Enzyme immunoassay or ELISA analysis: what is it and what diseases does it determine? Luis ifa what is Anti luis what.

ELISA or enzyme immunoassay refers to serological research and is intended for the detection and diagnosis of pathological microorganisms in the blood serum.

Through analysis, various immunoglobulin classes to bacteria: IgM- in acute pathological process, and IgG at the stage of recovery, which in some cases persist throughout life.

Through enzyme immunoassay, diseases of various etiologies are detected:

Also, ELISA is indicated for diagnosing syphilis when positive results are confirmed by the RV method, and allows you to monitor the effectiveness of treatment in a pathological process.

Enzyme immunoassay is prescribed according to indications (if infection with microorganisms is suspected), it is mandatory during pregnancy and before surgical operations to detect the carriage of various bacteria.

Positive ELISA

A positive ELISA result is confirmed by the presence of IgG and IgM immunoglobulins. Detected titers in the blood IgM always indicate disease in the progressive stage, in a healthy person, these antibodies are absent.

And IgG indicates a previous infection, or the carriage of pathogenic microorganisms, some of which are regarded in small quantities as normal. For example, the bacteria streptococci and staphylococci are present in every human body.

Syphilis

IgM antibodies in syphilis are detected from 2 weeks after infection and indicate the presence of a primary, secondary or congenital episode, with treatment they disappear after about six months, without therapy - after 18 months. If both types of immunoglobulins are detected at the same time, syphilis in the acute phase is confirmed. In sick people IgG antibodies to syphilis remain in serum for life.

Viral hepatitis

IgM to viral hepatitis are often detected in incubation period diseases, before the appearance of the first manifestations and persist during the course of the disease, after treatment - are not determined. The exception is viral hepatitis C, in which IgM is detected both at its active and latent or chronic stage

IgG antibodies for hepatitis A may be present even in healthy people, which is caused by an infection or a decrease in immunity, and the presence of IgG to hepatitis B, C and D in healthy people is not observed.

CMVI

CMVI is distributed almost everywhere and does not pose a threat to public health. However, it carries a deadly danger to the newborn and fetus during intrauterine infection.

Detection of IgM antibodies to cytomegalovirus indicates primary infection or activation of the latent phase. IgG titers persist in recovered people for 10 years.

Herpes

Antibodies to the herpes virus in healthy people are normally absent. The content of IgM indicates the acute phase of the disease, IgG indicates the latent (in this case, the person is the carrier of the infection). With the content of IgG to herpes, you should know that the virus can at any time be activated from a latent stage to a progressive one.

Chicken pox

At chicken pox and within 2 years after treatment, immunoglobulins of the IgM class remain in the blood. Normally, in healthy people, antibodies to chicken pox are not detected.

Diseases caused by staphylococci and streptococci

All people have immunoglobulins to staphylococci and streptococci. Therefore, it is possible to diagnose a pathological process caused by these groups of bacteria by conducting a double enzyme immunoassay. If there is an increase in titers with repeated ELISA (one week after the first), then the analysis is considered confirmed.

Chlamydia

A positive result for chlamydia is indicated by the detection IgM titers 1:8 and above and class IgG - 1:64 and above, which increase during the course of the disease and reach high values. For example, in children with chlamydial pneumonia, titers rise to 1:2000 - 1:4000. The presence of IgM indicates the activity of chlamydia, some time after infection, IgG globulins are detected in the blood.

A negative enzyme immunoassay is indicated by no IgM antibodies. Determined IgG is not in all cases regarded as confirmation of a diagnostic study, they often persist for several years after the infection, sometimes for life.

after syphilis, infectious mononucleosis IgG immunoglobulins persist throughout life and are determined in blood serum. For 10 years, the microorganisms of CMVI, measles, rubella, toxoplasmosis remain.

Titles to amoebiasis persist from several months to several years. Antibodies to the bacteria of staphylococcus and streptococcus are determined in absolutely all people in a small amount.

In the above cases, the detection of IgG immunoglobulins after past illnesses allows us to regard the results of ELISA as negative.

Norm during pregnancy

During the first trimester of pregnancy, every woman goes through full examination organism, which includes enzyme immunoassay.

Studies on toxoplasmosis, CMVI, chlamydia, type 2 herpes (genital), rubella, ureaplasma and mycoplasma are mandatory, as these diseases pose a serious threat to the development of the fetus. They are able to penetrate the child's body, bypassing the placental barrier.

And they pose a particular danger during first trimester of pregnancy and almost always lead to intrauterine fetal death and spontaneous miscarriage.

The problem with the above diseases is that they often occur in an asymptomatic stage, without visible symptoms. clinical manifestations, and it is possible to reveal them only by enzyme immunoassay.

Title detection IgG for rubella indicates a previous disease and is the norm during pregnancy. IgG to CMVI also do not pose a particular threat to the fetus, however, the possibility of exacerbation is not excluded (frequency of manifestation, approximately 1-2%).

