Description of STD diagnostics: enzyme-linked immunosorbent assay (ELISA). Enzyme immunoassay blood test Interpretation of the analysis using the IFA method
The list of available diagnostic methods has been rapidly expanding in recent decades; diagnosticians are trying to combine the advantages of all previous tests in new methods, getting rid of all their previous disadvantages.
Recently, the list of diagnostic procedures increasingly includes an enzyme-linked immunosorbent assay - a modern and fairly new test, which is little known to the average person not involved in medicine. Nevertheless, this technique is rapidly gaining ranks of its adherents among qualified medical personnel. You can try to figure out what it is and in what cases it should be used by familiarizing yourself with its features and main characteristics.
Enzyme immunoassay is an instrumental laboratory technique based on the molecular “antigen-antibody” reaction, which allows the detection of specific proteins in biological materials (samples for research). Such proteins can be enzymes, various microorganisms (viruses, bacteria, fungi), protozoa, etc.
After the discovery of the method, it was given the name ELISA test, which has nothing to do with the name of the discoverers, but is an abbreviation of the full name in English - enzyme-linked immunosorbent assay. Doctors all over the world use this name; some doctors in Russian-speaking countries also call this type of research this way.
The main principle of the method is the molecular “antigen-antibody” reaction.
An antigen is any foreign molecule that enters the human body as part of the microbe that causes the disease. Antigens are usually protein molecules. In addition to microorganisms, such a “stranger” can be foreign blood cells that do not match the group or Rh factor.
In response to the entry of such an antigen into the body, an immune reaction is launched, which is aimed at protecting against any foreign molecules. This occurs due to the synthesis of special agents of the immune system - antibodies (immunoglobulins). Each antibody is suitable only for a specific antigen and neutralizes the pathogenic “stranger” by binding to it into a single complex. It is the process of such binding that is called the “antigen-antibody” reaction.
Types of antibodies
All antibodies (immunoglobulins) are divided into 5 types depending on at what stage of the immune response they appear:
For ELISA diagnostics, the levels of immunoglobulins IgG, IgM and IgA are most often important. By their titers, you can find out whether a person has had the disease before or became infected recently, whether he has developed immunity or whether his body is defenseless against pathology.
Advantages and disadvantages of enzyme immunoassay
At the moment, ELISA is one of the most accurate and sensitive methods. It has been adopted by specialists in various fields of medicine and continues to expand its scope of application.
Advantages of the method
- High accuracy of the obtained data.
- Sensitivity (allows you to detect the required substance even with minimal presence of the pathogen in the sample).
- Possibility of diagnosis in the first days of illness or in the incubation period.
- Speed of data acquisition compared to other methods of similar accuracy.
- High automation of the process and minimal human participation, which reduces the error of the performer.
- Obtaining data on the stage of the pathological process and the effectiveness of the chosen therapy.
- Painless and minimally invasive when collecting material.
Disadvantages of the method
- In most cases, it allows you to determine the body's response to a pathogenic agent, and not the pathogen itself.
- Before the test, the suspected disease must be known exactly, since the test is highly specific.
- The likelihood of false indicators caused by technical issues, taking medications, the simultaneous presence of several chronic diseases or metabolic disorders in the patient’s body.
- The interpretation of the results should only be done by a highly qualified specialist, since to interpret the data obtained it is necessary to have special training and a large supply of medical knowledge in a certain area.
- ELISA is a fairly rare test, so it is not performed in all diagnostic laboratories.
- The method is quite expensive, since in addition to reagents, the laboratory must have numerous expensive equipment and samples of antigens produced in special institutes.
In what cases is enzyme immunoassay used?
The full list of indications for enzyme immunoassay is very wide; it includes almost all branches of medicine.
Most often, ELISA is used for the following purposes:
- detection of infectious diseases;
- diagnosis of sexually transmitted diseases;
- determination of immune status or individual autoimmune diseases;
- identification of tumor markers;
- determination of hormones.
In the case of infectious and viral diseases, the technique allows us to identify the following pathologies:
In addition, ELISA allows you to quickly and effectively determine the state of infarction, assess the reproductive potential of the body, identify allergies, their source, etc.
The immunoenzyme technique is used to conduct clinical studies during the development of new drugs and in assessing their effect on the human body.
Types of samples and methods of their selection for research
Most often, the test material for enzyme immunoassay is blood, which is taken from the patient’s antecubital vein. Sample collection is carried out on an empty stomach, mainly in the morning. After sampling, those formed cells that interfere with the research are separated and removed from the blood, leaving only serum.
When diagnosing genitourinary infections, the material often becomes smears from the mucous tissues of the genital organs, mucus from the urethra or cervix, samples from the rectum, scrapings from erosions or ulcers in the groin area and from other parts of the body. Swabs can be taken from the oral cavity, as well as from the nasopharynx.
