ELISA test systems "ds-ifa-hbeAg" and "ds-ifa-anti-hbe. Test systems produced by LLC "npo" diagnostic systems Requirements for washing the plate

1 Set assignment

1.1 The kit is intended for the simultaneous detection of antibodies to the human immunodeficiency virus of the first and second types (HIV-1 and HIV-2) and the HIV-1 p24 antigen in human serum and plasma “in vitro” by indirect enzyme-linked immunosorbent assay.

2 Characteristics and principle of operation of the set

2.1 Set composition:

Component name	Quantity
Immunosorbent	2 tablets
Positive control sample AT (K + AT)	1 vial, 3 ml
Positive control sample AG (K + AG)	3 vials, lyophilized preparation
Negative control sample (TO -)	2 bottles of 3 ml
Conjugate solution #1 (RK-1)	1 vial, 12 ml
Conjugate #2 (Kg-2)	1 vial, 1 ml
Conjugate dilution solution No. 2 (RR-K2)	2 bottles of 18 ml
Substrate Buffer ( BRS)	2 bottles of 18 ml
Chromogen TMB	1 vial, 1 ml
Tween Phosphate Buffer Concentrate (FSB-T×25)	2 bottles of 50 ml
Stop Reagent	1 vial, 12 ml
Reagent bath kit with tips for multichannel pipettes	1 kit
Adhesive film

2.2 The main components of the "ELISA-HIV 1.2 AGAT" kit are immunosorbent, conjugate solution No. 1 and conjugate No. 2.

Immunosorbent is a polystyrene tablet, in the wells of which a mixture of recombinant HIV-1 (gp41) and HIV-2 (gp36) antigens and monoclonal antibodies to HIV-1 p24 antigen is adsorbed.

Conjugate solution #1 is a mixture of biotin-labeled monoclonal human antibodies against HIV-1 p24 antigen and biotin-labeled recombinant HIV-1 and HIV-2 proteins.

Conjugate #2 is streptavidin conjugated with horseradish peroxidase.

Positive control sample AT- human blood serum containing antibodies to HIV-1 and HIV-2, not containing antibodies to the hepatitis C virus and Treponema pallidum, HIV-1 p24 antigen and HBs antigen, inactivated by heating for 3 hours at a temperature of 56 ºC.

Positive control sample AG– human blood serum containing the native p24 HIV-1 antigen, not containing antibodies to HIV-1, HIV-2, hepatitis C virus and Treponema pallidum and HBs antigen, inactivated by heating for 3 hours at a temperature of 56 ºC.

Negative control sample– human blood serum that does not contain antibodies to HIV-1, HIV-2, HCV, HIV-1 p24 antigen and HBs antigen, inactivated by heating for 3 hours at a temperature of 56 ºC.

The principle of operation of the set. When a solution of conjugate No. 1 and serum samples of infected blood are introduced into the wells of the tablet, the p24 antigen binds both to specific antibodies on the solid phase and to monoclonal biotinylated anti-p24 antibodies that are part of the conjugate solution No. 1; HIV-specific antibodies bind both to the recombinant HIV-1 and HIV-2 antigens adsorbed on the solid phase, and to the antigens included in the conjugate solution No. 1, forming antigen-antibody complexes. Immune complexes of anti-p24 specific antibodies and p24 antigen are detected by conjugate No. 2. After washing off the unbound components, a solution of peroxidase substrate (hydrogen peroxide) and TMB chromogen is added to the wells of the plate.

The peroxidase reaction is stopped by adding a stop reagent (0.9 M sulfuric acid solution) and the color intensity of the solution in the wells is measured on a spectrophotometer as an optical density (OD) value at a wavelength of 450 nm.

The OD value is directly proportional to the concentration of specific antibodies and/or p24 antigen in a serum or plasma sample. The higher the content of antibodies and/or p24 antigen in the serum sample, the higher the staining intensity.

2.3 The set is designed to hold to hold 24 productions ELISA: 1 setting - 1 strip (8 holes). Total - 192 definitions including control samples.

3 Precautions when using the kit

3.1 All components of the kit in the concentrations used are non-toxic. However, work with all tested samples of human serum (plasma) that should be considered as potentially infected, able to store and transmit HIV, hepatitis B virus or any other pathogen of viral infection, with spent solutions and liquids, various equipment that may be contaminated in analysis process requires certain security measures when using the kit:

Work must be carried out in a specially equipped room;

It is necessary to work with the use of personal protective equipment and with the observance of precautions in accordance with the requirements of , , and .

