ELISA is a reliable method for diagnosing HIV. How and where to take the ELISA test. ELISA diagnostics: what is the essence, determination of antibodies, how is it carried out and for what diseases is it effective? The most exciting of the enzyme immunoassays is the HIV ELISA

HIV is the scourge of our generation. What methods exist for diagnosing HIV, in-depth information about the ELISA test for HIV. How to take it, how to interpret the results of the analysis.

The human immunodeficiency virus is one of the most terrible infections for our generation.

HIV has a destructive effect on our immune system, and if the progress of the disease is not stopped in time, it can transform into acquired immunodeficiency syndrome. HIV spreads through direct contact between partners during sexual intercourse through the male and female genitals. Another cause of infection may be the virus entering the bloodstream. This occurs due to the use of unsterile instruments, transfusion of contaminated blood, and even across generations: to a child from an infected mother during breastfeeding.

Until now, scientists around the world are racking their brains to create a new generation of medicine that can cure AIDS, however, despite the rapid pace of scientific progress, the human immunodeficiency virus continues to spread across the planet.

ELISA – a method for diagnosing HIV

Scientists of the 20th century generation have developed several methods for diagnosing HIV, the most important of which is considered to be enzyme immunoassay. HIV ELISA began to be carried out relatively recently - in the early nineties. Over time, the method was refined and acquired new details. Now the reliability of the ELISA method is one of the highest - about 98%. Accordingly, this method is being used more and more often.

ELISA is the first test a patient encounters when testing for HIV. The ELISA method determines the presence of antibodies in the blood. If the test finds the presence of antibodies in the blood, then the virus is present. However, information on this test must be supported by other tests, such as immunoblotting (IB) and the latest generation method - polymerase chain reaction (PCR).

Antibodies are special protein compounds that are produced by plasma cells as a reaction to an infection entering the body (antibodies begin to form 1-2 months after infection).

Causes of HIV

Your doctor will write you a referral for testing if you have been in contact with someone infected with HIV or there is a risk of infection. Generations of people at risk of contracting HIV include:

• undergoing a course of injection treatment for a month;
• having sexual relations without special means of protection;
• carriers of infections transmitted during sexual contact;
• patients after undergoing a blood transfusion or injection to discover the cause of blood clotting in the eighties.

If you have doubts about your HIV status and concerns about the possibility of infection, it is recommended that you immediately seek help from any medical institution in our country to conduct a comprehensive HIV examination. Test results are received within a month. People at risk for HIV are recommended to be tested every 4 months.

How to get tested

There is no special preparation required for taking a blood test for HIV, but it is better to follow the simplest rules:

• It is better not to eat in the evening, but to take the test on an empty stomach in the morning;
• It is not recommended to overload the body with physical work;
• eat healthy foods, abstain from cigarettes and alcohol for a while;
• To exclude false test results, you should not take the test after treating serious diseases.

Human immunodeficiency virus infection is a serious diagnosis. Errors in diagnosis can lead to disastrous consequences. An ELISA test for HIV is the most accessible research method, but it is not informative in all cases.

ELISA – enzyme-linked immunosorbent assay. The purpose of the ELISA method is to detect antibodies to the immunodeficiency virus in biological material. Using the method, you can track the presence in the liquid not of the viruses themselves, but only of the antibodies produced in response to their presence. Enzyme immunoassay is widely used in the diagnosis of STDs. It helps to identify sexually transmitted diseases.

There are several types of ELISA: direct version, indirect version, sandwich method. In any case, the technique is based on determining the presence of antibodies, which serve as an indicator of the penetration of a foreign agent. To identify these “tags,” the biological component is treated with enzymes.

Enzyme immunoassay determines antibodies with an accuracy of 96–98%, the error is insignificant. It is 2 – 4%.

ELISA – a method for diagnosing HIV

ELISA test for HIV is the first stage of diagnosis. The antigens of the immunodeficiency virus are proteins p24, p15, p17, p31 and glycoproteins gp 41, gp55, gp66, gp120, gp160.