Sometimes enzyme immunoassay is used in obstetric and gynecological practice during pregnancy to determine the state of amniotic fluid. In this case, the amniotic fluid becomes the sample. To do this, a small amount of fluid is taken by puncturing the amniotic sac with a long needle. All manipulations are carried out with sterile instruments to minimize possible risks.
Often the material becomes cerebrospinal or serous fluid. This occurs with local anesthesia, which is administered by injection.
The specialist sending for the study should clarify what type of materials are needed for enzyme immunoassay. Often samples of several types or from different places are taken at once. The doctor issuing a referral for this type of examination should also tell the patient about preparations for donating biomaterial.
Preparation for enzyme immunoassay
To increase the accuracy of the data obtained after enzyme immunoassay, preparation for the selection of material should be as follows:
- 10 days before the study, exclude antibiotics, antiviral and antimycotic drugs;
- for a day it is necessary to exclude alcohol, smoking and taking narcotic substances;
- during the same period, it is necessary to avoid physical overexertion;
- Warn the prescriber about all medications the patient is taking;
- Notify your doctor if you are pregnant or suspect you are pregnant.
It is best if testing materials are collected in the morning on an empty stomach.
If the diagnosis aims to determine the state of hormonal levels, then it is important to ensure that you are in a calm state the day before and avoid nervous tension. For women, donating blood for hormones is clearly determined by the period of the monthly cycle, which will be discussed by the doctor at the time of appointment.
2-3 days before sampling, you need to exclude fried and fatty foods from the menu, and before testing for hepatitis, do not eat citrus fruits or any other orange or yellow fruits and vegetables.
Interpretation of enzyme immunoassay results
The result of a qualitative study is usually indicated by the signs “+” (detected) or “-” (not found).
Based on the presence or absence of certain groups of immunoglobulins, the following conclusions can be drawn:
- JgM (-), JgG (-), JgA (-) - immunity to the disease is completely absent (the body has not previously encountered this type of antigen);
- JgM (-), JgG (+), JgA (-) - there was a previous collision with this antigen or the vaccine;
- JgM (+), JgG (-/+), JgA (-/+) - acute pathological process (most likely primary);
- JgM (-), JgG (+/-), JgA (+/-) - chronic process;
- JgM (+), JgG (+), JgA (+) - relapse;
- JgM (-) - stage of recovery.
Quantitative values carry a large information load, but only the attending physician can interpret them, based on previous indications, the patient’s age, and the norms for each specific disease. It is for this reason that you cannot evaluate the results yourself.
How long do you need to wait for results?
This technique has many varieties, depending on which the period for obtaining data in hand is determined. The average duration of ELISA diagnostics is 4-6 hours, which allows you to provide results the very next day.
The longest methods take up to 10 days, for example, in the case of HIV infection.
In case of urgent need, it is possible to use express methods, in which the answer is obtained within 1-2 hours.
Where can I get ELISA testing?
Since equipment for this type of diagnostics is quite expensive, not all laboratories can purchase it. In addition, tests containing specific antigens have a limited shelf life (usually about 1 year) and therefore need to be constantly updated.
For these reasons, public medical institutions do not always have ELISA laboratories. Most often it is necessary to contact large private medical or large diagnostic centers.
To conduct an ELISA test, the laboratory must have a special license, and staff and laboratory assistants must undergo special training.
Most often, a specific diagnostic center or laboratory is recommended by the doctor who refers the patient for examination.
Cost of enzyme immunoassay
The price for this study depends on the region of the country and on the level of the clinic providing the service. In Moscow, the minimum price for determining one antigen starts from 700 rubles. If it is necessary to identify several immunoglobulins at once, the price will be added up.
In case of urgent analysis, the cost increases by 150-200 rubles. for each antigen.
Despite the rather high cost, enzyme immunoassay makes it possible to make the examination of the patient as informative and fast as possible, which reduces the time before the start of treatment and allows for faster stabilization of the person’s condition.
This video presents the film “Basics of enzyme immunoassay”.
Thanks to the development of modern medicine, when making a diagnosis, a doctor no longer needs to focus on indirect manifestations of diseases or conduct multi-stage laboratory tests. It is enough to conduct one analysis that will confirm or refute the alleged initial diagnosis.
This method is enzyme-linked immunosorbent assay (ELISA) - this study allows you to detect specific antibodies and antigens characteristic of various pathologies, which greatly speeds up the diagnosis.
ELISA analysis is a laboratory test (method) that helps determine the presence or absence of certain antibodies in the body to fight the virus and their quantity.
