JPH0740030B2 - Drying reagent for agglutination reaction - Google Patents

Description

TECHNICAL FIELD The present invention relates to a dry reagent for agglutination reaction. More specifically, the present invention provides a dry reagent for agglutination reaction in which particle dispersibility of the reagent is improved by using water-insoluble inorganic particles.

In the present invention, the average particle size () means that the particles are observed with a transmission electron microscope, the diameter in the longitudinal direction of the particles is measured, and the average diameter is calculated. Further, in the present invention, particle dispersibility is a property showing the degree of individual dispersion of particles, and the particle volume is The content ratio of the particles included in the range to all particles, that is, the particle dispersion value (%). The particle dispersion value is used for both water-insoluble inorganic particles as a carrier and a dry reagent for agglutination reaction prepared using the inorganic particles, as described later.

[Problems to be Solved by Prior Art and Invention]

In recent years, it has been widely practiced to measure biological components secreted in body fluids and urine for diagnosis of diseases and prognosis. For example, hepatitis virus, acquired immunodeficiency syndrome (AIDS), viral infections such as adult T-cell leukemia and rubella, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus and Hashimoto's disease, candida, mycoplasma, streptococcus and syphilis etc. Tests for bacterial infections such as insulin, human chorionic gonadotropin and estrogen,
Other examples include examination of tumor markers.

Diagnosis of these diseases has been extremely difficult with conventional chemical analysis because of the small amount of biological components to be tested, and immunoserologic tests using antigen-antibody reaction have generally been used. The most important feature of this test method is that biological components secreted in body fluids and urine can be measured with extremely high sensitivity and specificity.

One of the immunoserologic tests is the agglutination reaction method. The agglutination method is a method in which particles made of animal red blood cells, polystyrene latex, clay, etc. are used as a carrier, and an antigen or antibody is immobilized on these carriers (hereinafter referred to as sensitization), and a specimen (body fluid containing biological components). , Or urine) and measure the size of the agglutination product of the carrier or the intensity of the agglutination reaction image generated in the formation of the antigen-antibody complex, thereby measuring the target biological component. Is the way.

This agglutination reaction method does not require special and expensive equipment unlike labeling methods in immunoserologic tests such as radioimmunoassay (hereinafter abbreviated as RIA) and enzyme immunoassay (hereinafter abbreviated as EIA). It is highly useful because of its excellent features that it is easy to operate and allows quick diagnosis. Moreover, recently, the sensitivity of the agglutination method has been remarkably improved by the improvement of the purification method of the antigen and the antibody, the improvement of the sensitization method, and the improvement of the determination method, and it has become comparable to the sensitivity of RIA and EIA.

By the way, the reagent for agglutination reaction in which the carrier is sensitized with the antigen or the antibody has a drawback that the antigen or the antibody tends to be significantly inactivated in the state of the dispersion liquid, and the reagent performance deteriorates in a short period of time. Generally, for the purpose of releasing this, a method of using the reagent for agglutination reaction, for example, by heating or drying at room temperature and re-dispersing it in purified water, physiological saline or various buffer solutions after long-term storage is considered. However, there is a problem in that the activity of the biological substance sensitized to the carrier is lowered by the drying treatment, and there is a tendency that it is inactivated in a short time after redispersion. Therefore, various drying means have been studied, freeze-drying has been proposed, and further measures have been taken so that the performance does not deteriorate for a long period of time. Then, at the time of inspection, purified water, physiological saline, or various buffers are added to the freeze-dried agglutination reaction reagent to make an agglutination reaction reagent in which particles are completely dispersed, and measurement is performed. Even in this method, the performance of the reagent depends on the quality of the dispersibility of the reagent. That is, if the inspection is performed in a state in which the dispersibility of the work is poor and the particles are not completely dispersed, the presence of large aggregates not only significantly impairs the appearance of the aggregation reaction image, but also causes an erroneous determination.
Furthermore, in the case of a reagent having poor dispersibility, it takes time to uniformly disperse the reagent, which has a drawback that the feature of the agglutination reaction method of rapid diagnosis is significantly impaired.

Since red blood cells and polystyrene latex, which are conventional carriers, have poor dispersibility in the above-mentioned use, attempts have been made to devise the following freeze-drying method.

For example, as for red blood cells, JP-A-48-26913 and JP-A-54-23119 show improvements by devising pretreatment of red blood cells, and JP-A-50-13523 and JP-A-58-131566 show freezing. An improvement by devising the composition of the suspension during drying is described, and JP-A-59-169504 describes a method for improving the dispersibility of the reagent by improving the freezing method. Also, when polystyrene latex is used as a carrier, improvement by making the composition of the suspension liquid during freeze-drying multi-component has been reported (JP-A-59-109862).

As described above, the conventional carriers, erythrocyte and polystyrene latex, require a very complicated and precise freeze-drying method in order to maintain the performance of the obtained reagent in good condition.
Therefore, if the lyophilization conditions are adjusted incorrectly, the reagent performance will be extremely poor.