To detect the viral protein, a sample of blood is taken from a vein. A sample sent for ELISA blood testing is treated with enzyme immunoassay reagents. Serum is isolated from the blood. If during the study they are found, it means that the virus is already present in the blood.

Blood is donated strictly on an empty stomach. It is not recommended to eat fatty foods or drink alcohol 2 days before the test. You should stop taking antiviral drugs for 14 days.

Advantages of the method:

• relatively low cost;
• high stability of reagents;
• high sensitivity;
• short duration;
• minimal influence of the human factor.

Modern ELISA test systems are produced according to international standards. This increases the accuracy of the method.

Enzyme immunoassay does not always give reliable results. After the virus enters the blood, the latent (hidden) stage of development begins. The period before viruses begin to multiply and antibodies have not yet formed is called the “seronegative window time.” There is no point in doing ELISA at this stage. If infection occurs, the result will be . How quickly the virus reveals itself depends on how many dangerous cells have entered the body. In case of unprotected sexual intercourse or transfusion of contaminated blood, this period will be minimal.

For high reliability of HIV ELISA, the test is carried out three times. Deadlines for taking the ELISA test for human immunodeficiency virus:

• 6 weeks after probable contact,
• in 3 months,
• six months later.

4th generation ELISA for HIV is a method that is most informative in the early stages of infection. It can be performed as early as 1 month after the suspected infection. The test is expensive compared to the 3rd generation analogue. Therefore, in public medical institutions it is used as an additional diagnostic method. Test 3 is carried out free of charge. If based on its results a definite answer cannot be given, the patient is referred to a 4th generation ELISA.

Important! Immediately after infection, a person becomes contagious. He is dangerous to others, even when he does not yet know about his diagnosis!

If ELISA reveals antibodies to HIV antigens, additional studies must be performed. This includes . The reliability of this method is 80%. PCR tests blood, semen and vaginal discharge. The biological fluid is broken down in a medical reactor and then treated with enzymes. As a result, data is obtained on the concentration of HIV cells in the liquid medium. Due to the large error (20%), if the result is positive, immunoblotting is additionally performed.

The next stage of diagnosis is the Combo test (or immunoblotting). This is a highly sensitive test (98% confidence), which is carried out if ELISA results are ambiguous after 6 months.

Decoding ELISA results

Decryption time ranges from 24 to 48 hours. If it is necessary to obtain information urgently (surgery is required), decoding is carried out within 2 hours. Provincial medical centers do not always have the necessary reagents. The sample is taken at the place of application, then it is transferred to the regional center. Under such circumstances, the result can be found out in 1-2 weeks.

The result of an enzyme immunoassay can be either positive or negative; there are no other options.

Even if the result of the initial and repeated ELISA was positive, the patient cannot be recognized as HIV-infected. Subject to error. Reasons for a false positive result:

• chronic diseases;
• long-term infectious diseases;
• pregnancy.

Therefore, the result of the analysis should be clarified by additional studies.

If immunoblotting is HIV positive (reactive), the person is considered HIV-infected, which means they are healthy.

Over time, the virus cells adapt to the prescribed drugs. To control, the ELISA test is repeated periodically.

Sometimes immunoblotting shows a false negative result. It is extremely rare to have immunodeficiency virus for 6 months (or more). This is possible if a small amount of virus cells enters the blood. In 0.5% of the total number of cases, the infection can be diagnosed only after a year. In 99.5%, within six months after infection, ELISA will yield a reliable result.

Even with highly accurate studies, there is still a 2% chance of error. We should not forget about the human factor. To eliminate the possibility of error, the test can be performed in 2 different institutions.

S.N. IGOLKINA, V.F. PUZYREV, L.G. ZININA, N.M. DENISOVA, A.N. BURKOV,

A.P. RITE, T.I. ULANOVA
LLC "Research and Production Association "Diagnostic Systems",

Nizhny Novgorod
ELISA IMMUNO TEST SYSTEMS “DS-ELISA-HBeAg” and “DS-ELISA-anti-HBe” FOR SPECIFIC DIAGNOSIS AND PREDICTION OF OUTCOMES OF ACUTE AND CHRONIC HEPATITIS B
Hepatitis B is a viral infectious disease characterized by severe

inflammatory liver damage. About 1% of deaths occur in

acute period of the disease, in 4-10% of cases there is a transformation into chronic

process with the possible formation of subsequent cirrhosis of the liver and primary

hepatocarcinomas.

