Testing blood samples using ELISA for HIV infection is one of the earliest and most reliable diagnostic methods. The enzyme immunoassay test is one of the few studies, along with PCR, that with a high degree of probability confirm or refute such a terrible diagnosis as HIV infection or AIDS.
Since the detection of the first cases of infection with the immunodeficiency virus in the Soviet Union, and this is 1983. A medicine that would completely cure this infection, as well as a vaccine for it, have not been invented, but this process is under active development. In the meantime, only early diagnosis of HIV infection can keep the pathological process under control.
Information tour about HIV infection
The human immunodeficiency virus is a retrovirus that causes a slow course of the disease. Penetrating into the body, the immunodeficiency virus primarily affects lymphoid tissue. A healthy immune system produces T-lymphocytes in order to recognize the newly arrived viral agent. Then give information to the antibodies on what signs to recognize the immunodeficiency virus and destroy it. The process of “recognition” of the immunodeficiency virus occurs with the help of CD4 receptor proteins. But it is precisely at this stage that something strange is observed, something that for many years has not allowed us to defeat the plague of the 20th century.
Thus, human immunity falls into a vicious circle. The body can only be helped if highly active antiretroviral therapy (HAART) is administered. And also switch to a completely healthy lifestyle and diet, which will free up large immune resources from unnecessary work. Scientists around the world have joined forces and are constantly conducting research in search of an effective cure against HIV infection.
Not long ago, an experiment was carried out with the CCR5 protein, which ensures the innate immunity of a certain part of people to HIV infection. Scientists subjected the blood substance of human volunteers to gene therapy to introduce CCR5 into T helper cells. In all patients, this led to a large-scale decrease in the number of retroviral DNAs. And in one patient, the immunodeficiency virus was completely eliminated by this method. Based on this experiment, we can only assume in a single case a successful victory over the terrible plague of the 20th century.
Since 2016, this activity has advanced significantly. Staggeringly effective results of ninety percent destruction of the immunodeficiency virus were obtained using attacking antibodies secreted by muscle tissue, bypassing the captured immune system. To do this, a special gene was implanted into the muscle tissue of experimental living beings, producing these same killer cells to destroy HIV infection. But this method has not yet been used in humans, although it is planned in the coming years.
Moreover, there is no point in losing heart. It is better to identify the immunodeficiency virus as early as possible, which can be done using ELISA if there has been a risk of infection. And if the diagnosis is confirmed, maintain your immunity through HAART until a new cure for HIV infection is unveiled.
Diagnosis of HIV infection
The HIV ELISA method is aimed at detecting antibodies to the immunodeficiency virus in a human blood sample. Once in the body, retroviruses, a group of which includes HIV infection, do not immediately begin to actively capture CD4 immune cells (T-lymphocytes). But you shouldn’t wait until aggressive agents begin deadly activity in the body. It is no secret that with any disease, and HIV infection is no exception, the earlier therapy is started, the more effective the healing.
When it comes to the human immunodeficiency virus, cases of complete healing have not been officially recorded. But this does not mean at all that you need to give up and let the disease take its course. People with HIV infection live up to 75–80 years, provided that it is detected at an early stage and undergoes ongoing maintenance therapy.
The enzyme immunoassay diagnostic method for HIV infection has a high reliability of about 97–99%. But it is precisely this insignificant part of 1–3% that can give a false-positive or false-negative result in diagnosing HIV infection using ELISA. For these purposes, in order to definitively confirm or refute the indications of an enzyme immunoassay, an analysis called immunoblot (IB) is used.
What are ELISA and IB methods for HIV infection?
The 4th generation enzyme immunoassay method for HIV infection is aimed not only at searching and identifying pathogenic cells, but also at establishing their numbers. Therefore, one enzyme immunoassay test combines qualitative and quantitative research. The 4th generation ELISA method for HIV infection for a period of six weeks from the moment of suspected infection will show 100% high reliability of the results.
The basis of enzyme immunoassay is the interaction of specific antibodies and related antigens. Thanks to a special enzyme, the formation of an “antigen-antibody” complex occurs. To carry out this reaction to detect immunoglobulins for HIV infection, a blood sample is placed in a polystyrene panel with 96 wells, in which recombinant proteins (sorbed) are located in solid form. Antigens or antibodies found in a container with blood serum recognize an object known to them and form an “AT-AG” compound with it. To establish antibodies to immunodeficiency viruses of types 1 and 2, the DS IFF test for HIV AG-AT is also used.
Advantages of the 4th generation enzyme immunoassay method for HIV infection:
- High reliability, more than 96%. False results are often caused not by the test itself, but by improper preparation or a recent respiratory illness.
- Determination of both the presence and quantity of immunoglobulins.
- The ability to monitor the results of HIV therapy.