The basis of the study is the natural reaction antigen (an object harmful to the body) - antibody (a protein that destroys harmful objects), which makes it possible to detect the presence of various viruses and bacteria.
ELISA is a natural immune response of the body - the interaction of an antibody with the corresponding antigen. So, during ELISA, antigens or antibodies are added one by one to a test tube with the material, after which the concentration of the resulting antigen-antibody complexes is determined.
If matches are formed, then immune complexes arise, then an enzymatic reaction of the dye with the combined molecule occurs. It is thanks to the color change during enzymatic indication that the disease is identified after examining the level of the determined compound.
Types of immunoglobulins
Human immunoglobulins are differentiated into several classes that differ from each other in the properties, structure and antigenic characteristics of the heavy chains (H chain). All mammals, including humans, have five H-chains, which determine the belonging of immunoglobulins to the corresponding class: G, M, A, D, E.
Each class differs from each other in biological properties, and the ability to bind antigens, and the speed and strength of the connection with the molecule.
The functions of each immunoglobulin (Ig) are different:
| Quantity in the body | Functions | Half-life (days) | Meaning | |||||||||||||||||||||||||||
| G | 70% | Form passive immunity in the newborn; necessary for immune response improve phagocytosis, | 21-24 | provide long-term humoral immunity against infectious diseases | ||||||||||||||||||||||||||
| M | 5-10% | needed to activate phagocytosis, capable of binding 5 antigen molecules, | 5 | Provides the primary immune response | ||||||||||||||||||||||||||
| A | 10-15 | Neutralizes toxins and viruses needed for the formation of early immunity The emergence of immunoglobulins occurs along a peculiar “chain” - lgM lgG, this is how the body reacts to the appearance of an antigen in the body. During laboratory diagnostics, the concentration of three main immunoglobulins is assessed - G, M, A. Indications for immunoglobulin testingELISA analysis is becoming more and more popular every year. Such a study speeds up the diagnosis, and this is very important for the treatment of pathologies such as:
ELISA is a unique marker of autoimmune pathologies and malignant neoplasms. Preparing for the testWhen preparing for the study, you should adhere to the following rules: Doctors also recommend adhering to a special diet - excluding fatty and fried foods, and if the test is carried out for hepatitis, then it is necessary not to eat any orange vegetables and especially citrus fruits. You should donate blood in the morning on an empty stomach. False-positive analysis occurs due to unfulfilled recommendations, especially the consumption of fatty foods, which leads to too high a concentration of triglycerides in the plasma, which causes the ELISA conductivity to decrease. Sample collection procedureWhole blood, serum or venous blood plasma can be used as the test material. The material is collected, usually from the ulnar vein, using a disposable needle and a vacuum tube; 5-10 ml of blood is required. To ensure the accuracy of the result, it is important to adhere to the correct sampling technique - the puncture of the vessel itself and the surrounding tissues must be performed in one manipulation, therefore a short needle with a large diameter is used, so that the opposite wall of the vein is not injured and red blood cells are not damaged.
During storage of the material, possible ionization should be avoided; in addition, the material should not come into contact with residual disinfectants, therefore only a disposable plastic tube is used, marked with the patient’s name, date and time of delivery of the material. If short-term storage of the test material is necessary, then a refrigeration chamber with a temperature of 2-4 o C is used, if longer storage is necessary, then the material is frozen at a temperature of -20 o C. How the analysis is doneAfter preparing the material under study, the laboratory assistant begins the necessary manipulations. For this purpose, a number of special sets of antigens are used, which have the ability to provoke the body’s response to an irritant, these are various infections, hormones, and allergens. The expected antigen-antibody reaction looks something like this:
At the moment, many different ELISA options have been developed, but there is no clear classification of them. Typically, methods are considered based on their division into hetero- and homogeneous - all phases of analysis occur using a solid phase or using only a solution. Modern clinical diagnostic laboratories usually use heterogeneous (solid-phase) ELISA, in which the solid phase means the absorption of antigens or antibodies on the solid surface of special wells located on a polystyrene microplate; the method is divided into direct and indirect ELISA. With direct ELISA, the introduced antigen is fixed during the incubation process on the surface of empty wells; for this, the test material is placed in clean wells for 20-25 minutes, this is necessary for the antigen to attach to their surface. After this, the required antibody is added. Next, the material remains for a certain time to form bonds. Antibodies are always added in excess, so even if they are present, unbound antigens remain in the sample, and if there are no antigens at all, then there will be no connections. To remove “extra” antibodies, decantation is performed, after which only those antibodies that have created a bond with the antigen remain.