A particular threat is the presence of IgG to the herpes virus type 2 or genital (HSV2), since the risk of exacerbation increases significantly during childbirth. In the gestational period, the incidence of the acute phase occurs in 0.9% of cases. The defeat of the fetus by the herpes virus during the passage of the genital tract occurs in 40% of cases and leads to death in 50%.

With past toxoplasmosis, IgM can persist up to 2 years after treatment. The risk of infection of the fetus in this case is 17% in the first trimester and increases to 60% by the third, since the main route of infection is transplacental. Revealed IgG to toxoplasmosis many experts tend to evaluate it as a negative result, which practically does not pose a threat to the course of pregnancy.

It is very important to choose exactly those tests, methods or diagnostic methods that will really help to make an accurate diagnosis, and not confuse the doctor even more. An enzyme-linked immunosorbent assay has long established itself as a method that allows not only to get a positive or negative answer to the question - is a person sick with any sexual infection, but also to determine at what stage of development the disease is, and also to understand whether the patient had a similar problem in anamnesis. ELISA for syphilis today is one of the most sensitive and specific tests, while its accuracy is about 90%.

Test systems that use the enzyme immunoassay method are designed to detect not only syphilis, but also many other infections: hepatitis, HIV, CMV, herpes, toxoplasmosis, etc. An ELISA test for syphilis allows you to determine antibodies (IgG, IgA, IgM) to pale treponema contained in the blood of a sick person. This method allows you to see the interaction of antibodies with special preparations that contain antigens that, in combination with antibodies, can form a sign of the disease, which is clearly visible in various diagnostic methods.

The enzyme-linked immunosorbent assay for syphilis is based on the method of interaction of antibodies detected in the blood with the antigen of the infectious agent. Diagnosis will depend on which antibodies were used: either antibodies of a certain class or in general can be detected. To diagnose this method, blood is taken on an empty stomach from a vein.

ELISA blood test for syphilis involves the use of three classes of immunoglobulins: G, M, A. These immunoglobulins have one important property for diagnosing syphilis by ELISA: they are produced in a strictly defined sequence, due to which ELISA allows you to determine at what stage the process is and what dynamics of its development:

• Immunoglobulin M in the blood shows how much time has passed since infection. However, the positive results of its presence do not always mean the presence of a sexually transmitted disease: it is possible that a person has experienced an exacerbation of some chronic disease that caused such a reaction.
• The presence of IgA antibodies means that at least a month has passed since the infection.
• Since immunity to this disease is not developed, a positive IgG test result can show both the peak of the disease and the period after treatment.

What does ELISA for syphilis positive mean?

It should be noted that an ELISA positive for syphilis is not a cause for panic. The fact is that this method allows you to determine only the presence of antibodies, but not the pathogen. Therefore, if the ELISA result for syphilis is positive, this is not yet a basis for making a diagnosis. However, in the scientific world, test systems are being improved daily, and today this means that in most cases, the results of analyzes can still be trusted.

There are only two diagnostic results: “positive” and “negative”. In the first case, positive data means that the patient has a disease in one of the stages, or that he has recently completed a course of treatment. A negative result of an enzyme-linked immunosorbent assay means either the absence of the disease, or a very early stage of its development.

The clinical picture of the first stage of syphilis is represented by the detection of a hard chancre in various areas.

The first symptoms of syphilis appear 3-5 weeks after infection. Up to this point, the disease proceeds without any external manifestations.

Very often, after unprotected intercourse, a person who suspects syphilis, HIV or hepatitis.

Reviews and comments

The husband was tested for syphilis, ELISA for syphilis - positive, RMP for syphilis - negative. And what does that mean?

During pregnancy (the period was 37 weeks), the ELISA score was positive 2.1. After giving birth, I took tests 2 times - everything was negative. After 6 months, she passed again, ELISA - positive, CP=1.9. (IgM+IgG). What does it mean.

Hello! What does it mean: a blood test for syphilis ELISA (total at.) is positive?

Please tell me what rmp1+ means, rpga 1+ ifa in total is negative! Thanks in advance.

ELISA is false positive!

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ELISA blood test

For a comprehensive assessment of the state of the body (in particular, its protective functions), an enzyme-linked immunosorbent assay (ELISA) is prescribed. ELISA blood test is performed to diagnose infectious, autoimmune, hematological pathologies, primary and secondary immunodeficiencies. In this article, we propose to consider in more detail what an ELISA blood test is, as well as what indications exist for its implementation.