Despite the trend towards a decrease in the incidence of acute hepatitis B, an epidemiologically dangerous group of patients who are diagnosed with chronic viral hepatitis for the first time, as well as a group of carriers of the causative agent of the disease, continues to form. An unfavorable prognosis remains due to the high incidence of hepatitis B among the population of reproductive age, as well as among adolescents.

Therefore, the issues of treatment, prevention and diagnosis of hepatitis B are especially relevant now. Currently, enzyme-linked immunosorbent assay methods are widely used to detect markers of this infection. The most important serological markers of HBV include e-antigen (HBeAg) and antibodies to e-antigen (anti-HBe). HBeAg is associated with high blood contamination, indicating active replication of hepatitis B virus (HBV). It has been established that high titers of HBeAg correspond to high DNA polymerase activity and are always combined with the detection of complete Dane particles. When serum containing HBeAg enters the blood of a healthy person, the risk of infection is many orders of magnitude higher than after seroconversion occurs.

In acute hepatitis B, HBeAg is detected in the blood at the early stages of the infectious process, already at the first clinical manifestations of the disease, lagging behind the appearance of HBsAg by a week. Acute hepatitis B (AHB) of a cyclic course is characterized by short-term circulation of HBeAg. Soon, at 2-3 weeks of the icteric period, anti-HBe appears, which makes it possible to predict a favorable outcome of the disease.

Anti-HBe circulates in the blood for 2-5 years, less often for several months.

The onset of HBeAg-antiHBe seroconversion marks a sharp decrease in activity

infectious process. Detection of HBeAg in the blood of patients after 2 months of the disease indicates chronicity of the pathological process. In this case, anti-HBe may form many years after the appearance of antibodies to HBcAg or may not be detected at all.

The appearance of anti-HBe may have an unfavorable prognostic value in

acute period of HBV in severe forms, which corresponds to a mutation in the pre-core zone with

formation of HBV “e-” strain.

The goal of this work was to develop highly sensitive and specific

enzyme immunoassay test systems for the detection of HBeAg and anti-HBe and assessment of their main characteristics.

Materials and methods.

1. Enzyme immunosorbent test system “DS-ELISA-HBeAg”. Valid start of the test

are polyclonal goat antibodies to recombinant HBeAg, produced by NPO Diagnostic Systems, Nizhny Novgorod, adsorbed onto the solid phase, and a conjugate, which is polyclonal goat antibodies to recombinant HBeAg, labeled with horseradish peroxidase, produced by NPO Diagnostic Systems, Nizhny Novgorod. The analysis scheme is a one-stage “sandwich”. The total reaction time is 1.5 hours. The serum sample is analyzed undiluted.

2. Enzyme immunosorbent test system “DS-ELISA-anti-HBe”. The basis of the test is

recombinant HBeAg (AHBV 102), produced by NPO Diagnostic Systems,

Nizhny Novgorod, sorbed on the solid phase and anti-IgG conjugate with horseradish peroxidase, produced by Sorbent Service, Moscow. The reaction takes place in two stages. The test serum is added to the immobilized antigen in a 1/10 dilution and, after incubation and removal of unbound components, specific immunocomplexes are detected using mouse monoclonal antibodies against human IgG labeled with horseradish peroxidase.

3. To evaluate the developed test systems, 2178 serum samples were used

blood. Of these, 480 were serum samples from healthy donors. 1680 samples represent

are blood serum samples containing various markers of the hepatitis B virus.

Eighteen samples were obtained over time from patients with a clinical diagnosis of acute viral hepatitis B. Previously, all samples were tested for the presence of HBsAg, HBeAg, anti-HBe, anti-HBc using test systems produced by NPO Diagnostic Systems, Nizhny Novgorod: “ELISA-HBsAg/m”, “DS-ELISA-HBeAg”, “ DS-ELISA-anti-HBe", "ELISA-anti-HBc".