In the indirect ELISA method, antibodies are used that are pre-combined with the substrate of the enzymatic reaction; in this case, the connection of antibodies with the antigen occurs during the incubation process, after which the mobilization of bonds on the surface of the wells occurs, and the conjugate and substrate-chromogenic reagent added afterwards color the reaction. Thus, the main difference between the indirect and direct methods is not the adhesion of the material to the surface of clean wells, but the binding to the antigen immobilized on the plate. The reaction is stopped using specialized devices, then each well is subjected to a photometric process, followed by a comparative description of the result obtained with previously conducted control samples. If an increase in optical density is detected in the sample, then the concentration of specific antibodies in the test result is also overestimated. When will the analysis be ready?The study does not take much time; from blood sampling to receiving the result, it takes from 1 to 10 days, depending on the diagnostic measures. Test results and their interpretationThe diagnostic result form received by the patient indicates a negative or positive result for certain classes of immunoglobulins, and a quantitative indicator of various classes of antibodies is also indicated. Various interpretations of the results are possible:
So, for example, if IgG and IgM were detected, then the patient may have one of the following diseases:
Enzyme immunoassay is often prescribed for hormonal studies; the norms are shown in the table:
ELISA analysis is a diagnostic test that allows you to assess the likelihood of the formation of oncological pathologies. However, interpretation of the results is carried out only by the attending physician. Meaning of test resultsELISA is suitable for detecting various forms of sexually transmitted infections, including syphilis, and is often used for screening pregnant women. Thanks to this study, you can find out how long ago the infection occurred and the stage of the disease at the time of the study:
During the study, the wells on the plate with a negative indicator remain colorless, and the positive ones turn bright yellow. If the color of the positive wells does not match the color of the control, then the result is considered questionable and a repeat test is necessary. Enzyme immunoassay is the first step in diagnosing HIV. The analysis cannot be carried out immediately after suspected infection; it is necessary to wait until the end of the incubation period (from 14 days to 6 months). During the analysis, antibodies to HIV-1 and HIV-2 are determined, class G antibodies are searched for, which usually appears at a later stage, and class A and M antibodies are detected in the early stages (during the incubation period).
ELISA is also used as a diagnostic test for tuberculosis, but even if the patient has antibodies to this pathology, this does not always confirm the presence of tuberculosis, so ELISA is often used as a clarifying technique, or to diagnose a latent extrapulmonary form. IgG - chronic stage of invasion |
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| IgA – infection occurred more than 30 days ago | ||||||||||||||||||||||||||||||
| IgG – invasion is in the acute phase | ||||||||||||||||||||||||||||||
| IgG – confirms the diagnosis and shows the effectiveness of therapy |
Pros and cons of analysis
ELISA has many advantages, which explains its popularity among doctors and patients, these are:
- high accuracy of results,
- affordable price,
- quick result,
- identifying the stage of the disease,
- disease control over time.
However, along with the advantages, there is also a disadvantage - in rare cases, the analysis can be false positive or false negative.
Why the result may be unreliable
Errors as a result can occur due to technical violations; the analysis can also be unreliable in people with certain chronic diseases (rheumatoid factor) which are characterized by the production of specific antibodies.
The final result is also influenced by the patient’s use of medications and metabolic disorders. For these reasons, a positive result for HIV and oncopathology requires repeat samples.
Cost of the study
The price of ELISA varies depending on the direction of diagnosis (RUB):
- hepatitis 250 -900;
- viruses – 250 -1000;
- HIV – 250-350;
- helminthic infestations – 280 – 900;
- syphilis -150-250;
- fungal infections 400-500.
Article format: Lozinsky Oleg
Video about ELISA analysis
How is ELISA analysis performed:
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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Doctors
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The hemostasis laboratory of the MLC uses a new, highly sensitive ELISA method - ELISA (Enzyme Linked Immunosorbent Assay), a heterogeneous solid-phase immunoassay. During the study, the presence of antibodies in the patient’s blood to pathogens of various diseases is determined.
Cost of ELISA analysis*
Why do you take enzyme immunoassay?
ELISA is used in many areas of medicine. In people with coronary heart disease, atherosclerosis and hypertension, antibodies are detected to the proteins of the hemostatic system - thrombin, a2 macroglobulin and antithrombin 3, as well as to the peptide bioregulators of the coagulation system - angeotensin 2 and bradykinin.
ELISA has proven its effectiveness in prenatal diagnosis and in determining fertility - this is the AFP test (diagnosis of spina bifida, trisomy in the fetus), tests for the steroid hormones estadiol, estriol, progesterone.
Multiple complications of pregnancy, infertility and thrombophlebitis are provoked by a severe autoimmune disease - antiphospholipid syndrome. Diagnosis of APS at the Medical Women's Center is also carried out using enzyme immunoassay: antibodies to cardiolipin, b2-glycoprotein, prothrombin and annexin 5 are determined.