Indications for the appointment of a blood test by ELISA and the principle of its action

As we have already noted, an ELISA blood test is a laboratory test by which antigens or antibodies are determined in a blood sample. This analysis is used to detect the level of hormones, immunological complexes and immunoglobulins. There are the following indications for the delivery of an ELISA analysis:

• Allergy diagnosis.
• Diagnosis of diseases of viral origin - Epstein-Barr virus, herpes, hepatitis, cytomegalovirus.
• Diagnosis of sexually transmitted infections - mycoplasma, ureaplasma, syphilis, trichomonas, chlamydia.
• Definition of immunodeficiency.
• Diagnostics of oncological pathologies.
• Evaluation of the effectiveness of therapy.
• Determination of hormone levels.
• Preoperative comprehensive examination.

The principle of action of an enzyme immunoassay is based on a blood test for the presence of immunoglobulins (specific protein antibodies). Immunoglobulins are produced by the immune system when antigens (foreign microorganisms) enter the human body. These immune molecules bind to various infectious agents and neutralize them. An important distinguishing feature that immunoglobulins possess is their specificity. Due to these characteristics, they can form an antigen-antibody complex by binding to a specific antigen. During the ELISA blood test, it is this complex that is determined quantitatively and qualitatively.

For this study often used human blood. However, as a material for analysis, you can take the contents of the vitreous body, amniotic fluid, cerebrospinal fluid. The blood sample is usually taken from the patient's cubital vein. It is recommended to donate blood on an empty stomach (at least 12 hours must pass since the last meal). If the patient takes medicines must be reported to the doctor, as some of them may affect the result of the analysis. Also, the reliability of the test results is affected by the use of drugs and alcohol.

Deciphering a blood test for ELISA

The form of this analysis usually indicates a negative (-) or positive (+) result of the calculation of each class of immunoglobulins.

We propose to consider the interpretation of the probable decoding of a blood test for ELISA.

• IgG, IgA are not detected and the result of IgM is negative - complete recovery.
• The result of IgM, IgA, IgG is negative - there is no immunity to infection.
• IgG result, IgA positive and negative, as well as the result IgM positive- the presence of acute infection.
• Positive IgG result and negative IgA and IgM result - post-vaccination or post-infection immunity.
• A positive or negative IgG, IgA, and negative IgM result is a chronic infection.
• The result of IgG, IgM, IgA is positive - exacerbation of chronic infectious pathology.

In the enzyme immunoassay, in addition to clarifying the classes of antibodies, their quantitative indicators are indicated in the transcript. However, only the attending physician provides an extensive explanation of them.

Advantages and disadvantages of this study

• Relatively low cost.
• Ease of use.
• Possibility of diagnostics early stage suspected disease.
• Relatively high accuracy of the obtained data.
• A short period of time required to obtain the result of the study.
• The ability to track the dynamics of the development of the infectious process in the body.
• High level of unification, which makes it possible to conduct mass surveys.
• Automation of all stages of research.

The disadvantage of an ELISA blood test is that it can, in rare cases, give false positive or false negative results. Also, during the study, in addition to technical errors, the cause of false results in a patient can be a rheumatoid factor, the presence of chronic diseases(in which antibodies are produced), certain medications medical preparations, metabolic disorders.

• Giardiasis.
• Ascariasis.
• Cysticercosis.
• Amoebiasis.
• Trichinosis - the study is carried out more than once, at 4-12 weeks after infection, the maximum level of antibodies is determined.
• Teniasis.
• Opisthorchiasis - perform differential diagnosis between acute and chronic forms of the disease.
• Toxoplasmosis.
• Fascioliasis - in the acute stage of the disease, the presence of antibodies is determined.
• Cutaneous or visceral leishmaniasis.

When using materials from the site, the active reference is obligatory.

The information provided on our website should not be used for self-diagnosis and treatment and cannot be a substitute for consulting a doctor. We warn about the presence of contraindications. Specialist consultation is required.

What blood tests are given for syphilis: RW, RPGA, ELISA, VDRL, RPR, RIBT, decoding of test results

Syphilis is an infectious disease caused by the spirochete Treponema pallidum, prone to progressive chronic course, with a clear periodization of clinical symptoms.

The predominance of sexual transmission over contact and transplacental puts this disease in a number of sexually transmitted diseases (STDs, STIs). In addition to these methods of infection transmission, the artificial route (from the Latin "artificio" - artificially created) plays a special role.

It is typical for medical institutions, mainly implemented in a hospital setting. Infection occurs during blood transfusions, various surgical operations, invasive diagnostic methods.

Despite the quarantine of donated blood, the problem of detecting syphilis in donors is different stages disease is still present.

Therefore, diagnostic measures for syphilis require standardization, the introduction of new sensitive and informative methods of identification, as well as minimizing errors and misinterpretation of test results.

Classification of laboratory diagnostic methods

Diagnosis of syphilis has some features and differs from the diagnosis of other bacterial infections. The complex structure and antigenic properties of pale treponema cause errors in the interpretation of the results serological reactions.