- The speed of obtaining test results for HIV infection, which is several hours if carried out at a diagnostic center.
- Analysis for HIV infection four, the immunoenzyme reactivity of which is significantly higher than its predecessors, is informative during the serological window of the immunodeficiency virus.
Immune blotting is a screening test and is used to determine the type of antigen. IB screening study is used as a verification test for HIV infection 5–7 weeks after ELISA. The IB method is based on the use of electrophoresis to separate immunodeficiency virus antigens. The process of antigen separation makes it possible to detect specific immunoglobulins for each specific antigen of HIV infection.
Immunoblotting is used when an enzyme immunoassay test for HIV infection shows a false positive or false negative result twice in a row. Suspicions may arise in the absence or, conversely, presence of characteristic symptoms of HIV infection. Although here, too, various variations may arise.
Interpretation of ELISA results for HIV infection
In the case when ELISA plus is confirmed by positive IB screening, there is no doubt that the patient has an immunodeficiency virus in the body. But if, for example, the IFF for HIV is positive, and the immunoblot is negative, then a conclusion is drawn about the infection “minus”, that is, about its absence.
Only in such a situation, the patient is still sent for additional examination to definitely rule out the immunodeficiency virus.
And if the enzyme immunoassay for HIV 4th generation is “minus”, and immunoblotting is positive, perhaps there is another infection in the body. Perhaps a person has some kind of autoimmune disease, an oncological process, or psoriasis of the skin or joints gives false positive results for both ELISA and IB.
Another option is when an enzyme-linked immunosorbent test for HIV infection shows a “minus” result and an immunoblot also shows a “minus” value, here we can only rejoice due to the absence of the immunodeficiency virus. But if there are specific signs inherent in HIV infection or if the person is in close contact with an HIV-positive patient. Then it will also be necessary to carry out additional diagnostics, for example polymerase chain reaction (PCR). In addition, if the indicators are “minus” or “plus”, the patient is recommended to re-donate blood after 4, and preferably 6 months.
Norm and deviation of the number of T-lymphocytes in HIV infection:
- The norm of T cells is from 1000 to 2900 μl.
- Early AIDS from 500 to 200 µl.
- Late AIDS less than 200 µl.
It is easier to prevent a disease than to treat it - this is all well-known wisdom. Since the spread of free morals in society, a large number of different sexually transmitted diseases have appeared. HIV infections and AIDS are still considered incurable diseases. The mode of transmission is mainly through sexual contact and blood. It is impossible to protect yourself one hundred percent from any disease. There are a lot of unforeseen situations, even for people leading a moral and clean lifestyle. In situations of increased risk of contracting HIV and other dangerous infections, blood diagnostics are invaluable.
In contact with
1 Purpose of dialing
1.1 The kit is intended for the simultaneous detection of antibodies to human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and the HIV-1 p24 antigen in human serum and blood plasma “in vitro” using the method of indirect enzyme-linked immunosorbent assay.
2 Characteristics and principle of operation of the set
2.1 Set contents:
|
Component name |
Quantity |
|
Immunosorbent |
2 tablets |
|
Positive control sample AT (K + AT) |
1 bottle, 3 ml |
|
Positive AG control sample (K + AG) |
3 bottles, lyophilized preparation |
|
Negative control sample (TO -) |
2 bottles of 3 ml |
|
Conjugate solution No. 1 (RK-1) |
1 bottle, 12 ml |
|
Conjugate No. 2 (Kg-2) |
1 bottle, 1 ml |
|
Solution for diluting conjugate No. 2 (RR-K2) |
2 bottles of 18 ml |
|
Buffer solution for substrate ( BRS) |
2 bottles of 18 ml |
|
Chromogen TMB |
1 bottle, 1 ml |
|
Phosphate Buffer Saline Concentrate with Tween (FSB-T×25) |
2 bottles of 50 ml |
|
Stop reagent |
1 bottle, 12 ml |
|
Reagent bath kit with multichannel pipette tips |
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 plate in the wells of which a mixture of recombinant HIV-1 (gp41) and HIV-2 (gp36) antigens and monoclonal antibodies to the HIV-1 p24 antigen is adsorbed.
Conjugate solution No. 1 is a mixture of biotin-labeled human monoclonal antibodies against the HIV-1 p24 antigen and biotin-labeled recombinant HIV-1 and HIV-2 proteins.
Conjugate No. 2 is streptavidin conjugated to horseradish peroxidase.
Positive control sample AT– human blood serum containing antibodies to HIV-1 and HIV-2, not containing antibodies to 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 AG control sample– human blood serum containing the native HIV-1 p24 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.
