KR0160122B1 - Reagents for diagnosing haemorrhagic fever with renal syndrome - Google Patents

Abstract

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Description

Diagnostic reagents for renal hemorrhagic fever

The present invention relates to a reagent for diagnosing renal syndrome hemorrhagic fever, and more particularly, the present invention relates to a reagent for diagnosing renal syndrome hemorrhagic fever which can be usefully used for clinical examination. Hanta virus is a virus belonging to the Bunyaviridae family isolated from the lungs of Korean rats in 1978 (HW Lee et al ,; J. Infect. Dis., 137-). 298, 1978). The virus infects humans and becomes a hospital for renal syndrome hemorrhagic fever (hereinafter referred to as HFRS). HFRS has long been believed to have existed as endemic to northern Eurasia, but it occurred a lot in the Japanese army stationed in northeastern China around 138. I see it. Currently, this virus is known to be distributed over a wide range of the earth.

Although HFRS may have clinically typical symptoms of hemorrhagic fever, it is often associated with atypical clinical forms and is often difficult to diagnose based on clinical findings alone.

Conventionally, as a diagnostic method, HFRS employs a serum diagnostic method, such as a fluorescent antibody method and an enzyme antibody method.

However, although both methods are satisfactory in both specificity and sensitivity, they are not easy to apply to clinical examination because they are complicated to operate, require a lot of effort, and require special equipment and examination time.

Therefore, there has been a demand for the development of a technique for diagnosing whether Hanta virus is infected simply and quickly.

However, due to insufficient purification of the Hanta virus antigen, even if there is specificity as a diagnostic method, a diagnostic method better than the above is not yet developed due to insufficient sensitivity or vice versa.

The present inventors have been engaged in the development of various diagnostic drugs and diagnostic methods for a long time, and have been studying the development of diagnostic methods for infection with hanta virus. As a result, by infecting the mouse virus with the mouse or rat brain, a high titer antigen is obtained from it, and an aqueous suspension of sensitized insoluble particles purified from the Hanta virus antigen and sensitized to specific insoluble inorganic compound particles. As a result, it was found that the sensitivity was good and the diagnosis of renal syndrome hemorrhagic fever was possible quickly.

Accordingly, an object of the present invention is to provide a reagent for diagnosing renal syndrome hemorrhagic fever which can diagnose whether or not infected with Hanta virus easily and quickly without using a special device.

Another object of the present invention is to provide a reagent consisting of an aqueous suspension containing sensitizing insoluble particles in which Hanta virus antigens are sensitized to insoluble carrier particles.

Still other objects of the present invention will become apparent from the following description.

The reagent for use in diagnosing renal syndrome hemorrhagic fever provided by the present invention is an aqueous suspension containing sensitized insoluble particles in which the Hanta virus antigen is sensitized to insoluble carrier particles in a range of 0.005 to 2% by weight.

The hantavirus antigen used in the present invention may be prepared from an infected animal organ, an infected cultured cell, or the like, but is preferably prepared from cells of an infected mammalian mouse or rat, particularly brain cells, from the viewpoint of yield and stability. In particular, it is preferable to prepare from the cerebral cell of a rat. The preparation of the Hantavirus antigen is not particularly limited and can be prepared in basically the same manner as the method for preparing other antigens using the above-mentioned animal, especially a small mammal.

As a representative example of the method that can be most preferably employed, a method of preparing from brain cells such as a mouse or a rat is as follows.

For example, by inoculating Hanta virus into the brain of a mammalian mouse or rat, the supernatant is obtained by centrifuging the tissue emulsion obtained from the infected brain tissue at low temperature, which is treated with ethyl alcohol and protamine sulfate, The supernatant is again centrifuged at high speed, inactivated with formalin, and the formalin is dialyzed and removed to obtain a hanta virus antigen.

As said inorganic compound particle, well-known thing can be used suitably, For example, group III, group IV of periodic table, such as a silica, alumina, titania, zirconia, ferric oxide, iron trioxide, cobalt oxide, nickel oxide, etc. Oxides of metals of group VI; Hydroxides such as aluminum hydroxide, ferric hydroxide and chromium hydroxide; Halides such as silver bromide and silver chloride; Sulfides such as cadmium sulfide; Carbonates such as calcium carbonate and magnesium carbonate; Sulfates, such as barium sulfate and strontium sulfate, etc. are mentioned.

Among these, preferable is silica, alumina, titania, zirconia, or a composite oxide containing these as main components.

