The reagent for diagnosing infections caused by puumala virus |
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IPC classes for russian patent The reagent for diagnosing infections caused by puumala virus (RU 2218571):
 
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The invention relates to the diagnosis of the virus. Offers a reagent for the detection of Puumala virus infection through contact with the serum of a subject infected with Puumala virus, and observation of agglutination to determine whether serum antibodies against Puumala virus, with the specified reagent contains sensitized insoluble carrier particles, in which the antigens of the virus Puumala related to their external surface in the amount of 0.005 to 2 wt.%. The reagent is a simple and effective diagnostic tool Puumala virus. 8 C.p. f-crystals, 1 table.
The scope of the invention This invention relates to a diagnostic reagent for detection of viral infection. More specifically it relates to a diagnostic reagent for serological detection of infection caused by Puumala virus, which has a higher specificity and sensitivity, allowing you to accurately diagnose hemorrhagic fever with renal syndrome caused by Puumala virus.
Description of the prior art Hantaviruses (Hantavirus) are serologically related members of the family Bunyaviridae. The prototype Hantavirus , Isolation of the Etiologic Agent of Korean Hemorrhagic Fever," J. Infect. Dis. 137:298-307 (1978)). When the virus is transferred from mice to man, there is an acute disease known as hemorrhagic fever with renal (kidney) syndrome (HFRS, Korean hemorrhagic fever). Virus Seoul and Puumala virus, discovered later, are also members of the genus Hantavirus. Epidemic hemorrhagic fever, found in Asian countries, is caused by Hantaan virus or Seoul, while HFRS found in European countries, is caused by Puumala virus. The Puumala virus is transmitted through autochthonous European media-rodents, such as the Bank vole (Clethrionomys glareolus). Recently discovered Hantavirus pulmonary syndrome (HPS) in the United States, which is accompanied by high mortality. HPS is caused by a virus Sin Nombre the same phenotype as the Hantaan virus, but antigenically distinct from him.
The main clinical features of epidemic hemorrhagic fever, found in Asia and is caused by Hantaan virus or virus Seoul are fever, hemorrhage (in the digestive tract and subconjunctival) syndrome and renal failure. HFRS, found in Europe, is also characterized by fever, less severe hemorrhage and C is ndrama and pulmonary edema, leading to death, approximately weeks after the onset of the disease.
There are large antigenic and genetic differences between natural Hantavirus (hantavirus), so the vaccine is required for almost every individual. In addition, the high cross-reactivity between different species of the genus hantavirus difficult when diagnosing clearly distinguish one Hantavirus infection from others.
Infection caused by Puumala virus found in European countries and, in particular, are reported every year thousands of cases in the countries of Northern Europe. Because of its clinical symptoms are less severe than in the case of other Hantavirus infections, respiratory distress syndrome and the syndrome of renal failure complicates the difference of this infection from other cases of respiratory failure or acute nephritis. Therefore, we need a new, accurate, rapid and simple method for the serological diagnosis of virus Puumala.
The INVENTION is Therefore the purpose of this invention is to provide a simple, quick and effective diagnostic reagent for the detection of infections caused by Puumala virus.
Dunn is a water-insoluble particles of the carrier and the antigen of the virus Puumala, located on their outer surface.
Further, the present invention provides a reagent for the detection of infection with Puumala virus; the reagent is brought into contact with the patient's serum, presumably infected with Puumala virus, and observe agglutination to determine whether serum antibodies against Puumala virus, with the specified reagent is a water-insoluble sensitized carrier particles on the external surface of which are antigens of Puumala virus in the amount of from 0.005 to 2 wt%.
DETAILED description of the INVENTION For the purposes of this invention, the net Puumala virus antigen derived from tissue culture or animal origin, associated with a water-insoluble carrier particles, the result is sensitized carrier particles.
Net Puumala virus antigen can be obtained from various sources, such as organs, particularly the brain of animals or tissue cultures infected with a virus. In one typical embodiment of the invention the cells of the Vero E6 inoculant with Puumala virus ATCC VR-984 and cultivated properly, and infected cells are lysed. The virus antigen is isolated and purified from the lysate for ispolzoyovanija, where as the source of virus antigen using an appropriate animal, in the brain of a newborn hamster (e.g., age less than one day) is administered by injection Puumala virus ATCC VR-984, then multiply infected hamsters, dezintegrarea his brain, and separating and purifying the viral antigen ultracentrifugation. Viral antigen used for sensitization water-insoluble carrier particles with the purpose of preparation of diagnostic reagent according to this invention.
The virus antigen is subjected to the action of rays and chemically modified to increase its reactivity or sensitivity. For example, processing approximately 0.05% (vol/vol) formalin solution at a low temperature, for example at 4oWith in a few days, for example within 2 weeks, with the aim to modify the protein antigen. The thus treated antigen associated with the outer surface of the insoluble carrier particles, such as HDP (particles with high density) or erythrocytes, receiving the sensitized carrier particles. Diagnostic reagent according to this invention is an aqueous suspension containing the sensitized carrier particles.