Operating principle of the set. When a solution of conjugate No. 1 and samples of infected blood serum are added to the wells of a plate, the p24 antigen binds both to specific antibodies on the solid phase and to monoclonal biotinylated anti-p24 antibodies that are part of the solution of conjugate No. 1; HIV-specific antibodies bind both to recombinant HIV-1 and HIV-2 antigens adsorbed on the solid phase, and to 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 the 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 the serum or plasma sample. The higher the antibody and/or p24 antigen content in the serum sample, the higher the intensity of the staining.
2.3 The set is designed to carry out 24 productions ELISA: 1 set – 1 strip (8 holes). Total - 192 definitions, including control samples.
3 Precautions when working with the kit
3.1 All components of the kit are non-toxic in the concentrations used. However, work with all test samples of human blood serum (plasma), which should be considered as potentially infected, capable of storing and transmitting HIV, hepatitis B virus or any other causative agent of viral infection, with waste solutions and liquids, various equipment that may be contaminated in analysis process, requires certain safety measures when using the kit:
The work must be carried out in a specially equipped room;
It is necessary to work using personal protective equipment and taking 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 Disposal of medical waste and/or unused kits that have expired must be carried out in accordance with the requirements.
4 Rules for working with the set
4.1 To avoid 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 a temperature of (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 components of kits from different series;
A separate container must be used to prepare each reagent;
Do not treat all utensils used for preparing reagents with disinfectants and detergents. If necessary, rinse with drinking running water and then rinse five times with distilled water;
To work with TMB and PX chromogen, it is necessary to use separate containers for solutions, pipette tips, and dishes.
It is necessary to pay attention to thorough mixing of the reagents;
The time between filling and emptying the wells of the plate with solutions and reagents must be at least 30 s. Drying of the wells is not allowed at all stages of the ELISA;
When using the washer, monitor the condition of the container for the solution for washing the tablet and the connecting hoses: they should not show signs of bacterial or fungal growth;
It is necessary to use automatic pipettes with replaceable tips, certified for the average dose and the consistency of pipetting results (error no more than 3%);
Treat dispensers and working surfaces with a solution containing 70% ethyl alcohol by volume. Do not use chloramine or other chlorine-containing substances;
It is recommended to use disposable pipette tips for working with test and control samples. Each serum sample, as well as kit reagents, must be collected with a separate tip.
When adding RK-1 to the wells, do not touch the surface of the plate and the solution in the wells with the pipette tip.
During the analysis, direct sunlight should be avoided on the work surface.
4.2 When opening and dissolving lyophilized components, it is necessary to ensure that no dry matter remains on the lid and walls of the bottles.
5 Equipment and materials required for analysis
5.1 Vertical scanning spectrophotometer, allowing measurements of the optical density of solutions in the wells of a plate at a wavelength of 450 nm;
Semi- or automatic device for washing tablets (washer);
Dry air thermostat type TS-80 M2, maintaining a temperature of (37±1) °C, or characteristics similar to it;
Single-channel automatic pipettes with replaceable tips, allowing you to withdraw liquid volumes from 0.01 to 5.0 ml;
8-channel automatic pipettes with replaceable tips, allowing you to withdraw 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);
Medical hygroscopic cotton wool;
Filter paper;
Rubber surgical gloves;
A solution with a volume fraction of ethyl alcohol of 70%;
A 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 Before performing the analysis, remove the reagent kit from the refrigerator, open the lid of the box and keep the kit components at temperature (18-25) °C for 30 min.
Mix all serum (plasma) samples and reagents thoroughly before testing.
The consumption of reagents in 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 solution for washing the plate
Attention! Prepare the solution for washing the plate 15 minutes before the start of the analysis!
If a bottle with FSB-T×25 contains sediment, it must be heated before use at a temperature of (37±1) °C until the sediment is completely dissolved. Add the contents of the bottle with FSB-T×25 into a measuring cylinder with a capacity of 2000 ml, then add distilled water to the mark 1250 ml and carefully mix the solution. The solution can be stored at (2-8) °C for 72 hours.
If one or more strips are used, shake the contents of the bottle with FSB-T×25 vigorously for (20-30) s, take the required volume of 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 bottle at a temperature of (2-8) °C during the shelf life of the kit.
6.3 Preparation of immunosorbent
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 a temperature of (2-8) °C for 3 months.
6.4 Preparation of K + AT, K –, RK-1, RR-K2, BRS and stop reagent
K + AT, K – , RK-1, RR-K2, BRS and stop reagent are ready for use.
Attention! Precipitation may form in the bottle with RK-1. A supernatant liquid must be used for analysis.
Unused RK-1, RR-K2, BRS and stop reagent after opening the vials can be stored in closed vials at a temperature of (2-8) °C during the shelf life of the kit.
The remainder of K + AT and K - after opening the vial can be stored in closed vials at a temperature of (2-8) °C during the shelf life of the kit.