As the inorganic compound particles, at least one metal oxide selected from the group consisting of Groups I, II, III, and IV of the periodic table capable of bonding with silica and a composite oxide composed mainly of silica are used. It may be. Specific examples of the metal oxide include lithium oxide, sodium oxide, potassium oxide, magnesium oxide, calcium oxide, strontium oxide, barium oxide, aluminum oxide, titanium oxide, zirconium oxide, germanium oxide, hafnium oxide, tin oxide, and oxide oxide. Zinc and the like.

Moreover, the structure of the inorganic compound particle used for this invention is not restrict | limited, The particle | grains used as an electrical material may be sufficient, The particle | grain is made into the nucleus, The nucleus is 1 or 2 or more layers, for example, the dye mentioned later The insoluble inorganic compound particle which coat | covered the pigment layer to contain or coat | covered the pigment layer with silica or the composite oxide which has a silica as a main component may be sufficient.

The average particle diameter of the inorganic compound particles is 0.1-10.0 탆, preferably 0.8-5.0 탆. The specific gravity of the inorganic compound particles is usually 1.5-4.0, preferably 1.8 to 2.5.

Moreover, especially preferable among such inorganic compound particles is an electric material, for example, composite oxide which uses silica as a main component as a nucleus, and coat | covers the nucleus with the pigment layer containing the dye mentioned later, as needed. Inorganic composite particles having a reactor on the surface thereof by coating with an electric composite oxide and treating the surface with a silane coupling agent, a titanium coupling agent, etc., having an average particle diameter of 0.1 to 10.0 μm and specific gravity So-called high specific gravity composite particles (hereinafter referred to as HD) of 1.5-4.0.

Furthermore, in the present invention, the particle dispersibility of the electro-inorganic compound particles is preferably 80% or more, preferably 90% or more. The particle dispersibility referred to in the present invention refers to the ratio (%) of the number of non-aggregated particles, ie, the number of single particles, in all particles, and can be usually measured by Model ZD-1 manufactured by Coulter Counter. The shape of the inorganic compound particles differs depending on the crystal structure and production method of the inorganic compound to be used, and there are a polyhedron, a main body, a sheep body, a sphere, and the like, all of which are used. Although possible, preferably a sphere, particularly preferably a spherical body is preferred. Such an inorganic compound particle can be obtained by a well-known manufacturing method, for example, the method described in Unexamined-Japanese-Patent No. 52-138094, Unexamined-Japanese-Patent No. 61-149644, etc.

Such insoluble inorganic compound particles can also contain dyes.

The electric dye is not particularly limited, and known dyes can be used, and examples thereof include cationic drinks such as malachite green, rhodamine B, and methylene blue; Disperse dyes such as Dianics (registered trademark of Mitsubishi Mars), Disbazole (I. C., Isa registered trademark), and Myketone Polyester (registered trademark of Mitsui Toatsu Chemical Co., Ltd.); Direct dyes such as Congo red, Direct Deep Black E W and Chrysophenin G; Alizarin sharpol B; Acid dyes such as Alizarin Direct Blue A and Alizarin Cyanine Green G; Acid mordant dyes such as diamond black F, chrome fast navy blue B, palatin fast blue BN, and the like; Metal-containing dyes such as ashdol (registered trademark of BASF Corporation), Eisen Opal (registered trademark of Horoya Chemical Industries, Ltd.), oreosol (registered trademark of Taoka Chemical); Reactive dyes such as Diamira, Mikashiron (registered trademark of Mitsubishi Kasei), and Sumipix (registered trademark of Sumitomo Chemical Co., Ltd.); Fluorescent whitening dyes such as Mica White (registered trademark of Mitsubishi Mars) and Whiteux (registered trademark of Sumitomo Chemical).

Among these, preferred are metal-containing dyes and cationic dyes, and more preferably cationic dyes.

The solubility in 100 parts by weight of methanol in these dyes is preferably 1 part by weight or more, preferably 5 parts by weight or more, particularly preferably 10 parts by weight or more. The amount of the dye in the electrically insoluble inorganic compound particles is not particularly limited, but is usually 0.5-8% by weight, preferably 1.0-5.0% by weight.

By setting the amount of the dye in such a range, it is possible to easily determine the electrocoagulation reaction and to prevent the elution of the dye.

Moreover, red blood cells can be used as an insoluble inorganic compound particle used by this invention. The red blood cells are not particularly limited and have been used for a long time as antigen-supporting carriers in antigen-antibody reactions. In the present invention, such a known red blood cell can be used as it is.