3.2 Stop reagent containing sulfuric acid is irritating. In case of contact with skin and mucous membranes, rinse immediately with plenty of water.

3.3 When working with the kit, workplaces must be provided with supply and exhaust ventilation.

3.4 All persons working in the laboratory with kits must undergo a mandatory medical examination in accordance with the requirements.

3.5 Medical waste and/or unused, expired kits must be disposed of properly.

4 Rules for working with a set

4.1 To exclude false results, test samples must be prepared and stored under conditions that prevent bacterial growth. Serum samples containing aggregated serum components or sediment should be clarified by centrifugation for (5-10) minutes at 3000 rpm. Serum samples can be stored at (2-8) °C for no more than 5 days. Frozen samples (preferably to a temperature of at least minus 20 °C) can be stored for no more than 1 year. Repeated freeze-thaw cycles of samples should be avoided.

It must be remembered that samples with hemolysis, hyperlipidemia, bacterial growth, as well as those stored for a long time without freezing are not suitable for analysis.

The reliability of the results depends on the following rules:

It is not allowed to use the kit after the expiration date, as well as mixing the components of kits from different series;

A separate container should be used for the preparation of each reagent;

Do not treat all utensils used for the preparation of reagents with disinfectants and detergents. If necessary, rinse with drinking water, and then rinse five times with distilled water;

To work with the TMB and RX chromogen, it is necessary to use separate containers for solutions, pipette tips, and dishes.

Care must be taken to thoroughly mix the reagents;

The time between filling and emptying the wells of the plate with solutions and reagents should be at least 30 s. It is not allowed to dry the wells at all stages of the ELISA setting;

When using the washer, keep an eye on the condition of the container for solution for washing the plate and connecting hoses: they should not show signs of bacterial or fungal growth;

It is necessary to use automatic pipettes with interchangeable tips, certified for the value of the average dose and the convergence of the pipetting results (error not more than 3%);

Dispensers and working surfaces should be treated with a solution with a volume fraction of ethyl alcohol of 70%. Do not use chloramine and other chlorine-containing substances;

It is recommended to use disposable pipette tips for handling test and control samples. Each serum sample, as well as kit reagents, must be collected with a separate tip.

When introducing into the wells of PK-1, do not touch the surface of the tablet and the solution in the wells with the pipette tip.

During the analysis, avoid direct sunlight on the working surface.

4.2 When opening and dissolving the lyophilized components, it is necessary to ensure that no dry matter remains on the lid and walls of the vials.

5 Equipment and materials required for analysis

5.1 Vertical scanning spectrophotometer, which allows measuring the optical density of solutions in the wells of the tablet at a wavelength of 450 nm;

Semi- or automatic device for washing tablets (washer);

Dry-air thermostat type TC-80 M2, maintaining a temperature of (37 ± 1) ° C, or similar in characteristics;

Single-channel automatic pipettes with interchangeable tips, allowing to take liquid volumes from 0.01 to 5.0 ml;

Pipettes 8-channel automatic with interchangeable tips, allowing you to select liquid volumes up to 0.5 ml;

Measuring cylinder with a capacity of 2000 ml;

Laboratory flask with a capacity of 2000 ml;

Glass bottles with a capacity of 20 ml;

Trays for reagents or Petri dishes (diameter 100 mm);

Cotton wool medical hygroscopic;

Filter paper;

Rubber surgical gloves;

Solution with a volume fraction of ethyl alcohol 70%;

Solution with a mass fraction of hydrogen peroxide 6%;

Deionized or distilled water;

Container for collecting solid waste;

Container for draining liquid waste.

6 Preparing for analysis

6.1 Remove the reagent kit from the refrigerator before analysis, open the lid of the box and keep the components of the kit at a temperature (18-25) °C for 30 min.

Mix all serum (plasma) samples and reagents thoroughly before testing.

The reagent consumption of the test kit, which is determined by the number of strips used, is given in Table A.1 of Appendix A.

6.2 Preparation of the plate wash solution

Attention! Prepare the solution for washing the plate 15 minutes before the start of the analysis!

If the vial with FSB-T × 25 contains a precipitate, it must be heated before use at a temperature of (37 ± 1) ° C until the precipitate is completely dissolved. In a measuring cylinder with a capacity of 2000 ml, add the contents of the vial with FSB-T × 25, then add distilled water to the mark 1250 ml and gently mix the solution. The solution can be stored at (2-8) °C for 72 hours.