A blood test for syphilis is offered to 3 main groups of patients:

1. 1 Screening and prophylactic medical examination of population groups (including pregnancy, registration at the antenatal clinic, employment and registration of a medical book, and so on).
2. 2 Screening in risk groups (unprotected sex with a person infected with syphilis, persons after forced sexual contact, HIV-infected, and so on).
3. 3 Persons with symptoms or suspected syphilitic infection.

All laboratory methods conditionally divided into direct and indirect.

Direct Methods

1. 1 Identification of Treponema pallidum in the dark field (dark field microscopy).
2. 2 Infection of experimental animals (culturing in laboratory animals).
3. 3 PCR (polymerase chain reaction).
4. 4 DNA probe or nucleic acid hybridization.

Indirect Methods

Serological reactions are methods laboratory diagnostics based on the detection of antibodies (abbreviated as AT) to antigens of pale treponema (abbreviated as AG). They are essential for confirming the diagnosis.

1. 1 Non-treponemal tests:
   • Wasserman reaction (RSK);
   • Microprecipitation reaction (MR, RMP) and its analogues, which are given below;
   • Rapid plasma reagin test (RPR, RPR);
   • Test with red toluidine and serum (TRUST);
   • Non-treponemal test of the Laboratory for the Study of Sexually Transmitted Diseases - VDRL.
2. 2 Treponemal tests:
   • R-tion of immobilization of Treponema pallidum - RIBT / RIT;
   • R-tion of immunofluorescence - RIF, FTA (dilution of sera RIF-10, RIF-200, RIF-abs);
   • R-tion of passive hemagglutination (RPHA, TRPGA, TPHA);
   • ELISA (ELISA, EIA);
   • Immunoblotting.

Figure 1 - Syphilis serodiagnosis algorithm

Histomorphological methods

These methods are reduced to identifying the features of the histomorphology of syphilitic manifestations. Attention is paid to the subtleties of the structure of the hard chancre. However, differential diagnosis infection using histology is very difficult. Histomorphology is used with other laboratory and clinical tests.

Dark field microscopy of Treponema pallidum

This method is based on the direct detection of pale treponema in the material under study using a microscope and special devices (most often discharge of erosions and ulcers, less often cerebrospinal fluid and other substrates).

With the help of scarification, scraping, squeezing from erosions and ulcerative defects, exudate is obtained, then the prepared preparation is examined under a microscope.

Usually, pale treponemas are detected in the preparation obtained from the chancre, from the foci of secondary fresh, secondary recurrent syphilis, as well as punctate of the lymph nodes, placenta.

Based on the phenomenon of the glow of small particles in a dark field when hit by a beam of light (Tyndall phenomenon), the method perfectly allows you to differentiate the causative agent of syphilis from other treponemas based on morphological differences and differences in the ways the bacterium moves.

For microscopy, a special dark-field condenser of the appropriate optical resolution is used. The drug is obtained by the crushed drop method (a drop of material is applied to a clean, fat-free glass slide and covered with a very thin coverslip).

Immersion oil is dropped onto the coverslip. By turning the tube and turning the magnifying lens, the desired illumination is adjusted.

In the dark field of the microscope, blood cells are detected, epithelial cells and the causative agent of syphilis. Pale treponema looks like a spiral, very thin, radiating a silvery color, with smooth movements.

Figure 2 - Dark field microscopy as a way to visualize pale treponema in the material under study. Image source - CDC

Treponema pallidum must be distinguished from other treponemas, including Tr. refringens, which can be found in the oropharynx and genital mucosa. This bacterium makes chaotic movements, has wide and asymmetrical, rather coarse curls. In addition, Treponema pallidum is distinguished from Tr. Microdentium, Tr. Buccalis and Tr. vincenti.

Visualization of bacteria in a dark field is sometimes supplemented by a fluorescence reaction. For this, antitreponemal antibodies labeled with a fluorescent dye are added to the native material. This forms a complex called antigen-antibody (abbreviated as AG-AT), which is the object of study using a fluorescent microscope.

Polymerase chain reaction (PCR) method

PCR, developed in 1991 to detect the deoxyribonucleic acid (DNA) molecule of treponema pallidum, is highly sensitive and specific, allowing the detection of DNA fragments of the pathogen.

This analysis is based on copying short sections of spirochete pallidum DNA, which satisfies the specified parameters and is present in the sample. All this is done under artificial conditions (in vitro). The reaction is carried out in a device - an amplifier, which provides periodization of temperature cycles. Cooling occurs, followed by heating of the test tubes with an error of 0.1˚С.

The DNA template is heated for 2 minutes at a temperature of 92-98˚С ( Maximum temperature used if the polymerase is thermostable). When heated, the DNA strands diverge due to the breakdown of hydrogen bonds between them. In the annealing step, the reaction temperature is lowered to bind the primer to the single stranded template.

Annealing takes about 30 seconds, during which hundreds of nucleotides are synthesized. The newly synthesized molecules are copied by the polymerase, as a result, specific fragments of deoxyribonucleic acid increase many times over. The subsequent detection of fragments is carried out using agar gel electrophoresis.