[Means for solving problems]

The inventors of the present invention have conducted extensive studies based on such technical background, and as a result, by using water-insoluble inorganic particles having a specific contact angle to water as aggregating reactive particles, the dispersibility of the reagent for errands can be improved. They have found that an excellent dry reagent for agglutination reaction can be obtained, and have completed the present invention.

The present invention is a water-insoluble inorganic compound having a contact angle with water of 80 degrees or less, an average particle size of 0.1 to 10.0 μm, and a particle dispersion value of 80% or more of a particle sensitized with a biological substance on the surface of the aggregate. A dried reagent for condensation reaction obtained by freeze-drying a suspension of reactive particles.

The aggregating reactive particles have a contact angle with water of 80 degrees or less after sensitizing a biological substance to the surface of the water-insoluble inorganic particles,
Resuspend in an appropriate buffer. Here, water is a colorless and transparent liquid having a chemical formula H ₂ O and a molecular weight of 18.02, which is tasteless, odorless, and colorless, which is described in the Chemical Handbook (Basic Edition I, edited by The Chemical Society of Japan, Maruzen).

The water-insoluble inorganic particles of the present invention preferably exhibit insolubility particularly in physiological saline or a buffer solution. For example, the water-insoluble particles of the present invention are dispersed in physiological saline at a constant concentration, and the concentration of the inorganic compound eluted in physiological saline after standing for 24 hours is preferably 1000 ppm or less, particularly preferably 100 ppm or less.

The contact angle of the present invention refers to the angle formed by the tangent line drawn to the droplet and the solid surface at the intersection of the surface of the liquid droplet placed on the solid surface and the solid surface and containing the liquid. This contact angle is generally used when expressing wettability. The wettability is, for example, the energy difference (Δγ) before and after the liquid is placed on the solid surface.
Since it can be grasped as, it is expressed by the young expression below. Here, the liquid surface tension γl and the contact angle are obtained experimentally Δγ = γs-γsl = γl cosθ (1) γs: solid surface tension γl: liquid surface tension γsl: interfacial tension θ: contact angle Therefore, the wettability can be quantified by the above formula (1). When the contact angle is close to 0 degree, the solid and the liquid are completely wet, and when the contact angle is close to 180 degree, they are not wet.

The contact angle of the present invention can be measured by a commonly used contact angle measuring device (for example, goniometer, manufactured by Elma Co.).

The water-insoluble inorganic particles of the present invention may be used without particular limitation as long as the contact angle with water is 80 degrees or less. Specific examples include silica, alumina, titania, zirconia, ferric oxide, ferric tetroxide, cobalt oxide, nickel oxide, etc., Group III, Group IV or Group VIII metal or semi-group. Metal oxides; 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. And sulfuric acid such as barium sulfate and strontium sulfate. As the water-insoluble inorganic particles, complex oxides are also preferably used. For example, silica shown in British Patent No. 2115799 and I of the periodic table are used.
At least one metal oxide selected from the group consisting of Group III, Group II, Group III, and Group IV, and the silica is
Those containing 80 mol% or more are preferable.

Furthermore, the water-insoluble inorganic particles of the present invention may contain an organic compound externally or internally as long as the contact angle satisfies the condition of 80 degrees or less. For example, surface treatment of the water-insoluble inorganic particles is often a preferable means in order to further increase the poor solubility of the water-insoluble inorganic particles in physiological saline or a buffer solution and to improve the efficiency of sensitization. The surface treatment method is not particularly limited, and a known surface treatment method by a dry method or a wet method using a silane coupling agent or a titanium coupling agent is adopted. Alternatively,
Coloring the water-insoluble inorganic particles with a dye is also often a suitable means. This coloring is optimal when the end point of the agglutination reaction using the finally colored dry reagent for agglutination reaction needs to be accurately diagnosed with the naked eye. The dye is not particularly limited and may be selected from known dyes as necessary and used. Generally, cationic dyes are most effective, followed by metal-containing dyes, then reactive dyes and fluorescent brightening dyes. Further, depending on the application, there are cases in which the dyes are slightly inferior to the above-mentioned various dyes, but disperse dyes, direct dyes, acid dyes, acid mordant dyes and other dyes can be sufficiently used.

The average particle diameter () of the water-insoluble inorganic particles used in the present invention is not particularly limited, but is selected from the range of 0.05 to 200 μm, preferably 0.1 to 10.0 μm. When the average particle size is smaller than 0.1 μm, when the particles are used as a carrier for a dry reagent for agglutination reaction, the determination time tends to be long because the sedimentation speed is low. On the other hand, when the average particle diameter is larger than 10.0 μm, the agglomerated image tends to become unclear and the sensitivity tends to be lowered.