Insoluble in water dasticity animals. Particles of inorganic compounds may include, but are not limited to oxides of metals of the III, IV or VI group of the Periodic system of elements, such as silicon dioxide, aluminum oxide, titanium dioxide, zirconium dioxide, sesquioxide iron (F2O3), iron oxide (F3O4), cobalt oxide or Nickel oxide; hydroxides such as aluminum hydroxide, iron hydroxide (Fe(OH)3) or chromium hydroxide; halides such as silver chloride or silver bromide; sulfides such as cadmium sulfide; carbonates such as calcium carbonate or magnesium carbonate, or sulfates, such as barium sulfate or strontium sulfate. One of them is preferably used silicon dioxide, aluminum oxide, titanium dioxides, zirconium, or their mixed oxides.
Inorganic compounds in the form of particles may also include inorganic oxides, silicon dioxide and a metal oxide selected from the group consisting of metals of the II, III and IV groups of the Periodic system, in which the metal oxide can form a complex with silicon dioxide. Typical examples of inorganic oxides include lithium oxide, sodium oxide, potassium oxide, magnesium oxide, calcium oxide, strontium oxide, barium oxide, aluminum oxide, oxide UTSA.
For the purposes of this invention can also be applied particulate inorganic compounds coated pigments, if the pigments do not render harmful influence on the structure of inorganic compounds. Connections can be covered by one or more layers of pigments.
Particles of inorganic compounds can have an average diameter in the range from 0.1 to 10 microns and preferably from 0.8 to 5.0 microns and a density in the range of from 1.5 to 4.0 and preferably from 1.8 to 2.5.
Preferably can be applied multilayer coating particles of inorganic complexes. Complex inorganic particles can be obtained by coating particles of inorganic compounds, for example particles of inorganic compounds based on silicon dioxide, the layer of pigment and, if necessary, an additional layer of the above-mentioned complex oxides. Then, the resulting particles are treated with a binder, such as binder based on silicon and titanium, forming a reactive (reactive) surface. Thus obtained particles are usually referred to as "particles with high density (HDP), and their average diameter is from 0.1 microns to 10.0 microns and a density (specific gravity) from 1.5 to 4.0.
For the purposes of this izobreteny" in this context indicates the percentage of the number of individual deagglomerating particles from the total number of particles, suspended in the aqueous environment. Dispersibility can be measured with an ordinary counter, for example Coltercounter Model ZD-1.
The carrier particles may have different shapes depending on the crystal structure of inorganic compounds and the method of their derivation. They usually have the shape of a polyhedral, cylindrical, conical and spherical. Preference is given to the use of spherical particles, especially spherical shape. These particles can be obtained by the known methods described, for example, in JP 52-138094A or JP 61-149644A.
It is possible to use particles of media impregnated known dyes. The dyes may include, but are not limited to, cationic dyes, such as malachite green, rhodamine b or methylene blue; disperse dyes such as dianixTM(Mitsubishi Chemical Co., Ltd., Japan), DibazolTM(I. C. I., UK, U. K.) micropolyesterTM(Mitsui Toatsu K. K., Japan); direct dyes such as Congo red, direct deep black E W or chrysophanic G; alizarin-safrole; acid dyes such as alizarin direct blue or alizarin-cerenomy green G; acid dyes such as black diamond F (diamond black (F), chromfast In navyblue (chrome naval (BASF, Germany), eisen opal (Horoya Kagakusa, Japan) or oreosol (Taoka Kagakusa, Japan); active dyes, for example diamira, michishiron (Mitsubishi Chemical Co., Ltd., Japan) or sumifix (Sumitomo Chemical Co., Ltd. , Japan); fluorescent brightening dyes, for example mikiwhite (Mitsubishi Chemical Co., Ltd., Japan) or whitux (Sumitomo Chemical Co., Ltd., Japan). Preferred are metal-containing dyes and cationic dyes, and most preferred are cationic dyes.
Apply the dye having a solubility of 1 weight part or more, more specifically 5 weight parts, most preferably 10 weight parts in 100 parts of methanol.
The amount of dye in the particles of the medium may be from 0.5 wt.% up to 8 wt.%, and preferably from 1.0 wt.% to 5.0 wt.%, but is not limited to this. Taken in the above interval dyes facilitate observation of the agglutination antigen-antibody to detect infection with Puumala virus, and in this case prevents the outflow of the dye from the particles of the inorganic carrier.
Hematite (blood cells) can be used as a water-insoluble carrier particles for antibody detection of Puumala virus in this invention. The source or type of blood cells is not so limited and may viberates lower sensitivity, than HDP, they can mostly be used as a water-insoluble carrier particles for the purposes of this invention.