In the case of using one or more strips, shake the contents of the vial with FSB-T × 25 vigorously for (20-30) s, take the required volume of the solution (Table A.1) into a measuring cup or cylinder, add the required amount of distilled water and mix the solution . Unused FSB-T×25 can be stored in a closed vial at a temperature of (2-8) °C during the expiration date of the kit.

6.3 Immunosorbent preparation

The immunosorbent is ready for use.

Open the package and install the required number of strips on the frame. Store the remaining strips in a tightly closed bag with a desiccant at (2-8) °C for 3 months.

6.4 Preparation of K + AT, K - , RK-1, PP-K2, BRS and stop reagent

K + AT, K - , RK-1, RR-K2, BRS and stop reagent are ready for use.

Attention! Precipitation is possible in a vial with RK-1. The supernatant must be used for analysis.

Unused RK-1, PP-K2, BRS and stop reagent after opening the vials can be stored in closed vials at a temperature of (2-8) °C during the expiration date of the kit.

The rest of K + AT and K - after opening the bottle can be stored in closed bottles at a temperature of (2-8) ° C during the expiration date of the kit.

6.5 Preparation of K + AG solution

Attention! K + AG solution should be prepared 15 minutes before the start of the analysis!

To restore lyophilized K + AG, before opening the vial, lightly tapping to shake off the particles adhering to the walls of the vial or stopper. Open the vial and place the cork upside down on a dry surface. Add 0.8 ml of distilled water to the vial. Close the vial with a cork, hold for 10 minutes at a temperature of (18-25) ° C and gently tilt and rotate the vial, mix its contents until completely dissolved, avoiding foam formation.

Reconstituted K + AG can be stored in a closed vial at a temperature of (2-8) ° C for one month, at a temperature of minus 20 ° C - for six months. A single freeze-thaw of the restored K + AG is allowed.

6.6 Preparation of Kg-2 solution in working dilution

Take the volume indicated in Table A.1 from the vial with Kg-2 and transfer it to the vial with PP-K2. Mix the contents of the vial thoroughly, avoiding the formation of foam.

If one or more strips are used, take the required amount of PP-K2 into a clean vial, add Kg-2 in accordance with Table A.1 and mix the solution without foaming.

Attention! Kg-2 solution in working dilution is prepared immediately before use! Kg-2 solution in working dilution can be stored for 15 minutes at a temperature of (18-25) °C. Use only a new reagent tray and new tips!

The rest of Kg-2 can be stored in a closed vial at a temperature of (2-8) °C during the expiration date of the kit.

6.7 Preparation of working substrate solution

The bottle with TMB chromogen must be heated before use at a temperature of (37±1)° C until complete dissolution of the crystals.

Take the volume indicated in Table A.1 from the vial with TMB chromogen and transfer it to the vial with BRS. Mix the contents of the vial thoroughly, avoiding the formation of foam.

If one or more strips are used, take the required amount of BRS into a clean vial, add the TMB chromogen in accordance with Table A.1 and mix the solution without foaming.

Attention! The working solution of the substrate is prepared immediately before use in a place protected from light! The solution can be stored for 20 minutes at a temperature of (18-25) ° C, protected from light.

The solution must be protected from light and contact with metals or metal ions. The substrate solution must be colorless before use. Dishes that will be in contact with the substrate solution during the reaction should be washed without the use of synthetic detergents. Use only a new reagent tray and new tips!

The rest of the TMB chromogen can be stored in a closed vial at a temperature of (2-8) °C during the expiration date of the kit.

7 Plate wash requirements

At all stages of washing, it is necessary to control the filling of all wells and the complete removal (aspiration) of liquid from them;

It is necessary at each washing to fill all the wells with a solution to the brim (0.30-0.35 ml per well), without overflowing and overflowing of liquid from neighboring wells;

Keep the wells filled with the plate wash solution for 30 s;

With each aspiration, carefully remove the remaining liquid from the wells by tapping the frame with the strips in an inverted position on the filter paper folded several times, placed on a sheet of polyethylene;

Poor washing of the plate leads to incorrect results.

8 Conducting analysis

8.1 In any two wells of the tablet add 0.07 ml(70 µl) K + AT, in the other two wells - by 0.07 ml(70 µl) K + AG, in three other holes - by 0.07 ml(70 µl) TO - .