PCR diagnostics of syphilis is still experimental in nature, but it is justified when a congenital infection is detected, in difficult diagnostic cases, or with a minimal content of pale treponema in the test material.

DNA hybridization

DNA hybridization is performed in vitro and is based on the complete or partial connection of two single-stranded DNA molecules into one molecule. In the case of a complete match of complementary fragments, the union is easy. If the complementary match is partial, then the association of DNA chains occurs slowly. Based on the time of chain fusion, the degree of complementarity can be estimated.

When DNA is heated in a buffer solution, hydrogen bonds are broken by nitrogenous bases that are complementary, as a result, DNA chains diverge. Next, a preparation is obtained from two denatured deoxyribonucleic acids. Upon cooling, the single-stranded regions renature. A so-called DNA hybrid is formed.

The method makes it possible to estimate and analyze the annealing rate, taking into account the features (similarities and differences) of DNA between species or within a species.

The use of a DNA probe consists in hybridizing a labeled DNA fragment with a specific DNA region to identify complementary nucleotide sequences. A group of unsaturated atoms (chromophores) or radioactive isotopes is used to label the probe.

The DNA probe is used for heterogeneous and homogeneous detection of nucleic acids. The role of the probe is to determine the areas where the target-probe fusion has occurred. Detection in a homogeneous system has the advantage of being able to track the hybridization of DNA molecules in real time.

The essence of the method is reduced to DNA denaturation and renaturation (reunification of DNA chains). The process of renaturation of nucleic acid and DNA probe ends with the formation of a "hybrid".

Specific nucleic acid sequences hybridize with a DNA probe and thus are detected and allow the amount of DNA in the test material to be estimated.

Infection of laboratory animals

The high sensitivity of rabbits to Treponema pallidum (about 99.9%) allows them to be used in the diagnosis of syphilitic infection.

Infection of rabbits is carried out in research centers and is the "gold standard" for assessing the sensitivity of other methods.

Let's return to treponemal and non-treponemal tests, as they are used most often. Consider their advantages and disadvantages, as well as errors in the interpretation of the results.

Non-treponemal tests

These are tests to determine IgG antibodies and IgM to the standardized cardiolipin antigen. Them significant disadvantage is relatively low specificity.

Low cost and ease of implementation make it possible to classify these tests as screening diagnostic tests necessary for establishing a preliminary diagnosis and screening among the population.

It is non-treponemal tests that are given when registering a medical book, applying for a job, registering in a antenatal clinic.

1. 1 Minimum sensitivity in the stage of primary syphilis - 70%;
2. 2 Minimum sensitivity in the stage of late syphilis - 30%;
3. 3 Possibility of false-negative and false-positive results;
4. 4 The complexity of the implementation of the RSC.

1. 1 Relatively low cost of test production;
2. 2 Get a quick response;
3. 3 Possibility of their application for screening.

Obtaining false-positive or weakly positive samples is possible in the following cases:

1. 1 Violation of the execution technology, when blocking the AG-AT complex.
2. 2 The patient has autoimmune diseases (rheumatoid arthritis, rheumatism, scleroderma, systemic lupus erythematosus, sarcoidosis, etc.).
3. 3 Malignant neoplasms.
4. 4 Viral and bacterial infections.
5. 5 Endocrine diseases ( autoimmune thyroiditis, diabetes).
6. 6 Pregnancy.
7. 7 Drinking alcohol.
8. 8 Eating fatty foods.
9. 9 old age.

As you can see from the list, there are enough reasons for an incorrect result. Therefore, it should be treated with great caution. Let us consider two more samples along with RSC. This is a microprecipitation reaction and VDLR (its modification).

Complement fixation reaction (RSK, Wasserman, RW)

This is a test based on the ability of complement to bind to AG-AT complexes. The resulting complex is identified using a hemolytic system. Cardiolipin antigen significantly increases the sensitivity of the sample.

Sensitive is the Colmer reaction, which consists in performing at different temperature conditions. Thus, the first phase of the Colmer reaction proceeds at a temperature of 20˚С for half an hour, the second phase at a temperature of 4-8˚С for 20 hours. During this time, complement binding occurs.

When performing RSK, it is possible to obtain sharply positive results. The reason is probably a large antibody titer in undiluted serum. In this case, the samples are placed with decreasing doses.

To differentiate the stages of syphilis and evaluate the effectiveness of antisyphilitic treatment, the amount of antibodies in serum is determined.

The positivity of the sample is assessed using crosses; also, in the Wasserman, Colmer and Cann reactions, serum dilution is indicated.

Microprecipitation reaction

Since the complexity of performing the above tests is high, an accelerated method for syphilis serodiagnosis, the so-called express method, the microprecipitation reaction (abbreviated MR, RMP), has been developed for the breadth of coverage of medical examinations of different population groups.