4. A comparative assessment of the developed test systems was carried out using

commercial drug "Monolisa HBe", produced by BIO-RAD, France;

Results and discussion. NPO "Diagnostic Systems" has developed 2 new diagnostic kits: "DS-ELISA-HBeAg" and "DS-ELISA-anti-HBe". The “DS-ELISA-HBeAg” test system is designed to detect the e-antigen of the hepatitis B virus in human serum (plasma) using an enzyme-linked immunosorbent assay and can be used for specific diagnostics, determining the activity of the infectious process, predicting the severity and outcome of hepatitis B.

The “DS-ELISA-anti-HBe” test system is designed to detect IgG antibodies to the e-antigen of the hepatitis B virus in human blood serum (plasma) and can be

used in predicting the course of the infectious process and monitoring therapy for hepatitis B.

To study the specificity of the new tests, the distribution was assessed

optical density (OD) of blood serum samples containing and not containing

HBeAg or anti-HBe. Blood serum samples from healthy donors and blood serum samples selected for various markers of the hepatitis B virus were used.

blood transfusion stations. The results of the study showed a significant separation of the two populations. The OD values ​​of serum samples not containing HBeAg ranged from 0.011 to 0.111, and the main peak of serum samples containing HBeAg ranged from 2.186 to 3.186 (Figure 1a).

Fig.1a. Distribution of OD of serum samples containing and not containing HBeAg in the “DS-ELISA-HBeAg” test system
The peak corresponding to the group of sera with a low OD (0.3-0.6) probably consists of serum samples collected at the early stages of the infectious process.

Already during the incubation period, HBsAg and HBeAg are naturally registered in the blood of patients, which confirms their potential epidemiological danger.

Optical density range of serum samples not containing anti-HBe

was in the range from 0.002 to 0.122, and the main peak OD of serum samples containing anti-HBe was in the range from 2.431 to 3.231 (Figure 1b).

Rice. 1b. Distribution of OD of serum samples containing and not containing anti-HBe in the “DS-ELISA-anti-HBeAg” test system
The distribution features of the OD of anti-HBe positive samples were studied

serums containing ( n=78) and not containing HBsAg ( n=56).


Rice. 2a. HBe-positive serum samples containing HBsAg in the “DS-ELISA-anti-HBe” test system.

Fig.2b. Features of the distribution of OD of anti-HBe-positive serum samples that do not contain HBsAg in the “DS-ELISA-anti-HBe” test system.
The optical densities of 87% of anti-HBe-positive serum samples that did not contain HBsAg ranged from 0.41 to 0.81 (Figure 2b). Moreover, only 14% of anti-HBe-positive serum samples containing HBsAg had an OD in this range (Figure 2a). It is known that in the phase of late convalescence, with negative reactivity to HBsAg, there is a gradual decrease in antibody titers to HBeAg (Figure 2b). Therefore, it is possible that the predominant concentration of anti-HBe-positive samples corresponded to an OD of less than 0.8.

The data obtained indicate the reliability of the separation of positive and

negative blood serum samples, regardless of the stage of the disease. The sensitivity and specificity of the “DS-ELISA-HBeAg” and “DS-ELISA-antiHBe” test systems were studied in comparison with the “Monolisa HBe” test (BIORAD, France) (Table 1).

Table 1
Comparative characteristics of sensitivity and specificity

test systems DS-ELISA-HBeAg and DS-ELISA-antiHB

Index	Determination of HBeAg		Determination of antiHBe
	NPO "DS", DS-IFA-HBeAg	"BIO-RAD" "Monolisa HВe"	NPO "DS" DS-ELISA-antiHBe	"BIO-RAD"
Quantity researched samples	67	67	32	32
Revealed positive samples	47	47	16	16
Revealed negative samples	20	20	16	16

The data presented in Table 1 shows 100% agreement between the results.