6.5 Preparation of K + AG solution
Attention! Prepare the K + AG solution 15 minutes before the start of the analysis!
To restore lyophilized K + AG, before opening the bottle, lightly tap to shake off particles adhering to the walls of the bottle or stopper. Open the bottle and place the stopper upside down on a dry surface. Add 0.8 ml of distilled water to the bottle. Close the bottle with a stopper, hold for 10 minutes at a temperature of (18-25) °C and carefully tilt and rotate the bottle, mix its contents until completely dissolved, avoiding the formation of foam.
Reduced K + AG can be stored in a closed bottle at a temperature of (2-8) °C for one month, at a temperature of minus 20 °C for six months. Single freezing-thawing of reduced K + AG is allowed.
6.6 Preparation of Kg-2 solution in working dilution
From the bottle with Kg-2, take the volume indicated in Table A.1 and transfer it to the bottle with PP-K2. Mix the contents of the bottle thoroughly, avoiding the formation of foam.
If you are using one or more strips, take the required amount of PP-K2 into a clean bottle, add Kg-2 in accordance with Table A.1 and mix the solution, avoiding the formation of foam.
Attention! The Kg-2 solution in working dilution is prepared immediately before use! The Kg-2 solution in working dilution can be stored for 15 minutes at a temperature of (18-25) °C. Use only a new reagent bath and new tips!
The remainder of Kg-2 can be stored in a closed vial at a temperature of (2-8) °C during the shelf life of the kit.
6.7 Preparation of working substrate solution
The bottle with TMB chromogen must be warmed up before use at a temperature of (37±1)° Until the crystals are completely dissolved.
From the bottle with TMB chromogen, take the volume indicated in Table A.1 and transfer it to the bottle with BRS. Mix the contents of the bottle thoroughly, avoiding the formation of foam.
If one or more strips are used, take the required amount of BRS into a clean bottle, add TMB chromogen in accordance with Table A.1 and mix the solution, avoiding the formation of foam.
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 in a place protected from light.
The solution must be protected from light and contact with metals or metal ions. The substrate solution should be colorless before use. Dishes that will come into contact with the substrate solution during the reaction must be washed without the use of synthetic detergents. Use only a new reagent bath and new tips!
The remainder of the TMB chromogen can be stored in a closed vial at (2-8) °C until the expiration date of the kit.
7 Requirements for washing the plate
At all stages of washing, it is necessary to control the filling of all wells and the complete removal (aspiration) of liquid from them;
During each wash, it is necessary to fill all wells with the solution to the brim (0.30-0.35 ml per well), without overflowing or leaking liquid from adjacent wells;
It is necessary to keep the wells filled with a solution for washing the plate for 30 s;
During each aspiration, carefully remove residual liquid from the wells by tapping the frame with strips in an inverted position on 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 Pour into any two wells of the plate 0.07 ml(70 µl) K + AT, in the other two holes - each 0.07 ml(70 µl) K + AG, in the other three holes - each 0.07 ml(70 µl) TO - .
Attention! When performing ELISA on one strip, it is allowed to use two wells for K – -, one well for K + AT, and one well for K + AG.
Pour into the remaining wells of the plate 0.07 ml(70 µl) of the studied samples of human blood serum (plasma).
Attention! Each sample must be collected with a disposable tip!
8.2 In all wells of the plate on top of control samples and test samples of blood serum (plasma) straightaway deposit 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!(1-2) minutes before the end of incubation, prepare a solution of Kg-2 in a working dilution (section 6.6).
8.4 Remove the contents of the wells using a washer, then wash the wells of the plate with a solution for washing the plate (section 6.2) seven times.
8.5 Pour into all wells of the plate 0.15 ml(150 µl) solution Kg-2 in working dilution (clause 6.6).
8.6 Cover the plate with film or close with a lid and incubate in a thermostat at a temperature (37 ± 1) ° From within 10 min.
8.7 Remove the contents of the plate wells using a washer, then wash the plate wells with a plate washing solution (section 6.2) seven times.
8.8 Pour into all wells of the plate 0.15 ml(150 µl) substrate working solution(clause 6.7).
When preparing a working solution of the substrate (clause 6.7) f The lacquer with TMB chromogen must be warmed up before use for (3-5) minutes at a temperature of (37± 1) ° C until the crystals are completely dissolved.
8.9 Cover the plate with film or close with a lid and incubate in a thermostat at a temperature (37 ± 1) ° WITH in a place protected from light 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 serum sample being tested.
8.10 Stop the peroxidase reaction by adding 0.05 ml(50 µl) stop reagent.
Attention! 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 blue to yellow of varying intensity.
8.11 No later than (1-2) minutes after stopping the reaction, determine the OD in the wells in single-wave mode at the wavelength 450 nm.
