RU2339694C2 - Diagnostic test system for revealing of virus of bird flu a/h5n1 - Google Patents

Abstract

FIELD: medicine, microbiology.

SUBSTANCE: invention concerns area of biotechnology and medical virology. The diagnostic test system for revealing of a virus of bird flu A/H5N1 is offered at enzyme immunoassay carrying out on a solidphase carrier with use of peroxidase conjugate. As a solidphase carrier the activated aluminosilicate matrix with a magnetic material with immobilized immunoglobulins against a virus of bird flu is used. The invention can be used for diagnostics of a virus of bird flu A/H5N1.

EFFECT: obtaining of diagnostic test system for revealing if virus of bird flu A/H5N1 at enzyme immunoassay carrying out on solidphase carrier with use of peroxidase conjugate.

Description

The invention relates to biotechnology, medical virology and can be used to design diagnostic test systems.

Known hemagglutination inhibition reaction (RTGA) and reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of avian influenza and detection of its causative agent in biological and field material (Lvov D.K., Shchelkanov M.Yu., Deryabin P.G. et al. Isolation of A / H5N1 influenza virus strains from poultry and wild birds during epizootics in Western Siberia (July 2005) and their deposition into the State Virus Collection (August 6, 2005) // Issues of Virology - 2006. - No. 1 . - S.11-14; Lvov D.K., Prilipov A.G., Shchelkanov M.Yu. and others. Molecular genetic analysis of biologists eskih properties of influenza virus highly pathogenic A / H5N1, isolated from wild and domestic birds during the epizootic in western Siberia (July 2005) // Problems of Virology -. 2006. - №2 -. S.16-19). An immunofluorescence method for the detection of influenza A viruses of various subtypes is also known (Methodological recommendations "Quick diagnosis of influenza and other acute respiratory viral infections by the immunofluorescence method", M., 2006).

The disadvantage of these methods is that difficulties arise when processing samples from environmental objects (soil, water, etc.), since in this case it is necessary to get rid of impurities and to concentrate the desired antigen of the bird flu virus.

A known method for detecting the Crimean hemorrhagic fever virus (CGF) in biological and field material (RF Patent No. 2276362. "Method for detecting the Crimean hemorrhagic fever virus (CGF) in biological and field material", authors Vasilchenko NF, Efremenko VI , Tyumentsova I.S. et al., - G01N 33/551, G01N 33/569, publ. 05/10/2006. Bull. No. 13).

In this invention, antibodies against the CHF virus are used as raw materials and, after selective concentration, RT-PCR and ELISA are performed.

Closest to the alleged invention is a method for identifying hepatitis A virus in samples using enzyme-linked immunosorbent assay (ELISA) (RF Patent No. 2065164 "Diagnostic test system for the detection of hepatitis A virus", the authors Vasilchenko NF, Efremenko V.I., Gnutova I.N.- G01N 33/53, published on 08/10/1996, Bull. No. 22).

The disadvantage of this invention is the use of polyacrylamide gel as a solid phase in the manufacture of magnetic immunosorbents. To obtain a polyacrylamide gel, imported expensive highly toxic reagents are used.

One of the most important conditions for the successful use of ELISA in the diagnosis of infectious diseases is the availability of effective methods for preparing samples tested in this reaction. The most promising for this purpose seems to be the use of selective cell concentration using magnetic immunosorbents (MIS) (Efremenko V.I., Tyumentseva I.S., Vasilenko N.F. et al. Development of a production line in the methods of immunoassay of microorganisms: Final report on research, Inventory No. 02.9.70001181. - Stavropol, 1996. - 96 p.).

Thus, neither the use of aluminosilicate magnesorbents as a solid carrier on which class G immunoglobulins against avian influenza virus are immobilized, nor the detection of the virus by the enzyme immunoassay using MIS in virology at the current level of research is known to us. Based on this, we can conclude that the invention meets the criterion of inventive step.

The technical result of the invention lies in the fact that the rapid analysis of the detection of avian influenza A / H5N1 virus is carried out using a diagnostic test system consisting of a set of basic drugs and ingredients: magneto-immunosorbents with immobilized antibodies against avian influenza virus, immunoperexidase conjugate against influenza virus birds, dry weighed phosphate-buffered saline, bovine serum albumin, citrate buffer, orthophenylenediamine, tween-20, hydroperite and stop reagent (4 N solution sulfuric acid). The main component of the test system is the aluminosilicate magneto-immunosorbent, on which class G immunoglobulins against avian influenza A / H5N1 are immobilized. After selective concentration of the studied material on it, the sensitivity and specificity of the detection of pathogenic viruses at their low concentration in the samples increases, the analysis is simplified.

Distinctive features of the claimed object: an immunological reaction on a solid support is carried out using aluminosilicate magnesorbents on which class G immunoglobulins against avian influenza virus A / H5N1 are immobilized; virus detection is carried out by ELISA with immunoperoxidase conjugate.

When a small amount of the virus in environmental objects (litter, bird droppings, solid wastes that are suspended in the liquid) 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 detection of avian influenza virus A / H5N1 is carried out in stages as follows.

