RU2065164C1 - Diagnostic test-system for hepatitis virus a detection - Google Patents

Abstract

FIELD: medicine, virology, applied immunology. SUBSTANCE: method involves immunoenzyme assay on solid carrier using peroxidase conjugate and magnosorbents sensibilized with antihepatitis (A) immunoglobulin. Peroxidase conjugate is designed on the basis of immunoglobulins to hepatitis A virus. Method is used for diagnostic of hepatitis A virus. EFFECT: improved method of diagnosis.

Description

 The invention relates to medical virology, in particular to applied immunology.

Known enzyme-linked immunosorbent assay in the increasing titer of I ₉ M-anti-HAV (antibodies to hepatitis A virus) in the prodromal period of the disease and the initial phase of the heat-up period (EP Shuvalova. Infectious diseases. M. 1990, p. 154).

 The disadvantage of this method is that it is aimed at identifying antibodies and does not allow to identify the causative agent of the disease hepatitis A virus or its antigens.

 Closest to the proposed invention is an enzyme-linked immunosorbent assay for determining antigens of arenavirus infections, which is based on the principle of labeled antibodies. An enzyme, such as horseradish peroxidase, is used as a label. The test result is taken into account by the degree of specific binding of the labeled antibodies to the antigen fixed on a solid support. The degree of binding of labeled antibodies is determined by enzyme activity - the destruction of the enzyme substrate that gives the stained product (Guidelines for the use of solid-phase immunosorbent methods (enzyme-labeled and radioimmunological analysis) for laboratory diagnosis of arena virus infections, Crimean hemorrhagic fever and hemorrhagic fever with 1982 renal syndrome. p. 21).

 The disadvantage of this method is the inability to detect the presence of the virus with its discrete entry and low concentration, selectively concentrate viruses directly from the secretions of patients or other contaminated samples and reduce the time for enzyme-linked immunosorbent assay (ELISA) from 4 to 1 h.

 The detection of pathogenic microorganisms in environmental objects is associated with great difficulties. One of the most specific methods for the concentration of microorganisms is the use of magnesorbents. These drugs and devices have proven themselves in identifying the causative agent of cholera from various environmental objects. However, to date, effective methods have not been developed for determining the hepatitis A virus from environmental objects. The use of magnetic sorbents and other devices will help to develop effective methods of environmental monitoring for the presence of the causative agent of viral hepatitis A. The use of granular magnetic sorbents as a solid phase during enzyme-linked immunosorbent assay to identify the causative agent of viral hepatitis A from environmental objects will increase the sensitivity of the analysis, significantly reduce the complexity and analysis time, improve the quality characteristics of the definitions.

 Thus, neither the use of magnetosorbents that are sensitized by antihepatitis (A) immunoglobulins as a solid carrier, nor the use of immunoglobulin-based conjugates of hepatitis A virus immunoglobulins, nor the detection of hepatitis A virus by the enzyme immunoassay using magnetoimmunosorbents in virology at an existing level . Based on this, we can conclude that the invention meets the criterion of inventive step.

 The essence of the invention lies in the fact that the detection of hepatitis A virus is carried out by conducting ELISA on a solid carrier using a peroxidase conjugate.

 Distinctive features of the claimed object: an immunological reaction on a solid carrier is carried out using magnesorbents that are sensitized by antihepatitis (A) immunoglobulins and placed in the test medium; the construction of the immunoperoxidase conjugate is carried out on the basis of immunoglobulins to hepatitis A.

 When the virus is discrete, and the low concentration does not allow to detect it by known methods, the emerging need to selectively concentrate the virus is achieved using a combination of features included in the claims.

 The diagnostic test system is as follows.