9 Processing of analysis results
9.2 The results are taken into account only if:
The value of OPsr K - does not exceed 0.2 OE;
Each individual value of OP K - should not deviate from OPsr K - by more than 30%. If one of the three values of OP K - goes beyond this limit, it should be excluded from the calculation of OPsr K - . If two of the three OD K - values are outside this limit, the analysis should be repeated using the reagents of a new set;
The value of OPsr K + AT is more than 1.0 OE;
The value of OPsr K + AG is more than 1.0 OU.
9.3 If the above conditions are met, calculate the critical value (OPcrit.), OE, using formula (1):
OPcrit. = OPsr K - + 0,14 (1).
10 Analytical and diagnostic characteristics
Sensitivity set of reagents ELISA-HIV 1.2 AGAT for detection of HIV-1 p24 antigen –
Specificity set of reagents ELISA-HIV 1.2 AGAT– 100% AT(-)HIV standard. Standard panel of sera that do not contain antibodies to human immunodeficiency virus types 1 and 2 (HIV-1,2) and HIV-1 (p24) antigen. OCO42-28-214-02P.CatNo.INS-20. CJSC "MBS"
11 Set release form
11.1 The set is available in five configuration options:
1 set of 1P – manual analysis. The kit is designed to carry out 12 ELISA setups on a collapsible plate: 1 run – 1 strip (8 wells). Total – 96 determinations, including control samples;
2 set 2M – manual analysis. The kit is designed to carry out 2 ELISA tests on monolithic plates: 1 test – 1 plate. Total – 192 determinations, including control samples;
3 set 2P– carrying out analysis manually. The set is designed to carry out 24 productions ELISA for 2 dismountable x tablets: 1 setting – 1 strip (8 wells). Total - 192 definitions, including control samples;
4 set A2M – analysis on an automatic analyzer. The kit is designed to carry out 2 ELISA tests on monolithic plates: 1 test – 1 plate. Total – 192 determinations, including control samples;
5 set A2P – analysis on an automatic analyzer. The kit is designed to carry out 24 ELISA setups on 2 collapsible plates: 1 run – 1 strip (8 wells). A total of 192 determinations, including control samples.
12 Conditions for storage and use of the kit
12.1 The kit should be stored in a clean room, protected from moisture and light, at a temperature of (2-8) ° C throughout 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 Expiration date of the set – 12 months.
Appendix A
Table A.1 - Consumption of reagents for the ELISA kit
|
reagent, ml |
Number of strips used, pcs. |
|||||||||||
|
Preparing a solution for washing the tablet |
||||||||||||
|
FSB-T×25 |
||||||||||||
|
Distilled water |
||||||||||||
|
Preparation of Kg-2 solution in working dilution |
||||||||||||
|
RR-K2 |
||||||||||||
|
Preparation of working substrate solution |
||||||||||||
|
Chromogen TMB |
||||||||||||
Bibliography
|
Sanitary rules |
Order of the Ministry of Health of the Republic of Belarus dated December 16, 1998 No. 351 On the revision of departmental regulations regulating issues related to HIV/AIDS |
|
|
Order of the Ministry of Health of the Republic of Belarus dated November 25, 2002 No. 165 On disinfection and sterilization by healthcare institutions |
||
|
Resolution of the Ministry of Health of the Republic of Belarus dated April 28, 2010 No. 47 On approval of the Instruction on the procedure for carrying out mandatory medical examinations of workers and the recognition as invalid of certain resolutions of the Ministry of Health of the Republic of Belarus |
||
Owners of patent RU 2515051:
The invention relates to the field of biotechnology and medicine. An enzyme-linked immunosorbent test system for determining the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in human blood serum (plasma). The test system includes an immunosorbent based on human immunodeficiency virus type 1 antigens (HIV-1 and HIV-1 group O env), a sample dilution solution (SRS) and detection reagents (conjugates, chromogen/substrate). The invention also relates to a method for determining the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in human blood serum (plasma) by examining the patient’s blood serum using the described enzyme-linked immunosorbent test system. The invention makes it possible to quickly and easily determine the likely timing of HIV infection. 2 n.p. files, 4 tables, 2 pr.
The INVENTION relates to the field of biotechnology and medicine. And it can be used to determine the likely timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in human serum (plasma).
The spread of HIV infection remains a real problem in the modern world. Since timely and high-quality diagnosis is a real means of reducing the spread of HIV infection, the development of an effective test system capable of determining the probable duration of infection is an important task of modern society.