The particles of the medium with increased thanks to the antibody of Puumala virus sensitivity can be obtained by bringing into contact with the virus antigen, obtained as described above, with the particles of the medium to bind the antigen with the outer surface of the particles. The method of binding antigen with a particle is not specifically limited, but you can apply the usual methods. For example, the particles come in contact with the antigen in an aqueous medium, such as saline solution or saline solution with phosphate buffer (PBS), when mixing the antigen with suspended particles in the aquatic environment.
The ratio of antigen to the carrier particles is in the range from 100 to 400 agglutinating units HDP and preferably from 150 to 250 agglutinating units HDP 1 g of particles. The contacting is carried out at pH 6.0-8.0 and at a temperature of 4oWith up to 20oC. Used herein, the term "agglutinating unit HDP" means the lowest amount of antigen required to detect agglutination HDP-serum.
After stirring unbound antigen is removed by laundering and the solution is further thermostatic with the Xia of the active sites of the external surface of the particles.
Diagnostic reagent according to this invention can be obtained by suspending sensitized particles in an aqueous solution to a concentration of from 0.005 to 2 wt.% and preferably from 0.05 to 1 wt.%.
Sensitized carrier particles can be modified with the aim to prolong the shelf life, although the particles themselves are sustainable, and can be used simply suspending particles of the modified media in an appropriate aqueous medium, such as distilled water, immediately before use.
Diagnostic reagent according to this invention is a reactive (reactive) with respect to the serum sample of the patient, preferably infected or prone to infection with Puumala virus that causes agglutination with a high degree of specificity and sensitivity.
Examples the invention will be described in more detail by using examples that do not limit the scope of the present invention. In particular, water-insoluble particles of the carrier receive, covering a core of silicon dioxide with a mixture of dye and silicon dioxide, and then treat the surface of a silicon binder in one variant embodiment of the method according to this invention, which is not the Dol is kamo Puumala virus ATCC VR-984 obtained from the American collection of cell cultures (MD, USA) and used as source of antigen Puumala virus.
The Puumala virus ATCC VR-984 subcultured cells Vero E6 (ATSS CRL 1586) up to 10 passages and for use as blanks virus collect clones, forming the largest and most transparent sterile spot. Getting viral antigen carried out using cells Vero E6 or newborn hamster aged less than one day as host cells. Preferably use a cell Vero E6.
Getting viral antigen using cells Vero E6 or newborn hamster explained below.
The cultivation of the virus in the cells of the Vero E6 Cells Vero E6 tissue culture inoculant with 0.1 ml of virus Puumala, obtained as above, and cultured in an incubator in an atmosphere of CO2at 37oC for about 10 days. Infected cells treated with 0.1% Triton-X and collect. The collected cells destroy ultrasound and centrifuged at 4oWith getting the supernatant containing the viral particles. The supernatant is treated with gamma rays (3000 rad) for the complete inactivation of the virus. An inactivated virus inoculant cells Vero E6 and analyzed by indirect immuno-fluorescence analysis of antibody (IFA) to confirm the inactivation of viral fluid, containing viral antigen, which is diluted PBS and filtered through an ultra-fine filter, receiving the purified antigen of the virus Puumala.
Thus obtained solution of Puumala virus antigen treated with 0.05% (vol/vol) formalin at 4oWith in 2 weeks for modification of the surface protein antigen with the purpose of increasing sensitivity. The antigen is subjected to dialysis against saline solution and used as the antigenic component of the diagnostic reagent according to this invention.
The cultivation of a virus-infected newborn hamster Injection of Puumala virus (0.01 ml) is injected into the brain of a newborn hamster (aged no more than a day). After 20 days, when paralyzed more than half of the animal, the animal brain removed aseptically and homogenized. To obtain emulsion (10-20% vol.) the homogenate add saline solution. The emulsion thus obtained from the brain, centrifuged at 600 rpm, 4oC for 30 minutes and the supernatant liquid add a 10-fold volume of ethanol, and then gently shaken at low temperature and dried in vacuum.
To the dried substance of the brain add saline solution to the original (brain) volume and 20% (objemnocti 60 minutes, getting the supernatant containing viral antigen, free from the basic protein of myelin. The supernatant was diluted with PBS and filtered through super-thin (ultra) filter, receiving the purified antigen of the virus Puumala.
Thus obtained solution of Puumala virus antigen treated with 0.05% (vol) formalin at 4oC for 2 weeks in order to modify the protein surface antigen to increase the sensitivity. The antigen is subjected to dialysis against saline solution and used as the antigenic component of the diagnostic reagent according to this invention.
The title thus obtained solution of the antigen detected by ELISA method so that a diagnostic reagent containing the antigen, could have a permanent title. The titer of the antigen solution obtained in (1) and (2) the case is 5-10 agglutinating units HDP/ml and 120 agglutinating units HDP/ml, respectively.