Attention! When setting up ELISA on one strip, it is allowed to use two wells for K - - one well, for K + AT - one well, for K + AG - one well.

In the remaining wells of the tablet add 0.07 ml(70 µl) of the studied samples of human serum (plasma).

Attention! Each sample must be taken with a disposable tip!

8.2 Into all wells of the tablet over control samples and test samples of blood serum (plasma) straightaway contribute by 0.05 ml(50 µl) RK-1. Mix the contents of the wells by gently tapping the edges of the plate.

8.3 (37 ± 1) ° From within 60 min.

Attention! For (1-2) minutes before the end of incubation, prepare a Kr-2 solution in working dilution (section 6.6).

8.4 Remove the contents of the wells with the washer, then wash the wells of the plate with the plate wash solution (step 6.2) seven times.

8.5 Add to all wells of the plate 0.15 ml(150 µl) solution Kg-2 in working breeding (clause 6.6).

8.6 Seal the plate with a film or cover with a lid and incubate in a thermostat at a temperature (37 ± 1) ° From within 10 min.

8.7 Remove the contents of the wells of the plate with a washer, then wash the wells of the plates with the solution for washing the plate (section 6.2) seven times.

8.8 Introduce into all wells of the tablet 0.15 ml(150 µl) substrate working solution(clause 6.7).

When preparing a working solution of the substrate (clause 6.7) f lacon with TMB chromogen must be heated before use for (3-5) minutes at a temperature of (37± 1) ° C until the crystals are completely dissolved.

8.9 Seal the plate with a film or cover with a lid and incubate in a thermostat at a temperature (37 ± 1) ° WITH in a dark place for 15 minutes.

Attention! At the end of incubation, in wells with serum samples containing antibodies to HIV-1 and/or HIV-2 and/or HIV-1 p24 antigen, the color of the solution will change from colorless to blue of varying intensity depending on the concentration of antibodies and/or antigen in the test serum sample.

8.10 Stop the peroxidase reaction by adding to all wells 0.05 ml(50 µl) stop reagent.

Attention! Wells with serum samples containing anti-HIV-1 and/or HIV-2 antibodies and/or HIV-1 p24 antigen will change the color of the solution from blue to yellow of varying intensity.

8.11 Not later than (1-2) minutes after stopping the reaction, determine the OD in the wells in the single-wave mode at a wavelength 450 nm.

9 Processing of analysis results

9.2 Results are only considered if:

The value of OPsr K - does not exceed 0.2 OE;

Each individual value of OP K - should not deviate from OPs K - more than 30%. If one of the three values of OP K - goes beyond this limit, it should be excluded from the calculation of OPs K - . If two of the three ODK values are outside this limit, the assay should be repeated on a new set of reagents;

The value of OPsr K + AT more than 1.0 OE;

The value of OPsr K + AG is more than 1.0 OE.

9.3 If the above conditions are met, calculate the critical value (OPcrit.), OE, according to the formula (1):

OPcrit. = OPsr K - + 0,14 (1).

10 Analytical and diagnostic characteristics

Sensitivity reagent kit ELISA-HIV 1.2 AGAT for detection of HIV-1 p24 antigen –

Specificity reagent kit ELISA-HIV 1.2 AGAT– 100% Standard AT(-)HIV. Standard panel of sera that do not contain antibodies to the human immunodeficiency virus of the first and second types (HIV-1,2) and HIV-1 antigen (p24). CJSC MBS.

11 Kit release form

11.1 The set is available in five configurations:

1 set 1P - manual analysis. The kit is designed for 12 ELISA runs on a collapsible plate: 1 run - 1 strip (8 holes). In total - 96 determinations, including control samples;

2 set 2M - manual analysis. The kit is designed for 2 ELISA runs on monolithic plates: 1 run - 1 plate. Total - 192 determinations, including control samples;

3 set 2P- manual analysis. The set is designed to hold 24 productions ELISA for 2 collapsible x tablets: 1 setting - 1 strip (8 holes). Total - 192 definitions, including control samples;

4 set A2M - analysis on an automatic analyzer. The kit is designed for 2 ELISA runs on monolithic plates: 1 run - 1 plate. Total - 192 determinations, including control samples;

5 set A2P - analysis on an automatic analyzer. The kit is designed for 24 ELISA runs on 2 collapsible plates: 1 run - 1 strip (8 holes). A total of 192 determinations, including control samples.