It is known that the combined indication of HBeAg and anti-HBe, especially their quantitative assessment, has additional prognostic significance. A rapid increase in anti-HBe titer characterizes an active humoral immune response and virtually eliminates the threat of chronicity. We analyzed changes in the HBeAg/anti-HBe content in blood serum samples from patients with a clinical diagnosis of “acute viral hepatitis B” (Table 2).

table 2
Dynamics of HBeAg/anti-HBe seroconversion in patients with HBV

Subject sick	Blood collection day*	HBeAg	anti-HBe O.F./O.C.		Anti-HBc	Anti- HBc IgM
	1	1,4+	2,1+	+	+	+
	12	0,5-	1,0+	+	+	+
	21	0,3-	1,3+	+	+	+
	1	1,2+	1,2+	+	+	+
	5	0,4-	1,0+	+	+	+
	13	0,2-	2,5+	+	+	+
	1	1,2+	0,6-	+	+	+
	21	0,2-	4,6+	+	+	+
	30	0,2-	9,7+	+	+	+
	1	2,1+	0,5-	+	+	+
	8	0,6-	1,1+	+	+	+
	28	0,3-	2,8+	+	+	+
	1	0,7-	2,2+	+	+	+
	8	0,3-	2,2+	+	+	+
	15	0,3-	2,8+	+	+	+
	38	0,2-	3,0+	+	+	+
6	1	1,1+	4,4+	+	+	+
	14	0,5-	3,8+	+	+	+

* from admission to hospital
All serum samples were tested for the presence of serological markers

OGV: HBsAg, anti-HBc, anti-HBc IgM. The observation period ranged from 13 to 38 days. In patients No. 1, No. 2 and No. 6, anti-HBe was detected against the background of a decrease in the HBeAg titer. In patients No. 3 and No. 4, anti-HBe appeared after HBeAg disappeared from the blood serum on days 21 and 8, respectively. Analysis of HBeAg detection in the blood serum of patient No. 5 upon admission to the hospital showed a negative result. At the same time, conversion to anti-HBe was registered already on the first day of the examination.

All subjects showed a tendency towards an increase in the titer of antibodies to HBeAg in

blood serum, which makes it possible to predict favorable dynamics of clinical

manifestations of acute respiratory syndrome and rapid recovery.

The study of the new test systems showed their high diagnostic reliability. The results of comparison of DS-ELISA-HBeAg and DS-ELISA-antiHBe showed 100% agreement with the Monolisa HBe test.

When studying serum samples from patients with hepatitis B in dynamics, tests

confirmed high sensitivity and specificity.

The short incubation time of the test samples and the conjugate (1 hour) makes it possible to determine the correct tactics for carrying out therapeutic measures in the shortest possible time. The created test systems are easy to use and economical (50 μl of serum is required to determine HBeAg and 10 μl of serum to determine anti-HBe). The high quality characteristics of the tests allow them to be successfully used in predicting the course of the infectious process and monitoring therapy for hepatitis B.
LITERATURE

1.Mayer K.P. Hepatitis and consequences of hepatitis / K.P. Mayer. – M., GEOTAR, Medicine, 1999.- P.720

2.Onishchenko G.G. Spread of viral hepatitis as a threat to national security / G.G. Onishchenko, L.A. Dementieva // Journal of Microbiology. –2003.-No. 4.- P.93-99.

3.Sorinson S.N. Viral hepatitis / S.N. Sorinson. - St. Petersburg, Teza, 1997.- 306 P.

4. Baumeister M. Hepatitis B Virus e Antigen Specific Epitopes and Limitations of Commercial Anti-HВe Immunoassays / M. Baumeister // Journal of Medical Virology.- 2000.-N 60.- R. 256-263.

5.Kane M. Global program for control of hepatitis B infection / M. Kane // Vaccine. – 1995.-N131(Suppl. 1).- R.47-6. Shunichi SatO. Hepatitis B Virus Strains with Mutations in the Core Promoter in Patients with Fulminant Hepatitis / Shunichi Sato, Kazuyuki Suzuki // Annals of Internal Medicine. – 1995.- N122.- R.241-248.

7. Ou J.-N. Molecular biology of hepatitis B virus e antigen./ J.-N.Ou // Journal of Gastroenterology and Hepatology. – 1997.- No. 12 (Suppl.1) – R. 178-187.