 1. Get serum from people with viral hepatitis A, with high titers of specific antibodies.

, конъюгированными с пероксидазой хрена. Конъюгат вносят в каждую лунку в объеме 0,05 мл и инкубируют в течение 2 ч при 37^oС. Затем трижды промывают по 3 мин. 0,05% -ным раствором твин-20 в ФСБ и вносят по 0,1 мл субстрат-индикаторного раствора (0,01 М лимонная кислота с 0,2 М Na₂HPO₄ рН 5,0 в присутствии 0,006% -ной перекиси водорода и ортофенилендиамина в концентрации 0,4 мг/мл), инкубируют 15 мин при комнатной температуре (20-22)^oС в темноте. Для остановки реакции вносят 0,05 мл 2 М серной кислоты. В лунках, где присутствуют специфические антитела, раствор приобретает коричневое окрашивание. Контроль конъюгата и контроли специфичности остаются бесцветными. Высокотитражные сыворотки (1:600000-1:800000) используют для выделения специфических иммуноглобулинов.In patients with viral hepatitis A in a hospital, blood is taken from a vein in the last days of the icteric period or in the first days of the convalescence period. Sera obtained from blood inactivate at 56 ^o C for 30 minutes To determine the titers of antibodies using the three-phase enzyme-linked immunosorbent assay (TIMP). The polystyrene microplate wells are sensitized with rabbit anti-human immunoglobulins G (IgG) at a concentration of 10 μg in 0.1 ml of 0.05 M carbonate-bicarbonate buffer (KBB) pH 9.5. Incubated for 18 h at 4 ^o C. Unsorbed IgG is removed, 0.05 ml of 0.5% bovine serum albumin (BSA) is added to the microplate wells, incubated for 30 min at 37 ^o C. Then BSA is removed and the test serum in a volume of 0.1 ml, starting with a dilution of 1: 400, and titrated in 0.1 ml of 0.1 M phosphate-buffered saline (FSB) pH 7.4 to the 11th hole. The last well without serum (conjugate control). To control the specificity of the diagnosticum, sera obtained from patients with brucellosis, salmonellosis or other infectious diseases, and normal human serum are used. After the addition of the sera, the plate was incubated for 1 h at 37 ^° C. Then the contents of the wells were removed, the wells were washed with a 0.05% solution of tween-20 in PBS. The presence of specific antibodies in the studied sera is determined using a commercial diagnosticum of the Soviet-Swiss company Diaplus: hepatitis A virus antigen (from cell culture) associated with monoclonal antibodies to hepatitis virus

conjugated to horseradish peroxidase. The conjugate is added to each well in a volume of 0.05 ml and incubated for 2 hours at 37 ^° C. Then it is washed three times for 3 minutes. 0.05% solution of tween-20 in the FSB and add 0.1 ml of substrate-indicator solution (0.01 M citric acid with 0.2 M Na ₂ HPO ₄ pH 5.0 in the presence of 0.006% peroxide hydrogen and orthophenylenediamine at a concentration of 0.4 mg / ml), incubated for 15 min at room temperature (20-22) ^o C in the dark. To stop the reaction make 0.05 ml of 2 M sulfuric acid. In wells where specific antibodies are present, the solution becomes brown. Conjugate control and specificity controls remain colorless. High-volume sera (1: 600000-1: 800000) are used to isolate specific immunoglobulins.

 2. Obtaining specific antihepatitis (A) immunoglobulins.

Immunoglobulins are isolated using double salting out with ammonium sulfate. An equal volume of distilled water is added to the serum, then a weighed portion of ammonium sulfate to 40% saturation of the solution is added slowly while stirring the solution on a magnetic stirrer for 30 minutes. It is then left at 4 ^{° C.} for 18 hours to stabilize the protein and form a precipitate. Centrifuged for 30 minutes at 6,000 rpm. The supernatant is removed and the precipitate resuspended in distilled water to a double volume of the original serum. Ammonium sulfate was weighed to a solution up to 40% saturation, incubated for 30 min on a magnetic stirrer at room temperature, and then 18 hours at 4 ^° C. It was centrifuged by the above method, the supernatant was removed, and the precipitate was resuspended in distilled water to the original volume taken in serum work.