12 Storage and use conditions of the kit

12.1 The kit should be stored in a clean, protected from moisture and light room at a temperature of (2-8) ° C during the entire shelf life. Do not freeze kit components.

12.2 To obtain reliable results, strict adherence to the instructions for use of the kit is necessary.

12.3 Expiry date of the kit 12 months.

Annex A

Table A.1 - Consumption of reagents of the ELISA kit

reagent, ml

Number of used strips, pcs.

Preparation of solution for washing the tablet

FSB-T×25

distilled

water

Preparation of Kg-2 solution in working dilution

RR-K2

Substrate working solution preparation

Chromogen TMB

Bibliography

	Sanitary regulations	Order of the Ministry of Health of the Republic of Belarus dated December 16, 1998 No. 351 On the revision of departmental regulations governing issues related to the problem of HIV / AIDS
	Order of the Ministry of Health of the Republic of Belarus dated November 25, 2002 No. 165 About carrying out disinfection and sterilization by health care institutions
	Decree of the Ministry of Health of the Republic of Belarus dated April 28, 2010 No. 47 On the approval of the Instruction on the procedure for conducting mandatory medical examinations of employees and the invalidation of certain resolutions of the Ministry of Health of the Republic of Belarus

To conduct a comprehensive assessment of the state of the body, an ELISA diagnostic method is used. ELISA blood test is designed to diagnose infectious, hematological, primary and secondary immunodeficiencies.

What is ELISA analysis

Many patients are interested in the ELISA method: what is it, what is the study for. Immunoenzymatic analysis began to be used relatively recently. Initially, it was used to study antigenic structures, and it was carried out only for scientific purposes. Then the scientists came to the conclusion that with the help of enzymes it is possible to identify specific antibodies that arise in response to an ongoing disease.

Initially, this technique was used only by narrow-profile medical institutions, mainly at blood transfusion stations. Of particular importance is the ELISA method for detecting HIV infection.

Today, this method has a wide scope of application. Modern laboratories use it to diagnose:

tumors;
hormonal disorders;
infections;
chronic or previously transferred infectious processes;
helminths.

If an infectious process occurs in the body, then this type of diagnosis is considered the most optimal for determining the type of disease.

The essence of the method and its types

ELISA method - what is it, what is the essence of this type of research? This and many other questions are of interest to patients. The basis of this diagnostic method is the binding of the body's immune cells to the antigens of infectious agents. The resulting complex is determined using a special enzyme.

To understand the principle of the ELISA method, you need to know how the antigen-antibody reaction proceeds. An antigen is a protein molecule foreign to the body that enters along with the infection. Particles of foreign blood that do not match in group are also considered antigens. In the body, they provoke an immune response aimed at protecting against foreign substances. Therefore, the human body produces antibodies - immunoglobulins that can attach to antigens, forming an immune complex. Such compounds are much easier to recognize and destroy by immune cells.

The reaction for the presence of such immune complexes is carried out in the laboratory, using ready-made compounds to determine whether there are similar ones in the blood.

The essence of the ELISA method is quite simple, however, due to the fact that a blood test is performed to detect many infections and diseases, there are several of its varieties. Each differs in the scheme of conduct and scope. May be direct or indirect ELISA. The direct method implies that immobilized antibodies that react with antigens are used. The main advantage of this method is that all processes can be automated, which means that diagnostics takes a little time.

The indirect method implies that secondary antibodies are used. And on the solid phase, the antigen is immobilized. The analysis allows you to determine antibodies to various antigens. This helps to achieve a more accurate result, but the method is complex.

Study Benefits

The ELISA method has many advantages over other diagnostic methods. The main ones include:

high sensitivity;
stability during storage of ingredients;
speed of diagnostics;
a small amount of test material can be used;
it is possible to automate all processes;
infection can be detected at an early stage.

This diagnostic method is universal, therefore it is suitable for mass examination. With the help of analysis, it is possible to trace the dynamics of the course of the infectious process.

Indications for analysis and material sampling

Conducting a study using the ELISA method can be prescribed if a variety of diseases are suspected:

Venous blood is examined for the presence of antibodies. Before analysis, elements that can complicate the study are isolated from it. Other biological fluids may also be sampled.