8.Tiollais P. The hepatitis B virus. / P. Tiollais, C. Pourcel, A. Dejean // Nature. – 1985. – P.317, 489-495
Published: J. “Clinical laboratory diagnostics” - 2005.-No. 6-P.-34-37

ELISA (enzyme-linked immunosorbent assay, ELISA) entered the life of practical medicine somewhere in the 60s of the last century. Its initial task was histological research for scientific purposes, which boiled down to the search and identification of the antigenic structure of cells of a living organism.

The ELISA method is based on the interaction of specific (AT) and related antigens (AG) with the formation of an “antigen-antibody” complex, which is detected using an enzyme. This fact prompted scientists to think that the method can be used for diagnostic purposes to identify specific immunoglobulins of various classes involved in the immune response to a particular infection. And it was a breakthrough in clinical laboratory diagnostics!

The method began to be actively used only in the early 80s, and then mainly in specialized institutions. The first immunoenzyme analyzers were equipped with blood transfusion centers and stations, infectious diseases and venereology hospitals, since the formidable AIDS, born on the African continent, appeared on our horizon and immediately joined the “old” infections, required immediate diagnostic measures and the search for therapeutic drugs affecting him.

Scope of application of the ELISA method

The possibilities of enzyme immunoassay are truly extensive. Now it is difficult to imagine how one can do without such research, which is used in literally all branches of medicine. It seems, what can ELISA do in oncology? It turns out it can. And a lot. The ability of the analysis to find markers characteristic of certain types of malignant neoplasms underlies the early detection of a tumor, when it is not yet determined by any other method due to its small size.

Modern clinical laboratory diagnostics (CDL), in addition to tumor markers, has a significant arsenal of ELISA panels and uses them to diagnose various pathological conditions (infectious processes, hormonal disorders) and monitor pharmaceutical drugs in order to identify their effect on the patient’s body and, by the way, not only human. Currently, enzyme immunoassay is widely used in veterinary services, because “our little brothers” are also susceptible to many diseases, from which they sometimes suffer greatly.

Thus, ELISA, due to its sensitivity and specificity, can determine from a blood sample taken from a vein:

• Hormonal status (thyroid and adrenal hormones, sex hormones);
• The presence of viral and bacterial infections (HIV, B and C, chlamydia, syphilis, and, and, as well as many other diseases caused by pathogenic microorganisms);
• Traces of the vital activity of microorganisms that initiated the infectious process, which successfully ended and moved into the stage of forming an immune response to this pathogen. Such traces, that is, antibodies, in many cases remain circulating in the blood for life, thereby protecting a person from re-infection.

What is the essence of ELISA?

The enzyme immunoassay method allows one to determine not only the presence of the pathogen itself (qualitative analysis), but also its quantitative content in the patient’s blood serum.

The viral or bacterial dose significantly influences the course of the infectious process and its outcome, therefore quantitative analysis plays an important role in the diagnosis and treatment of diseases in various forms and stages.

However, knowing enzyme immunoassay studies as the ELISA method, we don’t even think about how it manages to cover such a wide range of microorganisms inhabiting our planet, many of which pose a direct threat to the health and life of humans and animals. But the fact is that ELISA has many options (non-competitive and competitive - direct and indirect), each of which solves its own problem and, thus, allows for a targeted search.

To identify immunoglobulins of one class or another, a traditional 96-well polystyrene panel (plate) is used, in the wells of which sorbed recombinant proteins are concentrated in the solid phase. Antibodies or antigens that get into the well with blood serum find a “familiar” object and form a complex with it (AG - AT), which, fixed by an enzyme conjugate, will be manifested by a change in the color of the well when reading the results.

Enzyme immunoassay is carried out using test systems of a certain specificity, manufactured in special laboratories and equipped with all the necessary reacting components. Research can be carried out using washing machines (“washers”) and reading spectrophotometers, which mostly involve manual labor. On full automatic machines, which free the laboratory assistant from monotonous instillation, washing and other routine tasks, of course, it is faster and more convenient to work, but not all laboratories can afford such luxury and continue to work the old fashioned way - on semi-automatic machines.