An analysis of the state of the art has shown the existence of a number of methods and tests proposed by different authors to determine the stages of HIV infection using various methods. For example, a molecular genetic test is known, based on determining the viral load of peripheral blood using the polymerase chain reaction (PCR) method (Kravchenko A.V., Serebrovskaya L.V., Golokhvostova E.L. et al. Timazid in combination with chivid and invirase in complex therapy of patients with HIV infection - Epidemiology and infectious diseases - 1998. - No. 5 - p.51-52). There is a known method for assessing the stages of HIV infection (Clinical Immunology and Allergology, edited by G. Lawlor Jr. - M.: Praktika, 2000. - P. 585-587), which consists in determining the level of serum β 2 -microglobulin. A method for diagnosing the stages of HIV infection is described (V.V. Pokrovsky et al. “Clinical diagnosis and treatment of HIV infection.” - M.: GOU VUNMC RF, 2001. - P. 11), which consists in determining the absolute number of CD4 + lymphocytes peripheral blood. There is also a known method for diagnosing the stages of HIV infection, based on the fact that when examining a patient’s blood serum using an enzyme-linked immunosorbent assay (ELISA), the optical density (OD) of the original blood serum sample is determined, reflecting the level of HIV-specific antibodies, and the OD of the original serum sample incubated for 10 minutes with 100 μl of 3.5 M sodium isothiocyanate solution. When the OP decreases by 40% or less, stage A is diagnosed - generalized lymphadenopathy; when the OP decreases in the range of 40-60% - stage B - bacillary angiomatosis, oropharyngeal candidiasis, etc., and when the OP decreases by 60% or more - stage C - AIDS (RU 2251701).
Thus, the analysis of the state of the art showed that none of the existing sources can serve as the closest analogue to the claimed invention, since all of these documents relate to methods and tests that determine the stage of HIV infection, and none of the described sources contains technical decisions aimed at determining the likely time interval of infection. The claimed invention relates to a test system that determines the probable duration of HIV infection. Analysis of the level of technology did not reveal similar solutions aimed at solving the problem.
The present invention claims a set of reagents “DS-ELISA-HIV-AT-SROC” for determining the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in human blood serum (plasma). The test is intended for additional testing of HIV-positive samples with a confirmed positive result in the immunoblot. It is possible to study samples with an indeterminate or negative immunoblot test result, in which the presence of HIV RNA and/or HIV-1 p24 antigen is confirmed.
The present invention also claims a method for using this set of reagents “DS-ELISA-HIV-AT-SROC” to determine the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in serum (plasma ) human blood.
The technical result of the claimed invention is the determination of the likely timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, the ease of performing the test, which allows the use of the claimed invention for epidemiological surveillance of the dynamics of the spread of HIV infection, the dynamics identification of seroconverters in a certain population (sex, age, social groups of the population), as well as during epidemiological investigation of cases and foci of HIV infection.
The essence of the proposed technical solution is that the determination of the probable period of HIV-1 infection is carried out using a test system, which includes an immunosorbent based on human immunodeficiency virus type 1 antigens (HIV-1 env and HIV-1 group O), a dilution solution samples (SRS) and detection reagents (conjugates, chromogen/substrate). The test samples are tested native and diluted in a sample dilution solution. Based on the testing results, samples are divided into “early” and “late”. “Early” samples - the probable period of infection is up to 9 months from the moment of infection, “late” samples - the probable period of infection is 9 or more months from the moment of infection. Whether a sample belongs to “early” or “late” is assessed by the percentage drop in optical density (OD) of the diluted sample relative to the undiluted one.
These examples serve to illustrate specific embodiments of the invention and are not intended to limit the rights of the applicant. The scope of the applicant's rights covers all possible variants of the invention, including those not listed in this section.
Recombinant antigens containing immunodominant env regions of HIV-1 and HIV-1 group O are used as immunosorbents. Conjugate-1 is a mixture of recombinant antigens HIV-1 gp41 and HIV-1 group O gp41 conjugated with biotin. At the first stage, the test samples, control samples and their dilutions of 1:101, as well as RRS, are incubated with conjugate-1. Specific antibodies present in the test and control positive samples bind to antigens sorbed in the microplate wells and antigens conjugated with biotin and form stable complexes. Excess samples and conjugate-1 are removed by washing. At the next stage, streptavidin conjugated with horseradish peroxidase (conjugate-2) is added to the wells. Conjugate-2 binds to the antigen-antibody-antigen complex present in the well. Excess conjugate-2 is removed by washing and then chromogen/substrate is added to the wells. In wells with bound conjugate-2, a blue color develops, which changes to yellow when a stop solution is added. The results are recorded using a spectrophotometer.