To obtain the most accurate information, blood sampling is carried out on an empty stomach. If the procedure was prescribed to determine a latent infection, then a few weeks before the analysis, you should stop taking antibacterial and antiviral drugs. Depending on the equipment of the laboratory where the material was taken, the result can be obtained within a day. In emergency cases, this time is reduced to a few hours.

Analysis for syphilis

Using the ELISA method helps to determine the presence of many infections in the body, in particular syphilis. For the study, blood is taken from a vein on an empty stomach. Then a study is carried out that helps to determine not only the presence of the disease in the body, but also the exact timing of its onset, since during the course of the disease some antibodies are replaced by others in a strictly defined order.

In the acute phase, indicating a prolonged course of the disease, or during an exacerbation of a chronic infection, type M immunoglobulins will be detected in the blood. The presence of type A immunoglobulins indicates that the infection lives in the body for more than 4 weeks. Group G immunoglobulins indicate the height of the disease or previous therapy.

According to the degree of color of the wells, the intensity of the course of the infectious process is assessed, since its saturation depends on the number of immune complexes formed.

HIV test

The ELISA method is also used to analyze for in this case, it has certain features that are associated with the course and progression of the disease. This research method is considered the most acceptable for determination, however, it should be carried out no earlier than a month after exposure to risk factors. This is due to the presence of an incubation period that runs from 45 days to 6 months. That is why the analysis must be repeated after six months.

ascariasis;
giardiasis;
toxoplasmosis, etc.

Despite all the advantages, there are also disadvantages of the ELISA method. The main disadvantage is that when conducting a study, the doctor must have an assumption about the disease in advance.

When there is no way to accidentally find the pathogen and determine its enzyme immunoassay properties. The test only indicates the presence of antibodies in the patient's blood. In addition, this is a rather expensive analysis.

Deciphering the analysis

The result of a qualitative ELISA will be either the presence of antibodies, or their absence in the blood. If a quantitative analysis is carried out, then the concentration of antibodies can be expressed either in a numerical value or in a certain number of + signs.

In addition, indicators such as:

The IgM indicator indicates the course of an acute infectious process in the body. Its complete absence may indicate the absence of the causative agent of the disease or its transition to the chronic stage.

An IgA reading with a negative IgM test indicates a chronic or latent infection. The simultaneous presence of IgM and IgA indicates that the disease is in an acute stage. The presence of IgG indicates the transition of the disease to the chronic stage or complete recovery and the development of immunity.

Now there are special ELISA tests that you can do yourself.

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

A.P. OBRYADINA, T.I. ULANOVA
LLC "Scientific and Production Association "Diagnostic Systems",

Nizhny Novgorod
ENZYME IMMUNE TEST SYSTEMS "DS-ELISA-HBeAg" and "DS-ELISA-anti-HBe" FOR SPECIFIC DIAGNOSTICS AND PREDICTION OF OUTCOMES OF ACUTE AND CHRONIC HEPATITIS B
Hepatitis B is a viral infectious disease characterized by severe

inflammatory liver disease. Approximately 1% of deaths occur in

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

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

hepatocarcinomas.

Despite the downward trend 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 associated with a 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 now particularly relevant. Currently, enzyme immunoassay 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 infection, indicating active replication of the hepatitis B virus (HBV). It has been established that high HBeAg titers 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.

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, a week behind the appearance of HBsAg. Acute hepatitis B (AHB) of 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 circulate in the blood for 2-5 years, less often for several months.

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

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

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

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

the formation of HBV "e-" strain.

aim this work was the development of highly sensitive and specific

ELISA test systems for detecting HBeAg and anti-HBe and assessing their main characteristics.

Materials and methods.

1. Enzyme immunoassay test system "DS-ELISA-HBeAg". Valid test start

are polyclonal goat antibodies to recombinant HBeAg, produced by NPO Diagnostic Systems, Nizhny Novgorod, adsorbed on 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. ELISA test system "DS-ELISA-anti-HBe". The basis of the test is

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

Nizhny Novgorod, adsorbed 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 at a dilution of 1/10 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 samples were 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 manufactured by NPO Diagnostic Systems, Nizhny Novgorod: "ELISA-HBsAg / m", "DS-ELISA-HBeAg", " DS-ELISA-anti-HBe", "ELISA-anti-HBc".

4. Comparative evaluation of the developed test systems was carried out using

commercial drug "Monolisa HBe", manufactured 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 test system "DS-ELISA-HBeAg" is designed to detect the e-antigen of the hepatitis B virus in the serum (plasma) of human blood by enzyme immunoassay and can be used for specific diagnosis, determining the activity of the infectious process, predicting the severity and outcome of hepatitis B.