As the insoluble inorganic compound particles described above, in consideration of effects such as sensitivity and rapidity as reagents, inorganic compound particles having an average particle diameter of 0.1-10.0 µm and specific gravity of 1.5-4.0 are used as nuclei, and the surface of the nucleus is dyed. Or composite particles having a structure coated with a mixture of a dye and an inorganic compound, particularly, having a powder dispersibility of 80% or more.

Although it tends to be slightly inferior in sensitivity to the composite particles, red blood cells may also be a desirable insoluble inorganic compound particle.

The sensitizing insoluble particle which concerns on this invention can be obtained by sensitizing the said one-virus virus antigen to the particle | grains which are electrically insoluble. The method of sensitizing a hanta virus antigen to the insoluble inorganic compound particle is not specifically limited, It can employ | adopt for a well-known method. For example, it is preferable to contact the insoluble inorganic compound particles with the hanta virus antigen in an aqueous medium (for example, saline solution, PBS, etc.), and usually, an aqueous medium suspension of the insoluble inorganic compound particles and the hanta virus antigen are mixed, Sensitize by shaking.

As for the usage-amount of the hantavirus antigen at the time of sensitizing an insoluble inorganic compound particle, generally, 100 g of HDP aggregation units can be used normally with respect to 1 g of insoluble inorganic compound particles. Preferably in the range of 100-400 HDP flocculating units, in particular in 150-250 HDP flocculating units.

In the present invention, the HDP aggregation unit refers to the minimum concentration required for HDP aggregation. It is preferable to perform said sensitization at pH 6.0-8.0 and 4 degreeC-20 degreeC.

After the sensitization, washing with an aqueous medium removes the unsensitized Hanta virus antigen, and again saturates the remaining surface of the particles with a substance adsorbed to insoluble carrier particles, such as bovine serum albumin.

The diagnostic reagent of the present invention can be obtained by suspending the electrosensitized insoluble particles, for example, in an aqueous solution.

The content of the sensitizing insoluble particles contained in the reagent may be selected in the range of 0.005-2% by weight, preferably 0.05-1% by weight.

Moreover, although the sensitizing insoluble particle which concerns on this invention is stable, it can be preserve | saved longer by lyophilizing this. The freeze-dried product can be used in exactly the same way as a fresh product simply by adding pure water and dissolving it in use.

Since the diagnostic reagent of the present invention is aggregated by the Hanta virus antigen, the body fluid or dilution thereof, such as human or animal serum, and the diagnostic reagent of the present invention are brought into contact with each other on a plate for microtiter, and the sensitized insoluble particles of the sensitized insoluble particles are observed. By doing so, the presence of a hanta virus antigen can be determined.

Therefore, the determination of the presence of the hantavirus antibody does not require the use of a special device, and it is possible to diagnose whether or not the hanta virus is infected simply and quickly. Moreover, since there is no antibody to insoluble carrier particles and only the presence of antibodies of sensitizing insoluble particles is determined, it is not necessary to pretreat serum or the like as a sample. According to the proposal of the present invention, it is difficult to diagnose only conventional clinical findings, and furthermore, it is possible to easily diagnose the presence or absence of Hanta virus infection which could not be generally diagnosed because it uses a special device and requires the utmost effort by complicated operation. It became possible.

Therefore, the present invention is evaluated not only in the normal clinic but also in enabling accurate treatment by knowing the presence or absence of infection before the onset of disease.

Hereinafter, the present invention will be described in more detail with reference to Examples. Incidentally, the present invention is not limited by this embodiment.

Example 1

[Preparation of Hantavirus Antigen]

0.01 ml of 1% emulsion of Hanta virus-infected mammalian rat brain was inoculated into the rat rat brain at 1 day of age, and after 8 days, the brain was extracted and 20% emulsion was prepared with physiological saline.

The emulsion was centrifuged at 600 rpm at 4 ° C. for 30 minutes to obtain a supernatant, 10 volumes of ethyl alcohol was added thereto, stirred at low temperature, and dried under reduced pressure.

Subsequently, it was made up again with physiological saline, and 20% of protamine sulfate was added thereto, the mixture was stirred at a low temperature, centrifuged at 10,000 rpm for 60 minutes, and the supernatant was taken out. 0.05% by volume of formalin was added thereto, and the mixture was left at 4 ° C for 14 days, and then dialyzed with physiological saline to obtain Hantavirus antigen. The titer was 120 HDP aggregation units / ml.

[Production of HDP]

2800 ml of methanol, 616 ml of ammonia water (25 wt%) and 21 ml of aqueous sodium hydroxide solution (5 mol / L) were added to a glass flask with a stirrer and stored at 10 ° C., followed by 256 ml of a methanol solution of tetraethyl silicate (22 wt%). Dropwise at a rate of 25.5 ml / hr, silica particles (average particle diameter: 0.91 mu m) were produced.