Recombinant antigens containing the immunodominant env regions of HIV-1 and HIV-1 group O are used as an immunosorbent. One conjugate is used - a mixture of recombinant antigens gp41 HIV-1 and gp41 HIV-1 group O, conjugated with horseradish peroxidase. During the analysis, samples, controls and their dilutions, as well as RRS are incubated with the conjugate in antigen-coated wells of the plate. The resulting stable complex after removal of excess sample and conjugate is revealed by the addition of chromogen/substrate. In this case, a blue color develops, which changes to yellow when the stop solution is added. The results are recorded using a spectrophotometer.
Whether the studied samples belong to “early” or “late” is assessed by the % drop in OD when diluting the sample, which is calculated using the formula:
% drop O P = 100% − O P reconnaissance of sample − O P s r.
R R S O P WHOLE SAMPLE × 100% ,
where OPsr. RRS - average value of OP RRS (calculated based on several values of OP RRS).
The samples under study are regarded as “early”: if the drop in OD is >40%.
The samples under study are regarded as “late”: if the drop in OD is ≤40%.
It is convenient to use Excel to record and process the results. Using this application will allow you to perform calculations automatically. The program for processing test results is located on the disk included with the kit, or it can be found on the company’s website.
When developing and evaluating the test, samples from seroconversion panels (BBI, Inc., Zeptometrix, USA) and HIV-positive blood serum (plasma) samples with established probable timing of infection were used. The probability of correctly determining the duration of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, established by seroconversion panels, is 100% (data are presented in Table 2), based on serum samples from persons with a conditionally established period of infection (n=129) is 94% (Tables 3, 4; data shown partially).
| table 2 | ||
| Results of testing of HIV-positive serum/plasma samples established by seroconversion panels | ||
| Seroconversion panel | Days since first blood draw | Test result in “DS-IFA-HIV-AT-SROC” |
| BBI PRB 914 | 31 | "early" |
| BBI PRB 916 | 35 | "early" |
| BBI PRB 925 | 49 | "early" |
| BBI PRB 929 | 28 | "early" |
| BBI PRB 930 | 10 | "early" |
| BBI PRB 931 | 42 | "early" |
| BBI PRB 934 | 11 | "early" |
| BBI PRB 941 | 25 | "early" |
| BBI PRB 942 | 14 | "early" |
| BBI PRB 944 | 16 | "early" |
| BBI PRB 947 | 20 | "early" |
| BBI PRB 951 | 19 | "early" |
| BBI PRB 952 | 21 | "early" |
| BBI PRB 955 | 14 | "early" |
| BBI PRB 965 | 21 | "early" |
| BBI PRB 966 | 51 | "early" |
| BBI PRB 968 | 35 | "early" |
| BBI PRB 969 | 77 | "early" |
| ZMC HIV 6243 | 33 | "early" |
| ZMC HIV 6247 | 30 | "early" |
| ZMC HIV 9014 | 31 | "early" |
| ZMC HIV 9017 | 35 | "early" |
| ZMC HIV 9018 | 33 | "early" |
| ZMC HIV 9021 | 57 | "early" |
| ZMC HIV 9022 | 29 | "early" |
| ZMC HIV 9032 | 55 | "early" |
| ZMC HIV 9077 | 104 | "early" |
| ZMC HIV 9079 | 95 | "early" |
| ZMC HIV 12008 | 42 | "early" |
The “DS-ELISA-HIV-AT-TERM” test assessed the possibility of studying blood serum (plasma) samples containing antibodies to HIV-1 of various subtypes, including HIV-1 group O: reference panel QCS 42-28-327 -03Р (GISC) (subtypes A, B, C) (except for sample No. 24 containing antibodies to HIV-2), 1st international reference panel anti-HIV (NIBSC Code: 02/210) (subtypes A, B, C, E) (except for sample No. 5, containing antibodies to HIV-2), samples of blood serum (plasma) containing antibodies to HIV-1 group O (No. 1342, K00175, K00259; BioMex). The results indicate the possibility of studying blood serum (plasma) samples containing antibodies to HIV-1 belonging to various subtypes, including HIV-1 group O, in the “DS-ELISA-HIV-AT-SROC” test.
The specificity of the DS-ELISA-HIV-AT-SROC reagent kit was determined by studying donor blood serum samples (n=352), which demonstrated a negative result in the ELISA. Specificity was 100%. Additionally, 255 blood serum samples (negative in ELISA) were tested:
■ 167 blood serum samples from pregnant women and patients with infectious diseases;
■ 128 blood serum samples from patients with various non-communicable diseases.
The specificity was 100%.
The data obtained show the high efficiency of the developed test system “DS-ELISA-HIV-AT-SROC” in determining the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in serum (plasma) human blood.
1. Enzyme-linked immunosorbent test system for determining the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in human blood serum (plasma), characterized by the fact that it includes an immunosorbent based on virus antigens human immunodeficiency type 1 (env HIV-1 and HIV-1 group O), sample dilution solution (SDS) and detection reagents, which are conjugates, chromogen/substrate.
