and other diseases, as well as different substances altered cells produced during a particular disease (autoimmune diseases, oncology).
Immune complex - the antigen-antibody involved in the immune process.
What based ELISA method.
There are several IFA species (direct, indirect, blocking method, competitive), but in practice most often used a heterogeneous enzyme immunoassay, or ELISA (enzyme linked immunosorbent assay)
basis of enzyme immunoassay of the immune response of an antigen and an antibody to form an immune complex: antigen-antibody, which resulted in a change in the enzymatic activity of specific antibodies to surface markers.
The method IFA
simple language, this process can be divided into several stages:
1) on the surface of the doctor-hole plate, the examiner is purified antigen specific pathogen.When adding the biological material (blood serum) patient specific reaction occurs between the antigen and the desired antibody (immunoglobulin).This connection will act as a "special antigen" in the next stage.
2) At this stage, there is the formation IR (immune complexes) - the reaction between the "specific antigen" and conjugate (this immunoglobulin labeled with peroxidase enzyme).Adding special chromogen.The result of this enzymatic reaction is the formation of colored substances in the well plate, the color intensity of which depends on the number of patient immunoglobulins (antibodies) contained in the material.
3) Next comes Interpretation: photometry using a multichannel spectrophotometer, comparing the optical density of the material with the optical density of the control samples, mathematical processing of the results.The amount of antibodies in the patient depends on the height of the optical density of the wells.
common practice to use the 96 well plates.
amount (or concentration) of a specific antibody per unit volume in the measurement of optical density (OD) of the sample liquid is calculated.The result is then compared with a control sample.
must be remembered: for every test system developed by individual performance to account for the results, normal and pathological indicators (ie, the "reference value").This should be taken into account when evaluating the results of each case study.Incorrectly interpret the results of a laboratory "reference values" another laboratory.It is also incorrect to compare the results of different laboratories with each other.
When setting ELISA reactions is important, and such a thing as antibody avidity.
avidity antibody - this antibody the antigen binding strength and the amount of antigen in association with located immnoglobulinami (antibodies).Avidity is of great importance when assessing the alleged infection period, which is extremely important in the diagnosis of primary infection in pregnant women.Basis
antibody avidity test consists of treatment of an immune complex (antigen-antibody) urea solution in order to destroy the protein.Vysokoavidnye connection remain intact, and low avidity destroyed.The result is given in the form of avidity index, expressed as a percentage (%).
What diseases are detected by ELISA-diagnostics?
1. Infectious diseases
1.1 Viral hepatitis A (IgM, G), B antigens -HBsAg, HBeAg, antibody - 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 infection (an antibody IgM, G)
1.4 Epstein-Barr virus infection or mononkleoz (antibody IgM, G to EA, VCA, NA proteins of the virus)
1.5 Herpetic infection (an antibody IgM, G)
1.6 Toxoplasmosis (antibody 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 parasitosis (giardiasis, toxocariasis, trichinosis, opistorhoz, hydatid disease, alveococcosis and others), in which the detected IgG
1.10 STDs (sexually transmitted diseases through) - Mycoplasmosis (IgM, G), Ureaplasmosis (IgM, G), Chlamydia (IgA, M, G), syphilis (IgG)
1.11 Helicobacter infection (IgG)
2. Markers of autoimmune diseases and human immunity indicators (total IgE, total IgG, total IgA, total IgM, total IgD, secretory IgA,IgG 2, of IgG4, CEC, circulating immune complexes, IgA and IgG antibodies to gliadin and other)
3. Onkologichsekie markers (TNF - tumor necrosis factor, CEA - carcinoembryonic antigen, PSA - prostate specific antigen, hCG - human chorionic gonadotropin, CA 125, alveomutsin and many others)
4. Reproductive disorders I (estradiol, progesterone, prolactin, testosterone, AFP AFP-, FSH - follicle stimulating hormone and other)
5. thyroid disease (available andrelated T3, T4, thyroglobulin, thyroid peroxidase - TPO, thyroid stimulating hormone - TSH).
This list is by no means all diseases are diagnosed by ELISA.
material for ELISA analysis and sampling
rules 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 urethral swabs.
Preparing patients for the delivery of material for IFA
Blood is taken on an empty stomach.Before delivery of blood do not need to take any drugs.Specific treatment (antibacterial, antiviral, antiparasitic, etc.) should be stopped at least 2 weeks prior to the study.