Example 2. Obtaining particles with a high density In a glass flask with a stirrer pour 2800 ml of methanol, 616 ml of an aqueous solution of ammonia (25 wt.%) and 21 ml of an aqueous solution of sodium hydroxide (5M). At 10oTo the solution dropwise with a speed of 25.5 ml/hour add 256 ml (22 wt.%) tetraethylsilane in Meath is and in methanol, and 625 ml (1.25 wt.%) methylene blue in methanol simultaneously added dropwise with a speed of 25.5 ml/hour, you get a colored silica particles. The silica particles re-suspended and decanted to clean and wash.
Thus obtained two-layer particles of silica/dye have an average diameter of about 1.57 MK. The carrier particles suspended in methanol to a concentration of 0.5 wt.%, and to the resulting suspension add phenyltriethoxysilane to a concentration of 0.5 wt.%. The reaction is carried out for 16 hours at 10oWith and as a result, the silica particles surface modified.
Example 3. Getting HDP film-coated, containing the antigen of the virus Puumala To a solution of Puumala virus antigen obtained in example 1, add M/60 and PBS (pH 7.2) to dilute the solution to 2 agglutinating units HDP/ml of the Resulting solution of antigen (5 ml) is mixed with 0.5% HDP/PBS (5 ml) and carry out sensitization with gentle shaking for 60 minutes. The mixture is then washed three times by PBS and suspended at a dilution (5 ml) inactivated rabbit serum in PBS with 56oC for 30 minutes to a concentration of 1%, getting sensitized solution HDP. Example 4. Determination of the titer of antibodies using HDPA or IFA The titer of serum antibodies of patients HFR the initial analysis). Every 0.025 ml cultivation of example 3 are placed in the wells of V-shaped microtiter plate and the first hole add 0.025 ml of the test serum (dilution 1:10). The test sample is diluted serially (sequentially), and added dropwise sensitized solution HDP (0.025 ml) of example 3, and mix well. Leave at room temperature for 40 minutes, observe agglutination in each breed and the maximum dilution at which there is agglutination antigen-antibody, defined as the titer of antibodies HDP". This way, the titer of antibodies HDP determine for 43 subjects serum samples. In addition, carry out the appropriate IFA (indirect immunofluorescence assay antibody) in respect of 43 subjects serum samples, and the maximum dilution at which there is a fluorescent complex antigen-antibody, defined as the titer of antibodies IFA". For comparison, determine the sensitivity and specificity of IFA and HDPA against Hantavirus infection with diagnostic reagent disclosed in KR 90-16761A containing HDP sensitized virus Hantaan. The results are shown in the table. As can be seen from the table, the results of IFA and HDPA show that patients in Korea, I had ala. IFA and HDPA not differ significantly in sensitivity and specificity. Diagnostic reagent according to this invention makes possible a simple test to detect differences between infection with the virus Puumala and Hantaan virus with the highest available relevant IFA degree of sensitivity. Consequently, it is possible to diagnose the early stage of hemorrhagic fever with renal syndrome (HFRS) caused by Puumala virus, and to distinguish it from related syndromes, such as acute respiratory distress syndrome or syndrome renal failure, which are difficult to distinguish from HFRS in clinical trials. Example 5. Getting erythrocytes sensitized with the antigen of the virus Puumala To PBS containing 2.5% inactivated sheep erythrocytes, add an equivalent amount of a solution of tannic acid/PBS 1:100000 and the mixture is left to react at 20oWith 20 minutes. The reaction mixture was washed with PBS (a mixture of 1/60 M saline phosphate buffer (pH 7.2) and saline in a volume ratio 1:3) once, and then suspended in PBS, receive a 0.5% suspension containing the complex tannin-blood cell (gemalt). Suspension tannin-hematit mixed with 80-fold dilution of antigenemia within 60 minutes for the implementation of sensitization. The mixture is then washed three times by PBS and suspended in inactivated rabbit serum (1%) in PBS to achieve a concentration of 0.5%, get sensitized with a solution of red blood cells. Example 6. Determination of the titer of antibodies with the use of sensitized solution of red blood cells In wells V-shaped microplate is placed in 0.025 ml dilution of inactivated rabbit serum (example 3) and in the first hole add 0.025 ml serum (dilution 1:10; serum N 11 in the table). The test sample is diluted in series, are added dropwise sensitized solution of erythrocytes (0.025 ml) in example 3. After 3 hours at room temperature is observed agglutination. As a result, the antibody titer is equal to 40. Although preferred embodiments of the method according to this invention described above, it should be clearly understood that many variations and/or modifications of the basic concepts proposed in the invention that will be obvious to the experts, meet the spirit and are included in the scope of the present invention, as reflected in the claims.
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