To the reaction solution containing the silica particles, 624 ml of tetraethyl silicate (44 wt%) and 625 ml of methylene blue methanol solution (1.25 wt%) were added dropwise at the same time at a rate of 25.5 ml / hr, and this tetraethyl silicate was added. The dropwise addition of the methanol solution of and the methanol solution of the dye was simultaneously terminated to synthesize silica particles colored with the dye. The silica particles obtained were decanted with methanol to repeat purification and washing.

The average particle diameter of the silica / dye composite of the two-layer structure thus obtained was 1.57 µm.

Subsequently, the obtained silica particles were dispersed in methanol so as to have a concentration of 10% by weight, and phenyltriethoxysilane was added to 100 ml of the dispersion so as to have a concentration of 0.5% by weight, and reacted at 10 DEG C for 16 hours for surface treatment. Silica particles were obtained.

[Preparation of sensitization HDP]

The obtained Hantavirus antigen was made into a solution of 2 HDP aggregate unit using PBS of M / 60 and pH 7.2, 5 ml of this antigen solution and 5 ml of 0.5% HDP / PBS were mixed, and sensitized for 60 minutes, stirring at room temperature gradually with a stirrer. It was. After sensitization, the cells were washed three times with PBS and suspended in 5 ml of diluent to obtain sensitized HDP. The dilution is the addition of 1% non-synchronized rabbit serum to PBS.

[Measurement of Antibody Value]

0.025 ml of the dilution was dispensed into the V-type microplate, and 0.025 ml of 1:10 test serum was added to the first well.

After diluting this with a diluent, 0.025 ml of sensitized HDP was added dropwise to each well.

After mixing well, the mixture was allowed to stand at room temperature for 40 minutes to determine the coagulated aggregate phase, and the maximum dilution factor of the test serum showing aggregation was taken as the HDP antibody titer. The HDP antibody titer was calculated | required about 20 serum samples by the above operation.

Display the results in a table.

(-) In the column of the HDP antibody titer of a table | surface shows the case where the HDP antibody of a test serum is 50 or less.

[Comparative Example]

About the test serum 20 sample used for the said HDP method, the maximum dilution factor of detectable test serum was calculated | required as the FA antibody titer by the fluorescent antibody method (FA).

Display the results in a table.

In addition, in the column of FA antibody number of a table | surface, (-) shows the case where FA antibody of a test serum is 16 or less.

Example 2

[Preparation of sensitized red blood cells]

1: 100,000 tannic acid / PBS solution was added to PBs containing 2.5% immobilized sheep erythrocytes in the same amount, treated with tannic acid for 20 minutes at room temperature (20 ° C.), and washed once with PBS. A 0.5% tannin cell suspension was prepared by suspending (except with PBS, a mixture of 1/60 mol of phosphate buffer (pH; 7.2) and 3 physiological saline).

One dose of the prepared tannin cell suspension was mixed with one dose of the 80-fold dilution of the Hantavirus antigen obtained in the same manner as in Example 1, and sensitized for 60 minutes while slowly stirring with a stirrer at room temperature. The particles after sensitization were washed three times with PBS and suspended in dilution to 0.5% to obtain sensitized red blood cells.

The diluting solution is added with 1% non-assisted rabbit serum to PBS.

[Measurement of Antibody Value]

To the V-type microplate, 0.025 ml of the diluted solution was dispensed, and 0.025 ml of 1:10 test serum (the same as that of Example 1 serum 11) was added to the first well.

After diluting this with a diluent, 0.025 ml of sensitized red blood cells were added to each well. The mixture was mixed well and allowed to stand at room temperature for 3 hours, and then the coagulated aggregate phase was determined. As a result, the antibody value was 40.

Claims (2)

1 g of insoluble inorganic compound particles having an average particle diameter of 0.1 to 10.0 μm and specific gravity of 1.5 to 4.0 g / cm 3 as insoluble carrier particles in which Hanta virus antigen is sensitized to 100 or more HDP aggregation units A reagent for diagnosis by the microtiter method of renal syndrome hemorrhagic fever. The inorganic compound particle according to claim 1, wherein the inorganic compound particles are composed of a composite oxide containing silica or silica as a main constituent as a nucleus, and the nucleus is coated with a dye layer containing a dye, or the dye layer is composed mainly of silica or silica. A diagnostic reagent which is an insoluble inorganic compound particle coated with a complex oxide as a component.