Dates ELISA material held fast during the day.The delays can be in different laboratories due to the accumulation of a certain number of sera.
Possible results of ELISA diagnostic
In assessing the results of specific infection is important class of antibodies identified and their number.On this depends not only the question of etiology of infection (ie it or not), but also the estimated stage of the disease (acute and chronic), and the presence of active infection (acute or exacerbation of chronic) at the time of the survey.
What are the approximate time of the appearance of antibodies (immunoglobulins - Ig)?
The earliest antibodies are IgM .Identify they can be 1-3 weeks after a possible infection that characterizes the acute phase of infection.The second situation is the appearance of IgM antibodies - activation (or worsening) of chronic process.IgM antibodies circulate in the average 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, so that there are non-specific false-positive results (often in pregnant women).Therefore, a positive IgM in this group of patients should be rechecked!
IgA antibodies appear 2-4 weeks after infection, but in amount sufficient for detection - a month.Serum IgA synthesized by plasma cells of the spleen, lymph nodes and mucous membranes, secretory IgA concentrate on the mucous membranes to achieve their protective function - participate in local immunity.
On the 4th week post-infection begin to appear antibody IgG .In most infections titer of gradually increasing, with a maximum at different times (an average of 1.5-2 months), then the titer remained at a low level and indicates immunity.In some diseases (mycoplasmosis, chlamydiosis, trichomoniasis) IgG level is not high, is significantly reduced due to the lack of immunity in these infections.
Possible detection of antibodies of different classes:
- Isolated detection of IgM antibodies suggests the presence of a primary infection
- Simultaneous detection of blood IgM and IgG is typical for primary infection
in the previous 2-3 months, as well as exacerbation of chronic diseases.Therefore, the presence of IgM in pregnancy does not always indicate the initial infection.
- Detection of IgG in isolation may indicate how immunity to the disease,
and the chronic infection.In the second situation is set and the amount of antibody (titer) and the change in titer of dynamics.Typically, studies were performed with an interval of 2-4-6 weeks.
- IgA detection isolated IgM or indicate a primary infection.When
appearance of IgA with IgG is expected activation of chronic infection (an average of 2 weeks from the time of exacerbation).
Determination of avidity IgG antibodies is an excellent complement to the diagnosis of the primary stage of infection from the long-standing infection that has its clinical significance, especially when assessing the risk of intrauterine infection of the fetus.Detection of low avidity IgG indicates a primary infection and the average detected after 4-6 months after infection, sometimes longer.Low avidity IgG require other laboratory evidence of primary infection (IgM).High avidity antibodies are a sign of chronic disease and exacerbation of, or formed immunity.
features in infants: in children under one year, and sometimes even 1.5 years in the blood circulating maternal IgG antibodies to various infections (ie, there was their penetration through the placenta from mother to fetus in utero).She is not in itself a sign of infection in the present.If at the age of takjm detected IgM (recall that maternal IgM cross the placenta can not), it is a sign of infection or intrauterine infection acquired after birth.
Quantitative ELISA method
Result ELISA diagnostics (using enzyme immunoassay analyzer) issued in specific units:
- The optical density (OD) of the sample - the concentration of specific antibody per unit volume.The higher the OD of the sample, the higher the concentration of antibodies.Some results indicate factor positivity (CP) - this is also the optical density of the sample.
- Units of antibody concentration (ng / ml or ng / ml).
- in a serum titer: 1:20, 1:40, 1: 100, 1: 200, 1: 400, 1: 800, 1: 1200 and so on.Diagnostic titers (at which it is diagnosed the disease, rather than the fact of infection) for various different diseases.
- in the form of symbols - "+", "-" (+, ++, +++, ++++) "?".
- In a qualitative assessment on specific criteria (positive or negative).
correctly estimate the amount of antibody variant class detection of antibodies, and thus expose the stage of the disease and the need for only a doctor can treat.
must not forget that their "reference values" (variants of normal), above which is diagnosed and this or that disease (variants pathology) are designed for any test systems."Reference values" for different test systems different.Correct
comparing the ELISA results, taken over time, is only possible in the case of their production in the same laboratory.
infectious disease doctor Bykov NI