1. Preparation of avian influenza A / H5N1 antigen

The transplanted culture of the kidney cells of a pig embryo (SPEV) is grown using a roller method until a complete monolayer of cells is formed (within 2-3 days). Cell cultures are grown in 2 L roller bottles on medium 199 with the addition of 10% serum of cattle and antibiotics (100 IU / ml of penicillin and streptomycin). Before infection, the culture fluid is drained, washed with a monolayer of cells three times with Hanks solution, after which a virus-containing material is added in a volume of 20 ml at a dose of 0.1 TCD ₅₀ / cell. The virus is adsorbed onto cells within 1 hour of rotation on a roller unit at a temperature of 37 ° C. The non-adsorbed virus is then removed by washing the cell monolayer with Hanks solution, and a support medium consisting of 199 medium with antibiotics is added to each roller bottle. 24 hours after infection of the cells, distinct phenomena of virus-induced cell destruction occur. At this point, the culture fluid is drained, which is then clarified by centrifugation at 2000 rpm for 30 minutes. At the end of centrifugation, the supernatant liquid is drained and inactivated by ultraviolet rays in an ultraviolet installation of Electronic Medicine company (Moscow) as follows: 30 ml of virus-containing culture fluid in open Petri dishes exposed to UV rays for 4 min (the inactivation method and exposure time worked out we have previously and allow 100% inactivation of the infectious properties of the influenza A / H5N1 virus while maintaining the antigenic activity of the viral material).

Thus inactivated material is subjected to concentration by ultracentrifugation at 30,000 rpm for 2 hours in a centrifuge company "Vesktap", USA. The precipitate obtained is resuspended in a TNE solution, homogenized in a Downs homogenizer, the antigenic activity is checked in the hemagglutination reaction with chicken erythrocytes or human group 0 red blood cells, then it is poured into 1.5 ml tubes and frozen at 60 ° C for long-term storage.

The activity of the antigen of influenza A / H5N1 virus according to the hemagglutination reaction (RGA) is 1: 20,000.

2. Obtaining immune ascitic fluid (IAG) to the antigen of the virus A / H5N1

To obtain an IAI for influenza A / H5N1 antigens, white outbred mice weighing 10-20 g are used, which are administered intraperitoneally once a week with Freund's complete adjuvant in the ratio 1: 1 inactivated A / H5N1 influenza antigen. Immunization of animals is carried out for 4 weeks. At the 5th week, animals are bled, IAG is extracted, and in the reaction of inhibition of hemagglutination (RTGA) they are checked for the presence of antiviral antibodies.

The activity of IAH according to RTGA - 1: 640.

3. Obtaining specific class G immunoglobulins (IgG)

The method of isolation of immunoglobulins with caprylic acid is as follows: 64 ml of a 0.06 M acetate buffer solution and 2.8 ml of caprylic acid are added to 40 ml of ascitic acid, and they are intensively mixed on a magnetic stirrer, incubating for 30 min. It is centrifuged at 6000 g for 40 minutes, filtered, the precipitate is removed, and the supernatant is IgG. The pH was adjusted to 7.2 and placed on dialysis against a 0.1 M phosphate-buffered saline solution. The protein concentration after dialysis is 5-7 mg / ml, therefore, concentration is carried out by placing dialysis bags with immunoglobulins in polyethylene glycol (PEG-6000). The amount of protein is determined spectrophotometrically. The protein content during isolation in this way is 20-30 mg / ml.

The obtained immunoglobulins are used for the preparation of an immunoperoxidase conjugate and a magnetic immunosorbent.

4. Obtaining immunoperoxidase conjugate for detection of avian influenza virus

The immunoperoxidase conjugate is obtained by the periodate oxidation method according to P.K. Nakane, A. Kawaoi (1974) in our modification as follows.

For conjugation of IgG with the enzyme, horseradish peroxidase type VI, RZ-3 (Sigma, USA) 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.

0.25 ml of a 0.32% formalin solution is added, carefully mixed on a magnetic stirrer for 30 minutes. Then add 1 ml of 0.04 M sodium periodate, mix gently on a magnetic stirrer for another 30 minutes. The color of the solution acquires a greenish-yellow hue. 1 ml of 0.16 M ethylene glycol is added to the solution, stirred for 1 hour. All of the above operations are carried out at room temperature. The solution is 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, 5 mg / ml anti-avian influenza virus IgG is added in 1 ml KBB , stirred for 2 hours at room temperature. Add 5 mg of sodium borohydride, leave without stirring for 2 hours at 4 ° C. Then dialyzed against 1 l of 0.1 M phosphate-buffered saline (PBS) pH 7.4 for 18 hours at 4 ° C. After that, bovine serum albumin (BSA) is added at the rate of 10 mg per 1 ml. The conjugate is poured into ampoules, lyophilized and stored at 4 ° C.