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

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

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

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 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 containingHBeAgin the test system "DS-ELISA-HBeAg»
The peak corresponding to the group of sera with low OD (0.3-0.6) is likely to be sera samples taken at the early stages of the infectious process.

Already in the incubation period, HBsAg and HBeAg are regularly 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 test system "DS-ELISA-anti-HBeAg»
The features of the distribution of OD of anti-HBe positive samples were studied.

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

Rice. 2a. HBe-positive sera samples containingHBsAgin the test system "DS-ELISA-anti-HBe».

Fig.2b. Features of the distribution of OP anti-HBe-positive sera samples that do not contain HBsAgin the test system "DS-ELISA-anti-HBe».
Optical densities of 87% of anti-HBe-positive serum samples that did not contain HBsAg ranged from 0.41 to 0.81 (Figure 2b). At the same time, only 14% of anti-HBe-positive sera samples containing HBsAg had 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 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 study of the sensitivity and specificity of the test systems "DS-ELISA-HBeAg" and "DS-ELISA-antiHBe" was carried out in comparison with the test "Monolisa HBe" ("BIORAD", France) (table 1).

Table 1
Comparative characteristics of sensitivity and specificity

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

Index	Definition of HBeAg		Definition of anti-HBe
Index	NPO "DS", DS-ELISA-HBeAg	BIO-RAD, "Monolisa HBe"	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 shown 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 an additional prognostic value. A rapid increase in anti-HBe titer characterizes an active humoral immune response and virtually eliminates the threat of chronicity. We analyzed the change in the content of HBeAg/anti-HBe in blood serum samples of patients with a clinical diagnosis of acute viral hepatitis B (table 2).

table 2
Dynamics of HBeAg/anti-HBe seroconversion in patients with acute hepatitis B

Examined sick	Day of blood sampling*	HBeAg	anti-HWe Ref./Opt.		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 the hospital
All sera samples were tested for the presence of serological markers.

OGV: HBsAg, anti-HBc, anti-HBc IgM. The observation period varied from 13 to 38 days. In patients No. 1, No. 2 and No. 6, anti-HBe was detected already against the background of a decrease in the HBeAg titer. In patients No. 3 and No. 4, anti-HBe appeared after the disappearance of HBeAg from the blood serum on days 21 and 8, respectively. Analysis of the detection of HBeAg 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 trend towards an increase in the titer of antibodies to HBeAg in

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

manifestations of OGV and a speedy recovery.

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

In the study of serum samples of patients with hepatitis B in dynamics, tests

confirmed high sensitivity and specificity.

The short incubation time of the test samples and the conjugate (1 h) makes it possible to determine the correct tactics for conducting therapeutic measures in the shortest possible time. The created test systems are easy to set up, economical (requires 50 µl of serum for the determination of HBeAg and 10 µl of serum for the determination of anti-HBe). The high quality characteristics of the tests make it possible to successfully use them in predicting the course of the infectious process and monitoring ongoing therapy for hepatitis B.
LITERATURE

1.Mayer K.P. Hepatitis and consequences of hepatitis / K.P. Mayer.– M., GEOTAR, Medicine, 1999.- C.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-HBe Immunoassays / M. Baumeister // Journal of Medical Virology.- 2000.-N 60.- P.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.-H. Molecular biology of hepatitis B virus e antigen./ J.-H.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: Zh. "Clinical laboratory diagnostics" - 2005.-№6-S.-34-37

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, how to interpret the results of the analysis.

The human immunodeficiency virus is one of the most feared infections of 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 is spread through direct contact between partners during sexual intercourse through the male and female genital organs. Another cause of infection can be the entry of the virus through the blood. This occurs due to the use of non-sterile instruments, transfusion of infected blood, and even through generations: to a child from an infected mother while breastfeeding.

Until now, scientists around the world are puzzling over the creation of a new generation of drugs that can cure AIDS, however, despite the great pace of scientific progress, the human immunodeficiency virus continues to spread across the planet.

ELISA - a method for diagnosing HIV

Scientists of the generation of the 20th century developed several methods for diagnosing HIV, the most important of which is considered linked immunosorbent assay. ELISA for HIV began to be carried out relatively recently - in the early nineties. Over time, the method has been honed, 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 that 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 needs to be supported by other assays, such as immune blotting (IB) and the latest generation method, polymerase chain reaction (PCR).