The interpretation of ELISA results is within the competence of the laboratory diagnostics physician, and the inherent property of almost all immunochemical reactions to give false-positive or false-negative answers must also be taken into account.

Video: modern enzyme immunoassay

ELISA results using the example of syphilis

Enzyme immunoassay is suitable for identifying all forms, and, in addition, is used in screening studies. To carry out the analysis, the patient's venous blood taken on an empty stomach is used. The work uses tablets with a certain specificity (AB classes A, M, G) or total antibodies.

Considering that antibodies in syphilis are produced in a specific sequence, ELISA can easily answer the question of when the infection occurred and at what stage the process is, and the interpretation of the results obtained can be presented in the following form:

• IgM indicates the duration of the infectious process (may appear during exacerbation of chronic inflammatory diseases);
• IgA states that the infection occurred more than a month ago;
• IgG indicates that the infection is in full swing or that treatment has recently been carried out, which is easily determined by taking an anamnesis.

When testing for syphilis, negative wells (and the negative control) will remain colorless, while positive wells (and the positive control) will show a bright yellow color due to the color change of the chromogen added during the test. However, the color intensity does not always coincide with the control, that is, it may be slightly paler or slightly yellowish. These are dubious results, which, as a rule, are subject to re-examination with mandatory consideration of the quantitative indicators obtained on the spectrophotometer, but in general, the color is directly proportional to the number of immune complexes (associated Ags and ATs).

The most exciting of the enzyme immunoassays is the HIV ELISA

Analysis on is perhaps more interesting than others to a wide range of the population, because it is still impossible to say with confidence that many social problems have disappeared (prostitution, drug addiction, etc.). Unfortunately, HIV affects not only these layers of human society; you can become infected under various circumstances not related to sexual immorality or drug use. But if there is a need for an HIV test, you should not be afraid that everyone around you will know about your visit to such a laboratory. Now HIV-infected people are protected by law, and those who have doubts can turn to anonymous offices where they can solve the problem without fear of publicity and condemnation.

The enzyme immunoassay method used to diagnose HIV infection is one of the most important standard studies, which, however, requires special conditions, since the topic is very delicate.

It makes sense to carry out HIV ELISA after sexual contact, blood transfusion, other medical procedures suggesting infection, and at the end of the incubation period (“seronegative window”), but it should be borne in mind that this period of time is not constant. It can end in 14-30 days, or it can last up to six months, so the average value is considered to be an interval from 45 to 90 days. Blood is donated for HIV in the same way as for other infections - from a vein on an empty stomach. The results will be ready depending on the accumulation of material in the laboratory and its workload (from 2 to 10 days), although most often laboratories provide an answer on the same day or the next.

What can you expect from your HIV results?

ELISA for HIV infection detects antibodies to two types of the virus: HIV-1 (more common in Russia and other countries of Europe and Asia) and HIV-2 (more common in West Africa).

The task of the HIV ELISA is to search for class G antibodies, which are detected on all test systems, but at a later period, and class A and M antibodies, detected on new generation recombinant test kits, which make it possible to find antibodies at the earliest stages (incubation period - "seronegative window"). You can expect the following answers from the ELISA:

Primary positive result: the blood must be retested using a test system of the same type, but if possible of a different series and by another person (laboratory assistant);

Repeated (+) involves a new blood draw from the patient with its examination similar to the primary analysis;

Another positive result is subject to reference analysis, which uses highly specific test kits (2-3 pcs.);

A positive result in both (or three) systems is sent for immunoblotting (the same ELISA, but performed individually using test kits of particularly high specificity).

The conclusion about HIV infection is made only on the basis of immunoblotting. A conversation is conducted with the infected person in complete confidentiality. Disclosure of medical secrets in Russia, as well as in other countries, is subject to criminal punishment.

Tests for chlamydia and cytomegalovirus using the enzyme immunoassay method have also gained particular popularity, due to the fact that they make it possible to determine the time of infection, the stage of the disease and the effectiveness of treatment measures.