The shape of the water-insoluble inorganic particles used in the present invention is not particularly limited, and particles existing in the shape of a polyhedron, a column, a cone, a sphere or the like are adopted. In particular, spherical particles, especially spherical particles are preferable because they have excellent dispersibility.

The particle dispersion value of the water-insoluble inorganic particles used in the present invention is 80.
% Or more is preferable. 90% or more is more preferable. When the particle dispersion value is less than 80%, it tends to be difficult to determine the agglomeration image and the non-aggregation image when the dry reagent for the agglutination reaction produced using the water-insoluble inorganic particles is used in the agglutination reaction described below. . The particle size distribution of the water-insoluble inorganic particles, that is, the standard deviation of the particle size of each particle divided by the average particle size and multiplied by 100, the distribution value (%) of the particle size is 3
It is preferably 0% or less, and more preferably 20% or less. This particle size distribution is preferably as small as possible in order to obtain a high immobilization rate when sensitizing biological substances.

Considering the requirements of the water-insoluble inorganic particles used in the present invention, the particles have a contact angle of 80 degrees or less, an average particle size of 0.1 to 10.0 μm and a particle dispersion value of 80% or more, and a core part. A particle having a two-layer structure of a colored layer on the surface of the core, the core being formed of an inorganic compound and the coloring layer being formed of a dye or a mixture of a dye and an inorganic compound. Composite particles are preferably adopted. Alternatively, particles having an average particle diameter of 0.1 to 10.0 μm and a particle dispersion value of 80% or more and having at least a three-layer structure of a core portion, a colored layer on the surface of the core portion, and a fixed coating layer coating the colored layer. The core is formed of an inorganic compound, the colored layer is formed of a dye or a mixture of a dye and an inorganic compound, the fixed coating layer of a dye and an inorganic compound having a dye content less than the coloring layer. Water-insoluble inorganic composite particles formed of a mixture or an inorganic compound containing no dye are also preferable. Water-insoluble inorganic composite particles having such a specific structure are composite particles that have not been known so far, but the manufacturing method thereof is not particularly limited, and representative ones are as follows.

At least a part of the raw material to be hydrolyzed to be dyed and hydrolyzed to be an inorganic compound to be hydrolyzed (hereinafter also simply referred to as a hydrolyzable compound) is dissolved, but a reaction product is not dissolved. It is produced by allowing a finely divided inorganic compound, which is a core portion, that does not dissolve in the solvent to exist in a water-containing solvent, and adding a compound to be hydrolyzed and a dye to the solvent to perform hydrolysis.

In the method for producing the composite particles, a solvent in which the dye and the compound to be hydrolyzed which are the raw materials are dissolved but the hydrolyzate is not used is used. As the solvent, those having the above-mentioned properties may be previously determined and used according to the kind of the dye or the compound to be hydrolyzed. However, these raw materials do not have to be completely dissolved, and any material that can dissolve a part thereof can be used. Specific examples of the normally employed solvent include, for example, methanol, ethanol, isopropanol, butanol, isoamyl alcohol, ethylene glycol, and the like because of the reactivity, operability, and ease of availability described later. An alcohol solvent such as propylene glycol is preferably used. Further, an organic solvent such as an ether solvent such as dioxane or diethyl ether or an ester solvent such as ethyl acetate may be partially mixed with the alcohol solvent.

Since the compound to be hydrolyzed needs to be hydrolyzed as described below, the solvent must contain water. The content of the water varies depending on the kind of the compound to be hydrolyzed or the alkalinity of the solvent and cannot be unconditionally limited, but it is generally preferable to select it in the range of 0.05 to 5% by weight. Some of the solvents specifically exemplified above generally include water in the above range, such as alcohol, and it is not necessary to control the water content in such solvents.

In addition, the above-mentioned solvent is generally used in a neutral or alkaline state. When the solvent is acidic, the particle dispersibility of the specific composite particles obtained by hydrolysis is remarkably small, and in most cases it becomes a gel-like powdery substance and the desired particles cannot be obtained, which is not preferable. . Any means may be used for making the solvent alkaline, but in general, ammonia water or alkali hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide is added, or a method of using these compounds in combination is preferable. Is. The addition amount of these compounds is not particularly limited and may be appropriately determined and used. Generally, when ammonia water is used, the ammonia concentration is adjusted to 5 to 30% by weight, preferably 10 to 25% by weight. It is suitable to choose. Further, when ammonia water and alkali hydroxide are used in combination, the size of the particles obtained can be controlled by the amount of alkali hydroxide added, so this is often used as a preferred embodiment. For example, the concentration of alkali hydroxide in ammonia water is
It is used in the range of 0.05 to 0.15% by weight, and the particle size obtained tends to increase as the alkali hydroxide concentration increases.

In the method for producing the specific composite particles, in the water-containing solvent, an inorganic compound serving as a core that does not dissolve in the solvent is allowed to exist, and a compound to be hydrolyzed and a dye are added dropwise to the solvent to carry out a hydrolysis reaction. Is preferred.