It is performed with cardiolipin antigen and excipients. Its advantage is the collection of peripheral blood for research. This significantly speeds up both the technique itself and the work of laboratory assistants.

Figure 2 - Microprecipitation reaction (scheme)

MR requires plasma or inactivated serum of the patient (they contain antibodies). Next, the plasma is placed in the marked wells. Then, a drop of cardiolipin antigen is added to the test material, mixed and shaken. As a result, characteristic flakes appear in the serum of the infected person, varying in intensity.

This is a quality test. Quantification uses 10 dilutions of serum placed in 10 appropriately labeled wells. With qualitative MR, the response is indicated in the form of crosses (pluses) or minus, with quantitative MR, the antibody titer is indicated (1:2, 1:4, and so on).

The presence of flakes is regarded as a positive or weakly positive response. The appearance of a flocculate is also possible in the absence of a disease, therefore, the final assessment of the result obtained is carried out after a control study or other reactions (RIBT, RIF, ELISA, RPHA).

The method recommended by the World Health Organization for setting up a reaction with an antigen of a lipoid nature (AH) is rightfully considered the best among other standard non-treponemal tests. Developed in the USA, Georgia in the laboratory of venereal diseases (Veneral Diseases Research Laboratories).

The abbreviation of the institution served as the name for the sample - VDRL. VDRL is a modification of MP. Serum from a patient with syphilis is inactivated and placed on a glass slide. The antigen used consists of cardiolipin, cholesterol and lecithin in different percentages. The answer is registered almost immediately.

Distinct flocculation occurs in the presence of antibodies in the serum. Serum becomes reactive 4 weeks after infection. To assess the amount of antibodies, the serum is pre-diluted exponentially.

1. 1 relatively high sensitivity;
2. 2 relatively high specificity;
3. 3 ease of implementation;
4. 4 low cost of reagents;
5. 5 get a quick response.

The disadvantage of VDRL is the relatively high false positive rate.

Their causes are all the same diseases listed above.

Treponemal tests are performed with specific Treponema pallidum antigens. They are necessary and mandatory for establishing the final diagnosis. This is an immunofluorescence reaction (RIF), indirect hemagglutination reaction (IPHA), enzyme immunoassay (ELISA), etc.

After positive result non-treponemal test (RPR, MP, VDRL), treponemal tests should always be performed (more often a combination - RPHA, ELISA, RIF).

Treponemal tests are more difficult to perform than rapid tests and cost more money.

This reaction (abbreviated as RIF) is used to diagnose syphilis, including latent forms, and to recheck positive and false positive samples.

RIF is based on the glow of labeled antibodies when combined with an antigen-antibody complex under a quartz lamp. The method began to be used in the 60s and was distinguished by its ease of implementation and high specificity (which is slightly inferior to RIBT).

It has several modifications: RIF-10, RIF-200 and RIF-abs.

The most sensitive RIF in dilution is 10 times, and the rest are more specific. RIF is carried out in two phases. The patient's blood serum is added to the AG. An AG-AT complex is formed, which is investigated in the next phase. The fluorochrome-labeled complex is then identified by microscopy. If no glow is observed, this indicates the absence of specific antibodies in the blood serum.

RIF-200 is the most valuable of all dilutions. The method is intended for diagnostics various forms syphilis, especially latent syphilis, and cross-checking of positive samples.

Treponema pallidum immobilization reaction (abbreviated RIBT, RIT) is one of the complex serological tests that require significant effort and financial costs. RIBT is used less and less, but its relevance remains in the diagnosis of latent syphilis.

It is of great importance in the recognition of false positive results in pregnant women and is based on the immobilization of bacteria in the presence of immobilisins - late antibodies.

The result is evaluated on the percentage (%) of immobilized treponema using a special table:

1. 1 From 0 to 20 - negative test.
2. 2 From 21 to 50 - weakly positive test.
3. 3 From 50 prepositive reaction.

False positive results are also possible with RIBT. Thus, an incorrect answer is possible with infection with tropical trepanematosis, as well as with tuberculosis, cirrhosis of the liver, sarcoidosis and senile patients.

This blood test for syphilis is called a passive hemagglutination test (abbreviated as blood for TPHA, TPHA).

Antigen for TPHA is prepared from ram erythrocytes coated with fragments of pale treponemas (obtained from infected rabbits (see Figure 4)). For analysis, venous blood (plasma or inactivated serum) of the patient is used.

When the antigen is added to the serum of a patient with syphilis, an AG-AT complex is formed, which leads to agglutination of red blood cells. agglutination is determined subjectively by a laboratory assistant.

Figure 3 - Scheme of RPHA (passive hemagglutination reaction)

The sample is evaluated as positive when agglutinates of a uniform pink color appear. The color of the precipitate in red indicates the deposition of erythrocytes. RPHA is highly sensitive and highly specific.