During implementation, one can also observe the appearance of antibodies of various classes in different phases of the pathological condition caused by an infectious agent:

• IgM can be detected as early as seven days after infection;
• IgA indicates that the infection has been living in the body for more than a month;
• IgG confirms the diagnosis of chlamydia and helps monitor treatment and determine its effectiveness. It should be noted that class G antibodies remain and circulate in the body regardless of the duration of the disease, therefore, to correctly interpret the analysis, you need to take into account the reference values ​​(norms), which, by the way, are different for each CDL: taking into account the brand of the test system and the specificity of the reagents included in the set. The normal values ​​are entered in the form next to the ELISA result.

As for , this is somewhat different: class M antibodies appear after about a month and a half, that is, the result (IgM+) becomes positive in the phase of primary infection or during reactivation of a latent infection and remains so from 4 months to six months.

The presence of class G antibodies is characteristic of the onset of a primary acute infection or reinfection. The analysis states that the virus is present, but does not provide information on what stage the infectious process is at. Meanwhile, determining the normal IgG titer also causes difficulties, since it depends entirely on the immune status of a particular person, which, however, is established by identifying class G immunoglobulins. Given this behavior of antibodies, when diagnosing CMV, there is a need to assess the ability of class G antibodies to interact with CMV, in order to later “neutralize” it (AT avidity). At the initial stage of the disease, IgG binds very poorly to viral antigens (low avidity) and only then begins to show activity, therefore, we can talk about an increase in the avidity of antibodies.

We can talk a lot about the advantages of enzyme immunoassay, because this method has managed to solve many diagnostic problems using only venous blood. There is no need for long waits, worries and problems with collecting material for research. In addition, test systems for ELISA continue to be improved and the day is not far off when the test will give a 100% reliable result.

Video: educational film of Moscow State Medical University named after. Sechenov on the basics of ELISA

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Article: L-1823

Description

Enzyme immunoassay test system for detecting antibodies of classes G, M and A to Treponema pallidum (T. pallidum) in human blood serum (plasma) and cerebrospinal fluid for the purpose of diagnosing syphilis, a complete set of reagents for 96x2 determinations, suitable for manual methods and automatic testing analyzers. The method is based on the “trap” principle, which consists in the fact that specific antitreponemal antibodies of all classes present in the sample bind simultaneously to recombinant antigens - analogues of immunodominant proteins of T. pallidum, fixed in the wells of the plate, and to the same antigens labeled with peroxidase horseradish, when jointly incubating samples of human blood serum (plasma) or cerebrospinal fluid and the conjugate. A change in the color of the substrate-chromogenic mixture containing TMB when it is added to the wells of the plate indicates the presence of specific antibodies to Treponema pallidum in the test samples

Functional purpose

Detection of antibodies to Treponema pallidum plays an important role in the diagnosis of syphilis, since T. pallidum cannot be isolated from cell culture, and samples for direct determination of the pathogen are often unavailable in latent and late stages of the disease. In the serodiagnosis of syphilis, non-treponemal (RMP, RPR, VDRL) and treponemal (RPGA, RIF, ELISA) tests are used. Enzyme immunoassay tests for determining total antibodies are used for mass screening of donor blood and for diagnosing syphilis as part of a complex of serological tests. After successful therapy, the reactivity of treponemal tests in most cases remains

Specifications

Set contents:
1. Polystyrene 96-well collapsible plate - 2 pcs.,
2. The conjugate is a transparent yellow liquid,
3. Positive control - transparent red liquid,
4. Negative control - transparent green liquid,
5. Wash solution, concentrate, pH from 6.9 to 7.6,
6. TMB is a transparent colorless liquid,
7. Substrate solution - transparent colorless liquid, pH ~4.2.
8. Stop reagent (0.2M),
9. Instructions for use and quality certificate.
Storage conditions: at a temperature of +2...8°C, freezing is unacceptable.
Shelf life is 15 months from the production date indicated on the kit label.
Compliance with the requirements of TU 9398-182-05941003-2010.
Registered with Roszdravnadzor.