As the inorganic compound to be the core, the above-mentioned ones can be used, but generally, the compound to be hydrolyzed to be described later is previously hydrolyzed in a solvent to precipitate a compound that is not soluble in the solvent, and the precipitate is used as the core part. The method of is also preferably adopted. The concentration of the core portion present in the solvent is not particularly limited and may be determined and used as necessary, but it is generally preferable to select from the range of 0.1 to 10% by weight.

The dye and the compound to be hydrolyzed are added dropwise to the solvent containing the core. Therefore, the dye is preferably dissolved in a solvent in advance or used as a solution to be hydrolyzed in a solution. It is also preferable to similarly use the compound to be hydrolyzed in the form of a solution. Of course, the dye and the compound to be hydrolyzed may be dissolved in the same solvent and added dropwise to the water-containing solvent at the same time, or may be added separately to the water-containing solvent from different dropping devices as required.

In addition, a method in which the concentration of the solution in which the raw materials are dissolved is generally low is preferable, but if the concentration is too low, the amount of the solvent used remarkably increases, and if the concentration is too high, it becomes difficult to control the reaction or the handling becomes inconvenient. It may be appropriately determined in consideration of the above. Generally, the raw material concentration is 50% by weight or less, preferably 5
Most preferably it is used as a concentration in the range of up to 50% by weight.

Furthermore, since the dropping rate of the dye and the compound to be hydrolyzed affects the particle size and particle dispersibility of the obtained composite particles, it is preferable to determine and use it in advance according to other conditions. Generally, it may be convenient to add 0.5 to 10% by weight, preferably 0.5 to 2% by weight, of the amount of the water-containing solvent dropwise over 1 hour. For example, if two water-containing solvents are used, the raw material may be at a speed of 10 to 200 ml / hour.

The compound to be hydrolyzed to be used as the raw material is not particularly limited as long as a part of the compound to be hydrolyzed can dissolve in a solvent and can be hydrolyzed. The generally used hydrolyzable compound is a metal alkoxide.
That is, an alcoside compound represented by the general formula Mx (OR) x (wherein M represents a metal or a metalloid, and x represents a valence of M) is preferable. In the general formula, R is an alkyl group, and generally, a lower alkyl group such as a methyl group, an ethyl group, an isopropyl group, a butyl group or the like is preferably used. The following is an example of what is preferably used. That is, in general, the general formula M ³ (OR ¹ ) ₃ , M ⁴ (OR ¹ ) ₄ , M ⁸ (OR ¹ ) ₃ , (where R ¹
Represents an alkoxide of a metal represented by an alkyl group or one or two alkoxyl groups (OR
A compound in which ¹ ) is substituted with a carboxyl group is preferable.
Where M ³ is a Group III metal, M ⁴ is a Group IV metal, M ⁸ is a Group V metal
Group III metals, specifically, aluminum, titanium, silicon, zirconium, germanium, hafnium, tin, lead, iron, cobalt, nickel and the like are preferably used. Specific examples of the above-mentioned compounds that are preferably used generally include Al (OC ₂ H ₅ ) ₃ , Al (OC ₃ H ₇ ) ₃ , Al (OC
₄ H ₉ ) ₃ etc. and aluminum alkoxides such as Al and alkoxides of Group III metals substituted by B instead of Al; Ti (O-isoC ₃ H ₇ ) ₄ , Ti (O-nC ₄ H ₉ ) ₄ , Ti (O-CH _{2
CH (C 2 H 5) C} 4 H 9) 4, Ti (O-C 17 H 35) 4, Ti (O-isoC 3 H
_{7) 2} [OCH (CH ₃₎ CHCOCH _{3] 2, Ti (O-nC 4} H 9) 2 [OC _{2
H 4 N (C 2 H 4} OH) 2 ] _2, Ti _{(OH) 2} [OCH (CH ₃₎ COOH] _2, T
_{i (OCH 2 CH (C 2} H 5) CH (OH) C 3 H 7) 4, Ti (O-nC 4 H 9) 2
(OCOC ₁₇ H ₃₅ ) ₂ etc. Titanium alkoxide and the above Ti
On behalf of, Si, Zr, Ge, Hf , Sn, alkoxides of Group IV metals replaced by Pb or the _{like; Fe (OC 2 H 5)} 3, Fe (O-nC 3 H 7) 3, Fe
_{(O-nC 4 H 9)} 3, Fe (O-secC 4 H 9) 3, Fe (O-tertC
₄ H ₉ ) _3, etc., instead of iron alkoxide and Fe above, Co,
Examples include alkoxides of Group VIII metals replaced with Ni. The CaCl _2, Ca [HCO ₆ H ₄ COO] compounds such as ₂ · 2H ₂ O can be used as a mixture to the alkoxide of the metal.