Microhemagglutination reaction

It is a simplified version of the RPGA. Differs from the sample described above in having less antigen, diluent, and blood serum to carry out the reaction. The sample can be evaluated 4 hours after serum incubation. It is used in screening and mass examinations for syphilis.

Linked immunosorbent assay

Enzyme-linked immunosorbent assay (abbreviated as ELISA) is based on a specific antigen-antibody reaction. Biological material (patient's blood serum, cerebrospinal fluid) is introduced into the wells, on the solid surface of which antigens of pale treponema are fixed. The test material is incubated, then the antibodies that have not bound to the antigens are washed off (see Figure 5).

Identification of the resulting complex is carried out at the stage of fermentation using immune serum labeled with the enzyme. During a chemical reaction, the enzyme colors the resulting complexes. The intensity of staining depends on the amount of specific antibodies in the patient's blood and is recorded by a spectrophotometer.

Figure 4 - Scheme of ELISA (enzymatic immunoassay)

ELISA sensitivity is more than 95%. The method is used in an automated mode to study decreed population groups: donors, pregnant women and others, to clarify the diagnosis in case of positive and false positive non-treponemal tests.

Immunoblotting

Immunoblotting is a highly sensitive method, a modification of a simple ELISA. The reaction is based on electrophoresis with the separation of pale treponema antigens.

Separated immunodeterminants are transferred to nitrocellulose paper and shown in ELISA. Serum is then incubated and unbound antibodies are washed off. The resulting material is treated with enzyme-labeled immunoglobulins (IgM or IgG).

Clinical evaluation of the results of laboratory diagnosis of syphilis

In table 1 below, we have given the possible results of the analyzes and their interpretation. As you can see in the table, the main value in deciphering is the comprehensive assessment of tests.

Table 1 - Deciphering the results of serological reactions (blood tests for syphilis). Click on the table to view

The reactivity of tests is also evaluated by "crosses":

1. 1 The maximum response (strongly positive test) is indicated by 4 crosses.
2. 2 A positive sample is indicated by 3 crosses.
3. 3 A weakly positive reaction is indicated by two crosses.
4. 4 One cross indicates a dubious and negative result.
5. 5 A negative answer is marked with a minus sign.

The problem of optimizing the laboratory diagnosis of syphilis has not lost its relevance to date. Modern methods diagnostics, despite the desire of scientists to bring diagnostics to the highest possible sensitivity and specificity, require a control check and an individual approach.

A feature of syphilitic infection is the phenomenon of seroresistance, which has not received a scientific explanation. The diagnosis is made after a full examination of the patient by epidemiological, clinical, laboratory methods.

Against the background of the economic and technical development of medicine, there is also progress in the development of new criteria for diagnosing syphilis. All this will allow you to quickly, successfully and accurately treat patients.

1. 1 FEDERAL CLINICAL RECOMMENDATIONS FOR THE MANAGEMENT OF PATIENTS WITH SYPHILIS. RUSSIAN SOCIETY OF DERMATOVENEROLOGISTS AND COSMETOLOGISTS. Moscow 2013;
2. 2 MINISTRY OF HEALTH OF THE RUSSIAN FEDERATION ORDER of March 26, 2001 N 87 “ON IMPROVEMENT OF SYPHILIS SEROLOGICAL DIAGNOSIS”;
3. 3 Syphilis. Pranatharthi Haran Chandrasekar, MBBS, MD Professor, Chief of Infectious Disease, Department of Internal Medicine, Wayne State University School of Medicine. Medsacape.com;
4. Sexually Transmitted Diseases Treatment Guidelines (CDC).

(link to the 3rd article in this file), allowing to assess the body's ability to resist infectious diseases and showing the phase of the disease, an important place is occupied by enzyme immunoassay (ELISA). Conducting this study allows you to comprehensively assess the activity of the protective function of the blood and identify the state of immunodeficiency in infectious pathologies, as well as blood diseases, autoimmune processes, and hormonal problems.

How is it possible to cover so many targets in one analysis, and what are the indications for its implementation? Let's try to figure it out.

What is an ELISA blood test?

This is a laboratory test that allows you to determine the presence of specific antibodies (protective blood factors of a protein nature) to certain antigens (pathogenic agents). Among the antibodies of paramount importance are immunoglobulins, which can exist in the form of immunocomplexes.

Immunoglobulins are produced as a result of complex human neurohumoral reactions that occur as a response to the introduction of foreign antigens. Each type of pathogen produces its own specific antibodies. They act by “binding” an antigen or a pathological microorganism, forming an “antigen-antibody” complex compound, followed by neutralization, enzymatic lysis, phagocytosis reactions and excretion from the body.

Note:it is by the presence of certain complexes that the ELISA method determines the type of pathogen or harmful substance present in the patient.

You can learn the basic principles of the functioning of human immunity by watching this video review:

What are immunoglobulins

5 main classes of immunoglobulins have been discovered and studied - IgA, IgM, IgG, IgD, IgE. the role of the rest has not yet been fully elucidated and they are at the stage of scientific research.