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

Causes of HIV

The doctor will write you a referral for analysis if you have been in contact with a person infected with HIV or there is a risk of infection. Generations of people at risk of HIV infection include:

undergoing a course of injection treatment within 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 fears related to the possibility of infection, it is recommended that you immediately seek help from any medical institution in our country for a comprehensive HIV test. The results of the tests are received within a month. People at risk for HIV are recommended to be screened every 4 months.

How to take an analysis

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

it is better not to eat in the evening, but to take an analysis on an empty stomach in the morning;
it is not advised to load the body with physical work;
eat healthy food, refrain from cigarettes and alcohol for a while;
to exclude false test indicators, you should not take an analysis after the treatment of serious diseases.

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

ELISA - enzyme immunoassay. The task of the ELISA method is to detect antibodies to the immunodeficiency virus in biological material. Using the method, it is possible to track the presence in the liquid not of the viruses themselves, but only of antibodies produced in response to their presence. ELISA is widely used in the diagnosis of STDs. It helps to identify sexually transmitted diseases.

There are several types of ELISA: direct variant, indirect variant, sandwich method. In any case, the technique is based on the determination of the presence of antibodies, which serve as an indicator of the penetration of a foreign agent. To reveal these "marks", the biological component is treated with enzymes.

ELISA detects antibodies with an accuracy of 96 - 98%, the error is negligible. It is 2 - 4%.

ELISA - a method for diagnosing HIV

ELISA analysis 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 a viral protein, a portion 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, then the virus is already present in the blood.

Blood is given strictly on an empty stomach. 2 days before the analysis, it is not recommended to eat fatty foods and drink alcohol. You should stop taking antiviral drugs within 14 days.

Advantages of the method:

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

Modern test systems for ELISA are produced according to world standards. This improves the accuracy of the method.

ELISA does not always give reliable results. After the virus enters the blood, the latent (hidden) stage of development begins. The period until the viruses have begun to multiply and antibodies have not yet formed is called the “seronegative window time”. It is pointless to do ELISA at this stage. If an infection has occurred, the result will be . How quickly the virus detects itself depends on how many dangerous cells have entered the body. With unprotected intercourse or transfusion of infected blood, this period will be minimal.

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

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

4th generation ELISA for HIV is the most informative method in the early stages of infection. It can be carried out as early as 1 month after the alleged infection. The test is expensive compared to the 3rd generation counterpart. Therefore, in public medical institutions, it is used as an additional diagnostic method. Test 3 is carried out free of charge. If it is impossible to give an unambiguous answer based on its results, the patient is sent for 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 antibodies to HIV antigens are detected during ELISA, additional studies are necessary. It belongs to these. The reliability of this method is 80%. With the help of PCR, blood, semen and vaginal discharge are examined. The biological fluid is split in a medical reactor, then subjected to enzyme treatment. As a result, data are obtained on the concentration of HIV cells in a liquid medium. Due to the large error (20%), with a positive result, immune blotting is additionally performed.

The next diagnostic step is the Combo test (or immune blotting). This is a highly sensitive study (98% confidence) and should be done if ELISA results are inconclusive at 6 months.

Interpretation of ELISA results

Decryption time is from 24 to 48 hours. If it is necessary to obtain information urgently (surgical intervention is required), decoding is carried out in 2 hours. Provincial medical stations do not always have the necessary reagents. The sample is taken at the place of circulation, 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 was positive during the primary and repeated ELISA, the patient cannot be recognized as HIV-infected. Possible error. Reasons for a false positive result:

chronic diseases;
prolonged infectious diseases;
pregnancy.

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

If during immunoblotting for HIV (reactive), a person is considered HIV-infected, which means that he is healthy.

The cells of the virus eventually adapt to the prescribed drugs. To control, the ELISA test is periodically repeated.

Sometimes immunoblotting shows a false negative result. Extremely rare immunodeficiency virus 6 months (or more). This is possible if a small amount of virus cells has entered the bloodstream. In 0.5% of the total number of cases, infection can be diagnosed only after a year. In 99.5%, within six months after infection, an ELISA will give a reliable result.

Even with high-precision studies, there is still a margin of error of 2%. We should not forget about the human factor. To eliminate the possibility of error, the test can be carried out in 2 different institutions.