The resulting immunoglobulin solution is dialyzed against tap and then distilled water until NH ions disappear. $\genfrac{}{}{0ex}{}{\text{+}}{\text{4}}$ , which is checked using Nessler's reagent.

 The amount of protein is determined spectrophotometrically and the activity of immunoglobulins in TIMP. The protein content during the isolation of immunoglobulins in this way is 25-30 mg / ml, and the activity in TIMP is 1: 600000-1: 800000.

 The obtained immunoglobulins are used to obtain immunoperoxidase conjugate and hepatitis (A) magneto-immunosorbent.

 3. Obtaining immunoperoxidase hepatitis (A) conjugate.

 Hepatitis Immunoglobulins (A) labeled with horseradish peroxidase are obtained by periodically oxidizing horseradish peroxidase.

 For conjugation of immunoglobulin with an enzyme, horseradish peroxidase type VI, RZ-3.0 of the Scientific and Research Institute "Biochemical Reactor" of the All-Russian Research Institute of Applied Biochemistry (Olaine) is used, which is dissolved in an amount of 5 mg in 1 ml of a freshly prepared solution of 0.3 M sodium bicarbonate.

Adding 0.25 ml of a 0.32% formalin solution was carefully mixed on a magnetic stirrer for 30 minutes. Then add 1 ml of 0.04 M sodium periodate, carefully mix on a magnetic stirrer for another 30 minutes. The color of the solution takes on a greenish yellow hue. 1 ml of 0.16 M ethylene glycol is introduced into the solution, stirred for 1 hour. All of the above operations are carried out at room temperature. The solution is transferred into a dialysis bag, dialyzed against 1 liter of 0.01 M carbonate-bicarbonate buffer (KBB), pH 9.5 at 4 ^° C for 18 hours. The solution is transferred to a vial, hepatitic immunoglobulins (A) are added at a concentration of 10 mg / ml in 1 ml of KBB, stirred for 2 hours at room temperature. Add 5 mg of sodium borohydride, leave without stirring for 2 h at 4 ^o C. Then the solution is dialyzed against 1 l of 0.1 M phosphate-buffered saline (PBS) pH 7.4 for 18 h at 4 ^o C. From a dialysis bag the solution is applied to a pre-prepared column (1.5 cm • 90 cm) with Sephadex G-100, balanced with 0.1 M FSB pH 7.4. Elute with the same buffer at 4 ^{° C.} , collecting 3 ml fractions. Each fraction is photometric at a wavelength of 280 nm and 403 nm. The extinction ratio of 403 nm / 280 nm (RZ) should be at least 0.5 (in this case, one immunoglobulin molecule is associated with approximately one peroxidase molecule). In a fraction with an RZ of about 0.5 (peak fractions), 10 mg per ml of bovine serum albumin (BSA) is added. The conjugate is poured into ampoules, lyophilized and stored at 4 ^o C.

 4. Obtaining hepatitis (A) magneto-immunosorbent.

 Magnoimmunosorbent (MIS) is obtained by covalent cross-linking of immunoglobulin to hepatitis A virus on the surface of granules.

Magnosorbent is washed from the preservative with distilled water / 3-4 times for 1 min /. 1 g of magnetosorbent is poured into 10 ml of an 8% solution of glutaraldehyde, incubated for 18 hours at room temperature (20-22) ^o With shaking. Then washed with glutaraldehyde in ten volumes of distilled water and 3-4 volumes of 0.1 M phosphate-buffered saline (FSB) pH 7.6. The activated magnetosorbent is conjugated with 10 ml of antihepatitis immunoglobulins in FSB pH 7.6 with a protein concentration of 500-600 μg / ml. Conjugation is carried out for 18 hours at room temperature with shaking. Magnoimmunosorbent is washed with three volumes of 0.1 M FSB pH 7.6. Pour 10 ml of a 1% solution of bovine serum albumin (BSA), jutter for 3 hours at room temperature. Wash with three volumes of 0.1 FSB pH 7.6, add sodium merthiolate to a final concentration of 1: 10000 and store the drug at 4 ^o C. Shelf life of the drug 6 months (observation period).