Note:most important in practical medicine immunoglobulins of classes - A, M and G are important. The information content of the definition is based on different time intervals of their appearance, maximum and disappearance.

Let's consider this question in more detail.

The main task immunoglobulin A (IgA) is the protective function of the mucous membranes respiratory tract, gastrointestinal tract and urinary system. With an acute onset of the disease, it is impossible to identify them. These protective complexes appear only from the 2nd week of the onset of the disease, sometimes later. The bulk of immunoglobulin A is concentrated in the mucous tissues. Roughly about 80%. The remaining antibodies circulate in the blood. The main function is the neutralization and destruction of microorganisms. After the subsidence acute manifestations disease, the number of these immunoglobulins begins to decrease and completely disappears for up to 8 weeks after the onset of the disease. If IgA are found in more than late dates, then this indicates a chronization of the process.

The main and first markers of the acute phase developing pathology are class M immunoglobulins (IgM). They are found by the 5th day of the onset of malaise. You can determine their presence in the blood for about 6 weeks. Then they start to disappear quickly.

The residual immune response characterizes the presence in the blood class G immunoglobulins (IgG). The appearance of these factors in the blood is detected approximately one month after the onset of the disease. In the future, they can be determined for many months, years, and even a lifetime, performing a protective function against the return (relapse) of the disease, and in some cases making it impossible for the secondary development of pathology. If the amount of immunoglobulin G began to rise again, then re-infection can be suspected. A similar conclusion can be drawn by conducting two or three samples taken with an interval of 2 weeks.

Immunoglobulin D (IgD) is located on B-lymphocytes, is in a small concentration in healthy people. After 10 years of life reaches its maximum values. The amount of immunoglobulin D increases during pregnancy, in patients with systemic connective tissue diseases, diseases caused by an immunodeficiency state.

Indications for the appointment of enzyme immunoassay

Determination of antibodies to the presence in the body of pathogenic microbes that cause:

• trichomoniasis;
• ureaplasmosis and.

There is an increase in the number of immunoglobulins and with.

Diagnostics is carried out to detect:

• herpetic diseases;
• groups of viral hepatitis;
• Epstein-Barr virus;
• cytomegalovirus.

With the help of ELISA, it is possible to determine the presence of antibodies to 600 types of allergens, detect the state of immunodeficiency, conduct a comprehensive examination before transplant operations, and conduct a comprehensive analysis for the effectiveness of treatment.

ELISA is additional method detection of cancer cells.

How is a blood ELISA performed?

For enzyme-linked immunosorbent assay, in most cases, the blood of patients is used, sometimes vitreous tissue, spinal canal fluid, and amniotic fluid are taken.

Blood is drawn through an injection needle into a syringe from the cubital vein. The study is carried out on an empty stomach. It should be remembered that taking certain medications can affect the result of the analysis. Before donating blood, you must refrain from smoking, drinking alcohol. The use of narcotic substances can distort the results.

In the case of negative values ​​of immunoglobulins IgM, IgG, IgA, we can talk about the absence of the disease or its initial phase, and the result with minuses is also possible with complete recovery after a significant amount of time.

If IgA and IgM are not detected, and IgG is positive, then in all likelihood we are talking about the formed immunity after infectious disease or after vaccination.

In the case of a high titer of IgM with negative values ​​of IgG, IgA, it can be concluded that there is an acute infectious disease.

Simultaneous positive values ​​of the results of immunoglobulins - IgA, IgM, IgG are characteristic of the acute phase of the recurrence of an existing chronic disease.

For chronic infection ELISA, which is in the remission phase of the process, shows negative values ​​of immunoglobulin M (IgM), while the result of immunoglobulins G (IgG) and A (IgA) will be positive.

Benefits of the enzyme immunoassay method

The main advantages of the ELISA method are:

• low cost of analysis;
• diagnostic specificity, accuracy;
• dynamic control (repeat analysis to determine the effectiveness of treatment and stages of the disease);
• the possibility of conducting mass examinations in the foci of infection;
• the speed of obtaining the result;
• relative simplicity of analysis;
• the possibility of using information technology in processing;
• safety and painlessness for the patient.

Are there any disadvantages of blood ELISA?

The main negative point of the study is the possibility of obtaining false negative and false positive data. The cause of misunderstandings can be technical flaws, taking medications, which can distort the picture.

ELISA is used to detect:

• (ascaris, pinworms);
• sharp and chronic form opisthorchiasis;
• trichinosis;
• the presence of lamblia (as an additional analysis);
• forms of leishmaniasis;
• amoebiasis;
• the content of toxoplasma;

In conclusion, it should be noted that modern immunology is constantly in the development stage, searching for new methods for diagnosing and treating diseases.

Stepanenko Vladimir, surgeon

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