It is not necessary to use only one type of compound to be hydrolyzed, and it may often be a preferable embodiment to use two or more types at the same time. Further, for example, as described in British Patent No. 2,115,799, Examples, a low condensate of alkoxysilane and an alkoxide of another metal are mixed in advance to prepare a mixed solution, and the prepared mixed solution is used as a raw material. You can also

Part of the dye that is the other raw material of the specific composite particles is, for example, the solvent 10.
It is not particularly limited as long as it dissolves 1 part by weight or more, preferably 5 parts by weight or more, more preferably 10 parts by weight or more, relative to 0 parts by weight. Generally, the above-mentioned various dyes can be preferably used. The dye is generally 0.001 to 1
Preference is given to using concentrations of weight%. Furthermore, it may be chosen such that there is always 0.05 to 0.5% by weight, preferably 0.05 to 0.2% by weight, of dye in the solvent.

The above raw material immediately undergoes a hydrolysis reaction in a solvent to form a particulate insoluble precipitate. The conditions for the hydrolysis are not particularly limited, and any conditions can be selected, but it is generally preferable to carry out the reaction with or without stirring at a temperature in the range of 5 to 50 ° C, preferably 10 to 30 ° C.

The composite particles of the dye and the inorganic compound obtained by hydrolysis by the above method have a mean particle size () of 0.1 to 10.0 μm and are non-aggregating, and the particle dispersion value is 80% or more in most cases. 90% or more. As described above, it is quite surprising that the hydrolysis reaction proceeds in the form of forming a colored layer in the core even in the presence of a dye which is considered to hinder the growth reaction if the core is present in the solvent. It is a phenomenon. Moreover, the dye is firmly fixed to the colored layer to form a composite particle having colorability according to the dye. The composite particles thus obtained are composite particles in which a colored layer composed of a mixture of a dye and an inorganic compound is present in the core of the inorganic compound containing no dye.

Moreover, the following method is also suitable when another manufacturing method is illustrated. The metal oxide particles serving as the core of the specific composite particles have a good particle dispersion value, for example, 80% or more and a porous particle is selected by contacting the porous particle with a solution in which a dye is selected and the porous particle is contacted. Specific composite particles in which the dye is impregnated are obtained. In the method for producing the above-mentioned specific composite particles, selection of porous particles is an important requirement. Of course, the method for producing the porous particles is not limited. However, in general, porous particles obtained by the following method are particularly suitable. For example, a composite oxide consisting of a metal of Group III, IV or VIII of the Periodic Table and an alkali metal such as potassium, sodium or lithium is produced by the above-mentioned metal alkoxide hydrolysis method. Then, the obtained composite oxide was contacted with a mineral acid solution such as sulfuric acid, nitric acid or hydrochloric acid to elute the contained alkali metal component, and the obtained particles were porous and had an excellent particle analysis value of 80% or more. Will be things.
By using such particles, it becomes a specific composite particle in which the dye is impregnated, that is, the dye is present on the surface of the core. If necessary, the specific composite particles may be dried before use or subjected to post-treatment.

In some cases, the dye may be eluted from the colored layer composed of the dye or a mixture of the dye and an inorganic compound. In such a case, the colored layer is further coated with a fixed coating layer of an inorganic compound having a smaller dye content or an inorganic compound containing no dye, or a three-layer structure, or the above-mentioned lamination is repeated four times. The specific composite particle having the above structure can be obtained. The means for forming the multilayer structure of three or more layers is not particularly limited, and any means may be used. To give a typical example of methods that are preferably adopted, a dye and a compound to be hydrolyzed are dropped into a water-containing solvent in which the inorganic compound serving as the core is present, and the raw material is hydrolyzed to form particles having a two-layer structure. Or after producing dye-impregnated porous particles, a method of further dropping only the compound to be hydrolyzed into the same reaction vessel for hydrolysis, or the above-mentioned two-layer structure particles or dye-impregnated porous particles The unreacted or unimpregnated dye is separated from the production system, and then the compound to be hydrolyzed is further added dropwise to hydrolyze to obtain the specific composite particles.
When the former, that is, when the reaction is carried out in the same system, composite particles having a three-layer structure are obtained, but the third fixed coating layer has a residual dye that has been digested in a solvent when a colored layer is formed on the core. When the solution to be hydrolyzed to be dripped does not contain a dye even though it is dissolved, the composition has a remarkably small dye content. In this case, the amount is usually about 1/5 or less, preferably 1/10 or less of the dye contained in the colored layer formed directly on the core. This phenomenon is also a phenomenal phenomenon from a general reaction, but its action is not clear at present. Therefore, the manufacturing method of the above-mentioned specific composite particles does not need to take a special reaction form,
It can also be obtained by laminating more than one layer.
The composite particles thus obtained are excellent spherical particles in which the dye hardly elutes even if a small amount of the dye is contained in the third layer.