5. Obtaining aluminosilicate magnimosorbent

1.5 g of aluminosilicate and 3 g of magnetic powder are mixed, thoroughly triturated in a mortar. 30 ml of polyglucin (6% dextran) and 30 ml of distilled water are mixed. Then, the solution is added in portions to the resulting sample, mixing thoroughly. Incubated for 2 hours at room temperature or 18 hours at 4 ° C. The resulting sorbent is dried at a temperature of 100-110 ° C for 30 minutes, crushed, and fractions with a particle size of 80-120 microns are isolated by sieving through a sieve. Next, the sorbent is activated with sodium secondary alkyl sulfate. To do this, add 3 ml of PBS pH 7.2-7.4 and 0.2 ml of secondary sodium alkyl sulphate to 0.4 g of magnetosorbent. Incubated for 1 h at a temperature of 37 ° C. Then, IgG is immobilized against avian influenza virus on a magnetosorbent in a volume of 2 ml and a protein concentration of 2 mg / ml. Incubated for 2 hours at a temperature of 37 ° C. Washed with five volumes of distilled water and five volumes of FSB, holding the MIS with a permanent magnet.

Based on the above developments, a diagnostic test system for selective concentration and detection of the A / H5N1 avian influenza virus virus was assembled in an enzyme-linked immunosorbent assay (ELISA), consisting of a set of basic drugs and ingredients: magnetic immunosorbents with immobilized antibodies against avian influenza virus, positive control of avian influenza virus antigen , conjugate of immunoperoxidase against avian influenza virus, immunoglobulins fluorescent against avian influenza virus, dry weighed phosphate-saline buffer, bovine cheese orotochnogo albumin, citrate buffer, orthophenylenediamine, Tween-20, stop solution (4 N sulfuric acid solution) gidroperita.

6. Enzyme-linked immunosorbent assay (ELISA) for the detection of avian influenza virus A / H5N1

Laboratory experiments are carried out with an antigen of avian influenza virus with various concentrations, from 1000 μg / ml (1 mg / ml) to 10 ng / ml. In penicillin vials (with a capacity of 10 ml), 0.2 ml of a suspension of an appropriate concentration of antigen and 0.1 ml of a 10% suspension of a magnetic immunosorbent are added, a negative control is a bottle with 0.2 ml of FSB and 0.1 ml of a 10% suspension of MIS. Incubated for 30-60 minutes at room temperature, then washed thoroughly and 200 μl of working dilution of the immunoperoxidase conjugate were added to detect the avian influenza antigen, incubated for 20 minutes at 37 ° C. Wash with 0.1 M phosphate-buffered saline with tween-20 at least 6 times using a permanent magnet. Then, 200 μl of the chromogenic mixture are added to the bottles. After 1-2 minutes (when the supernatant begins to turn slightly yellow in the negative control), the contents of the vials are transferred to microplates and the reaction is stopped with 50 μl of stop agent (2M sulfuric acid solution). The results are taken into account on a Multiscan recording device at a wavelength of 492 nm. Samples are considered positive if their optical density (OD) is 2 or more times higher than the OD of the negative control. In the absence of a recording device, the results are recorded visually, while samples are considered positive, the color of which is significantly (50%) different from the control color.

An enzyme-linked immunosorbent assay is also carried out in model experiments on water contaminated with various concentrations of avian influenza antigen suspended in 500-3000 ml of tap water. Suspensions are passed through a device with magnetic immunosorbent. After contact with the sample, the magnetic immunosorbent is washed thoroughly, passing a ten-fold (relative to the sample suspension) volume of phosphate-saline buffer. MIS is removed from the "trap", placed in a bottle and conduct ELISA according to the method described above. A negative control is the magnetic immunosorbents that are not in contact with the antigen.

In both experiments, positive results were obtained in the presence of 10 ng / ml or more in the sample volume.

To conduct a comparative analysis of the modified ELISA proposed by us with other traditional methods of setting up this analysis, depending on the solid phase, ELISA was carried out with the same components, only polystyrene plates sensitized by immunoglobulins against avian influenza virus for 18 hours were used as solid phases, then analysis in accordance with the methodology of M.Clark and A. Adams in the sandwich variant of ELISA. The sensitivity of ELISA in this case was 100 ng / ml. The time of setting ELISA using MIS was 50-60 minutes, and the traditional method, taking into account the 18-hour sensitization, was 20-21 hours.

Thus, when using MIS in ELISA, there is no need to use polystyrene tablets, the time of sensitization of the solid phase is reduced by 15 times, the shelf life of the sensitized solid phase is significantly increased; the time of direct analysis is reduced by 1.5 times, and sensitivity is increased by several orders of magnitude compared with conventional ELISA due to the selective concentration of a specific antigen on the surface of the IIA.

All this can contribute to operational epidemiological analysis and the formation of targeted medical and veterinary measures aimed at the prevention of avian influenza.

Claims (1)

Diagnostic test system for detecting A / H5N1 avian influenza virus during enzyme-linked immunosorbent assay (ELISA), on a solid-phase carrier using a peroxidase conjugate, characterized in that an activated aluminosilicate with magnetic material matrix with immobilized immunoglobulins against avian influenza is used as a solid-phase carrier .