 5. An analysis aimed at identifying hepatitis A.

 Enzyme-linked immunosorbent assay using magnetic immunosorbet (MIS).

To carry out an enzyme-linked immunosorbent assay aimed at identifying hepatitis A virus or its antigens, the microplate is filled with reagents in accordance with the following reaction scheme: 5 mg MIS (or 50 μl of 10% suspension) are placed in the wells of the first row and the washing solution is phosphate Salt buffer (PBS), pH 7.2, 200 μl each; in the wells of the second row — analyzed samples of 100 μl containing antigens, in addition to the last well of the row, which is used as a control and filled with 100 μl of FSB; in the wells of the third row, 200 μl of FSB for washing; in the wells of the fourth row, 100 μl of the working dilution of the immunoperoxidase conjugate; in wells of the fifth, sixth and seventh rows of 200 μl of FSB for washing; in wells of the eighth row, 100 μl of a substrate-indicator solution (0.1 M citric acid with 0.2 M Na ₂ HPO ₄ pH 5.0 in the presence of 0.006% hydrogen peroxide and orthophenylenediamine at a concentration of 0.4 mg / ml) .

The prepared tablet is installed in the frame of the device for IVF using MIS and carry out operations in the following sequence:
washing MIS from the preservative in the wells of the first row 30 s with stirring;
transfer using a metal comb MIS in the holes of the second row. Incubation with samples for 20 minutes with stirring five times for 30 seconds;
the transfer of MIS in the holes of the third row. Rinse with stirring for 30 s;
the transfer of MIS in the holes of the fourth row. Incubation with immunoperoxidase conjugate for 20 minutes with stirring five times for 30 seconds;
transfer of MIS in the wells of the fifth, sixth and seventh rows with stirring in each row for 30 s for washing;
transfer of MIS into the wells of the eighth row for reaction with a substrate-indicator solution, incubation for 3-5 minutes with stirring 30 s.

 After the development of a color reaction, the MIS is removed from the wells of the eighth row. The reaction is stopped by adding 100 ml of 2 M sulfuric acid to the wells. The result of the reaction is evaluated visually compared with the control. Samples are considered positive, the staining of which exceeds the similar control indicator by 70-80%. The analysis lasts 45-50 minutes.

 Example.

 Detection of hepatitis A virus or its antigens in environmental objects.

 To detect hepatitis A virus or its antigens in the water supply network, sewage or open water bodies, magnetic traps were used, in which hepatitis (A) magneto-immunosorbent (MIS) was placed and left for a day at the research object: sewage from the Stavropol Infectious Diseases Hospital, water supply network , treatment facilities in the city of Essentuki, the sewer drainage of the resort area in the city of Pyatigorsk and other facilities. During this time, there was a selective concentration of hepatitis A virus or its antigens on the surface of hepatitis IIAs. Then the traps were removed, the MIS was washed several times in physiological saline, and then an enzyme-linked immunosorbent assay using magnetic immunosorbents was carried out. This version of the analysis excludes the moment the sample is connected to the MIS on the microplate, since this process occurs while the magnetic immunosorbent is on a magnetic trap. The analysis itself lasts 25-30 minutes.

 As a result of the studies, it was found that using the proposed test system, the hepatitis A virus was detected in the sewer of the infectious diseases hospital in Stavropol, in the sewage of the resort area of Pyatigorsk, the sewer of the infectious diseases hospital in Essentuki and other facilities.

Claims (1)

 Diagnostic test system for detecting hepatitis A virus by conducting an enzyme-linked immunosorbent assay on a solid support using a peroxidase conjugate, characterized in that the immunological reaction on a solid support is carried out using magnesorbents that sensitize with antihepatitis (A) immunoglobulins and place them in the test medium, and the design of immunoperoxidase conjugate is carried out on the basis of immunoglobulins to hepatitis A.