From the above, when the addition of the dye in the case of performing the production of the core and the coloring layer of the inorganic compound in a series of operations, in order to adjust the particle size of the resulting specific composite particles in a specific range and from the specific composite particles In order to prevent the elution of the dye, the total synthesis time required for the formation of the core and the colored layer is 0.1 to 0.
The target specific composite particles can be obtained by starting and completing the addition of the dye within the time indicated by 9, particularly preferably 0.2 to 0.8.

When the specific composite particles having a multi-layer structure of three or more layers are obtained, the unreacted or unimpregnated dye is separated from the particles of the two-layer structure coated with the colored layer on the surface of the core and used for the production of the particles. After washing several times with the same solvent as above, the hydrolyzable compound not containing the dye is dropped into the solvent containing the particles to hydrolyze the hydrolyzable compound, and the fixed coating layer of the third layer structure does not contain the dye. It is a layer that hardly contains it.

By subjecting the specific composite particles having the two-layer structure or the three-layer structure to the treatment as described above, composite particles having a multi-layer structure of three or more layers can be obtained. In the composite particles having a multi-layered structure, it is preferable to select the fixing coating layer from a light-transmitting material in order to display the coloring degree of the coloring layer more clearly. In this sense, the fixed coating layer is the Periodic Table III.
A group or group IV metal or a complex oxide of the metal and a group I, II or V metal of the periodic table is preferable.

The biomaterials to be sensitized to the water-insoluble inorganic particles described above are not particularly limited and known ones are used, but immunoactive substances such as antigens, haptens and antibodies obtained by immunization thereof are preferably used. . For example, immunoglobulin, albumin, fibrinogen (fibrin and their degradation products), α-fetoprotein,
C-reactive protein, beta ₂ - microglobulin, myoglobin, carcinoembryonic antigen, hepatitis virus antigen, human chorionic gonadotropin, human placental lactogen, insulin, digoxin, protein A, protein G, DNA, proteins such as RNA, hormones , Drugs, and their antibodies.

As a method for sensitizing the water-insoluble inorganic particles of the present invention with the above-mentioned biological substance, a known sensitizing method can be adopted. For example,
The above-mentioned protein and water-insoluble inorganic particles in an aqueous medium (for example,
It is preferable to bring them into contact with each other in water, physiological saline, various buffer solutions, etc. Generally, it is carried out by mixing and leaving still,
It may be stirred or shaken to improve the contact efficiency. After the sensitization, the biological material in the supernatant is removed by centrifugation and, if necessary, washing with the above aqueous medium.

In the present invention, the freeze-drying method is not limited and any ordinary method may be used. For example, the method and conditions adopted in the freeze-drying method of sensitized red blood cells are used. Preferably, a method of rapid pre-freezing and then vacuum freeze-drying is adopted.
The rapid pre-freezing is performed in liquid nitrogen, dry ice-methanol, dry ice-acetone, or fluorocarbon, etc., and a container such as a vial or ampoule containing a suspension liquid in which agglutination-reactive particles sensitized with the above-mentioned biological material are dispersed. Is achieved by dipping.

The liquid composition of the suspension is not limited, and the liquid composition used for rapid pre-freezing in the conventional field of producing agglutination reaction particles is adopted. For example, various buffers include saccharides such as glucose, serum albumin, amino acids or the like. And a mixture of the above is added. The concentration of the aggregation-reactive particles in the suspension is adjusted to 0.05 to 50% by weight, preferably 0.1 to 10.0% by weight.

The vacuum freeze-drying method is not limited, and for example, a vial containing a suspension liquid of the above-mentioned agglutination-reactive particles is rapidly pre-frozen, and then it is cooled to −40 ° C. to −60 ° C. on a shelf in a chamber of a freeze dryer. Place in a vacuum and freeze-dry for 24 to 72 hours. The pressure in the chamber at this time is 50-200 μHg,
The final drying temperature is 20 to 50 ° C. Then, it is vacuumed or filled with an inert gas and stored with a stopper.

There is no limitation on the agglutination reaction using the dry reagent for agglutination reaction of the present invention thus obtained, and the agglutination reaction method usually used for diagnosis is applied. For example, a plate method for qualitative diagnosis, a microtiter method for semi-quantitative method, a turbidimetric method for quantitative method, a particle number measuring method and the like. Preferably, the effect of the present invention can be remarkably exhibited by adopting the microtiter method, which is easy to operate and enables titer measurement.

[Action and effect]

The thus-obtained dry reagent for agglutination reaction of the present invention has extremely good dispersibility in use.

A dry reagent for agglutination reaction using water-insoluble inorganic particles having a contact angle with water of more than 80 degrees has very poor dispersibility at the time of use and causes agglomerates to cause misjudgment. or,
It takes a long time to stand until it has a good dispersibility, which is not preferable for rapid diagnosis, which is a characteristic of the agglutination reaction. On the other hand, the agglutination reaction dry reagent of the present invention using a water-insoluble inorganic particle having a contact angle to water of 80 degrees or less has extremely good dispersibility in the errand, and the particles constituting the reagent are instantly dispersed so that it is rapid. It is possible not only to perform a simple diagnosis, but also to significantly suppress the presence of aggregated particles that are not dispersed, so that it is possible to prevent erroneous determination.