2. A method for determining the probable timing of infection with human immunodeficiency virus type 1 (HIV-1), including HIV-1 group O, in human blood serum (plasma), based on different antibody contents depending on the timing of infection, including separation of the test sample into two parts and diluting one of them; examination of both (native and diluted) parts of the patient’s blood serum sample using the enzyme-linked immunosorbent test system described in paragraph 1; determination of optical density (OD) of native and diluted samples and RRS; calculation of the average value of OP RRS; calculation of the percentage drop in OD of a diluted sample relative to an undiluted one using the formula
% drop O P = 100% − O P reconnaissance of sample − O P s r.
R R S O P WHOLE SAMPLE × 100% ,
in this case, the probable period of infection is regarded as “early” if the drop in OD is >40%, and as “late” if the drop in OD is ≤40%.
Similar patents:
The invention relates to a computer method that uses biochemical databases in the development of new protein compounds. Design is carried out by the operator using a specially written PROTCOM program based on the use of a database of protein pentafragments.
The invention relates to the field of biotechnology and immunology. A monoclonal antibody and antigen-binding portions thereof are provided that specifically bind the C-terminal or central region of macrophage migration inhibitory factor (MIF).
The invention relates to the field of molecular biology and medical diagnostics. A method for detecting proteins in the amyloid state is proposed, in which a sample of lysate of a yeast culture or mammalian tissue is obtained, an ionic detergent is added to the sample, the proteins in the amyloid form are concentrated on a cellulose acetate membrane and they are detected using aptamers, their conjugates or antibodies specific to the amyloid form of proteins .
The invention relates to biotechnology, in particular to enzyme-linked immunosorbent assays, and is a method for obtaining incorporated control samples for enzyme-linked immunosorbent test systems for determining infections of the TORCH group.
The invention relates to medicine, namely to gynecology, and can be used for individual selection of drugs containing probiotic strains of lactobacilli for effective intravaginal therapy.
The group of inventions relates to a sensor-analyzer reagent composition adapted to assist in determining the concentration of an analyte in a liquid sample, to methods for determining the concentration of an analyte in a liquid sample, and to a method of applying the analyzer sensor reagent composition to a substrate by screen printing.
The group of inventions relates to veterinary medicine and concerns adjuvant compositions containing a combination of Quil A or its purified fraction, cholesterol, dimethyldioctadecylammonium bromide (DDA), polyacrylic acid (CARBOPOL) and CpG and/or N-(2-deoxy-2-L-leucylamino- β-D-glucopyranosyl)-N-octadecyldodecanamide acetate; a method for preparing such adjuvant compositions and the use of such adjuvant compositions in immunogenic and vaccine compositions with various antigens.
The present invention relates to the field of biotechnology and medicine. A method is proposed for creating an antibody and its functional fragments directed against a tumor antigen expressed on the surface of a tumor resistant to at least one antitumor compound, based on the use for immunization of a crushed homogenate and/or suspension and/or cell lysate originating from such tumors. Also discussed is the use of the method according to the invention for the production of monoclonal antibodies and their functional fragments, monoclonal antibodies obtained by this method, the nucleic acids encoding them, an expression vector, a host cell and a method for producing antibodies using them, as well as hybridomas capable of secreting these antibodies, the use of antibodies and their functional fragments for the manufacture of drugs, antitumor composition and its use as a drug. This invention may find further application in the treatment of resistant tumors. 16 n. and 26 salary f-ly, 7 ill., 4 pr., 6 tab.
The invention relates to the field of biotechnology and medicine. An enzyme-linked immunosorbent test system for determining the probable timing of infection with human immunodeficiency virus type 1, including HIV-1 group O, in human blood serum. The test system includes an immunosorbent based on human immunodeficiency virus type 1 antigens, a solution for diluting samples and detection reagents. The invention also relates to a method for determining the probable timing of infection with human immunodeficiency virus type 1, including HIV-1 group O, in human blood serum by examining the patient’s blood serum using the described enzyme-linked immunosorbent test system. The invention makes it possible to quickly and easily determine the likely timing of HIV infection. 2 n.p. files, 4 tables, 2 pr.
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.
Purpose This work was the development of 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 containingHBeAgin the test system "DS-IFA-HBeAg»
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 test system “DS-ELISA-anti-HBeAg»
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 containingHBsAgin the test system “DS-ELISA-anti-HBe».
Fig.2b. Features of distribution of OP anti-HBe- positive serum samples that do not contain HBsAgin the test system "DS-ELISA-anti-HBe».
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 already 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
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 ELISA 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 initial and repeated ELISA results were 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 he is 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, an ELISA will yield a reliable result.
Even with highly accurate studies, there remains 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.