The reason for this is not necessarily clear, but the contact water amount in the vicinity of 80 degrees and the amount of bound water on the particle surface of the water-insoluble inorganic particles dried and the conformation change abruptly. It is speculated that dispersiveness makes a difference. That is, the agglutination reaction dry reagent using a water-insoluble inorganic particles having a contact angle with water of 80 degrees or less, the surface of the particles constituting when contacted with water is more hydrophilic because the surface of the particles is more hydrophilic. A large amount of it works well in its conformation and quickly becomes compatible with water. As a result, the penetration of water into the interfacial space of agglomerated particles occurs promptly, and the particles are separated into individual particles, and finally one particle group is completely suspended in water and dispersion is very efficient. It is often done.

Further, in the present invention, as described above, since the inorganic particles are selected as the sensitizing carrier, it is possible to provide a carrier having a high specific gravity and a large particle size as compared with the case of using red blood cells and polymer particles, and the determination is easy and quick. become. Furthermore, since the surface has no antigenicity, nonspecific agglutination reaction based on the antigen-antibody reaction of heterophilic antibodies can be prevented. Furthermore, the control of surface characteristics is easy and a wide range of proteins can be immobilized, so that the range of use as a diagnostic reagent is expanded.

INDUSTRIAL APPLICABILITY The present invention provides a dry reagent for agglutination reaction in which the dispersibility of the affairs is remarkably improved as compared with the conventional dry reagent for agglutination reaction in immunoserologic tests, and makes a great contribution to the field of clinical examination.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples. The properties of the water-insoluble inorganic particles, the elution amount and the contact angle, and the particle dispersibility of the dry reagent for agglutination reaction in the following examples were measured by the following methods.

(1) Average particle diameter () The average particle diameter () was determined by observing the diameter of 100 or more particles in the longitudinal direction with a transmission electron microscope.

(2) Particle size distribution [Distribution value of particle size] (SD) Distribution value of particle size (SD) is a value obtained by dividing the standard deviation of the particle size of each particle by the average particle size and multiplying by 100. (3) Particle dispersion value (PD) The particle dispersion value (PD) is expressed as a value (%) obtained by dividing the number of particles having a volume in the range of π / 6 (± 0.5) ³ by the total number of particles and multiplying by 100. did. The particle volume was measured and the number of particles was counted using Model ZD-1 manufactured by Coulter Counter. This value is a value indicating the degree of individual dispersion of particles.

(4) Dry reagent particle dispersion value (RPD) for agglutination reaction As for the dry reagent particle dispersion value (RPD), 1 mg of the dry reagent for agglutination reaction was put in 5 ml of physiological saline, and after manual stirring, left still for 1 minute. It was measured in the same manner as the particle dispersion value of (3).

(5) Average Particle Diameter of Core Part () The inorganic compound particles forming the core part were measured in the same manner as in (1) above after the inorganic compound particles were produced.

(6) Elution amount of inorganic particles Each water-insoluble inorganic particle was dispersed in distilled water so as to be 10% by weight, and 1 ml of this dispersion was taken and allowed to stand for 24 hours, followed by ions derived from the eluted inorganic compound in the supernatant. It was quantified by the atomic absorption method.

(7) Elution amount of dye (SC) The concentration of the dye dissolved in methanol and the absorbance at the concentration are subjected to spectroscopic analysis to obtain the extinction coefficient (ε) of the dye. Separately, the inorganic compound / dye composite particles were dispersed in 5 ml of methanol to a concentration of 5% by weight, and allowed to stand for 1 week.
Is taken and spectroscopically analyzed to determine the absorbance (A) of the supernatant. The elution amount (C) of the dye was calculated by the following formula.

C = A / ε (8) Dye content in inorganic compound / dye composite particles (DC) First, the extinction coefficient (ε) of the dye is determined by the same method as in (5) above. Separately, inorganic compound / dye composite particles are dispersed in methanol at a concentration of 0.5% by weight, 4 ml of this dispersion is taken, and its weight and absorbance (A ') are spectroscopically analyzed. Separately, inorganic compound particles forming the core of the inorganic compound / dye composite particles and having the same average particle diameter as the composite particles are dispersed in methanol at a concentration of 0.5% by weight, and the absorbance (A ″) Is spectroscopically analyzed.

From the above measurement results, the weight (DW) of the dye contained in the composite particles is calculated by the following formula.

DW = (A′−A ″) / ε Then, the weight of the alcohol liquid is subtracted from the weight of 4 ml of the methanol dispersion liquid of the composite particles to calculate the weight (PW) of the composite particles in the 4 ml.

Finally, the dye content (DC) in the composite particles is calculated by the following formula.

(9) Proportion of Dye Contained in Colored Layer (CDC) Inorganic compound / dye composite particles are cut into ultrathin sections by an epoxy resin embedding method, and the ultrathin sections are observed with a transmission electron microscope. As a result of the observation, a darkened part (nucleus) of the central part,
When the composite particles are composed of a thinly visible portion (colored layer) on the outside and three or more layers, the outer layer portion of the third layer or more is analyzed with an analytical microscope. The number of carbon atoms was calculated from the intensity of the specific X-ray (289 eV) of carbon obtained by the analysis, and the ratio (CDC) of the dye contained in the colored layer in contact with the core was calculated by the following formula.

(10) Contact angle Water-insoluble inorganic particles are pressed at a pressure of 100 kg / cm ² to make a flat measurement sample, and then distilled by a predetermined method using a contact angle measuring device (goniometer, manufactured by Elma Co.). The contact angle with water was measured.

Examples 1 to 5 (1) Synthesis of water-insoluble inorganic particles In a glass flask equipped with a stirrer, 2800 cc of methanol, 616 cc of ammonia water (25% by weight) and 21 cc of aqueous sodium hydroxide solution (5 mol /) were added and kept at 10 ° C. Then, add 256 ml of a solution of tetraethyl silicate in methanol (22 wt%) to 2 ml.
Silica particles were prepared by adding dropwise at a rate of 5.5 ml / hr.
To the reaction solution containing the silica particles, 624 ml of a methanol solution of tetraethyl silicate (44% by weight) and 400 ml of a methanol solution of various dyes shown in Table 1 (1.25% by weight) were simultaneously added dropwise at a rate of 25.5 ml / hr. Then, it was colored with a dye to synthesize colored silica particles. The obtained colored silica particles were repeatedly decanted and purified with a large amount of methanol. No silica elution was observed at all when the silica particles containing this dye were dispersed in methanol.

Similarly, non-colored silica particles containing no dye were synthesized. This is the same condition except that the methanol solution of the dye is not added simultaneously in the above synthetic method.

The silica particles thus obtained are finally added to 10% by weight.
Disperse in methanol to make the concentration 100m
The surface-treating agents shown in Table 1 were added to 1 l so as to each have a concentration of 0.5% by weight and reacted at 10 ° C. for 16 hours to obtain surface-treated silica particles (colored and non-colored). . The characteristics of these silica particles are shown in Table 1.

(2) Synthesis of water-insoluble inorganic particles sensitized with heat-denatured human IgG Human Chon F II fraction (manufactured by Sigma) was adjusted to 1/150 M phosphate-saline buffer pH = 7.4 (hereinafter abbreviated as PBS). Heat-denatured human IgG was obtained by dissolving at 10 mg / ml and heating at 60 ° C for 10 minutes. 1 ml of the resulting heat-denatured human IgG diluted 50 times with PBS and the silica particles obtained in the previous section with PBS
1 ml of a solution diluted to weight% was sensitized for 2 hours while stirring at room temperature.

Then, centrifuge to remove heat-denatured human IgG in the supernatant,
The precipitate was redispersed with 2 ml of PBS containing a small amount of sucrose, bovine serum albumin and glycine. Thus, a suspension of agglutinating reactive particles using silica particles was obtained. The same operation was performed for the surface-treated silica particles.

(3) Freeze-drying method Transfer 1 ml of the flocculation reactive particle suspension obtained in (2) to a 3 ml vial and rapidly freeze the vial with liquid nitrogen.
It was placed on a shelf in the chamber of the freeze dryer that had been cooled to -50 ° C in advance. Then, under reduced pressure of 100 μHg, 4
Lyophilization was performed for 30 hours at a temperature rising rate of 5 ° C / hr until the temperature reached 0 ° C.

Thus, a dry reagent for agglutination reaction for semi-quantifying rheumatoid factor in rheumatoid patient serum was obtained.

(4) Dispersion of dry reagent for agglutination reaction and antigen / antibody reaction In a vial containing the dry reagent for agglutination reaction obtained in (3), add 2 ml of PBS containing a small amount of normal rabbit serum.

On the other hand, a pooled serum of rheumatoid patient serum is diluted 20-fold with PBS to prepare a stock solution of a rheumatic patient serum to prepare a diluted solution. Prepare a V-type microtiter plate to carry out antigen-antibody reaction, and add rheumatoid patient serum dilution to each well
Add 25μ. Then, 2 wells of dry reagent for agglutination reaction dispersed in PBS containing a small amount of the above normal rabbit serum was added to each well.
After adding 5 μ, the microtiter plate was shaken for 5 minutes and then left standing at room temperature. The dry reagent for agglutination reaction was added dropwise to the well 5 minutes after dispersion.

Then, the bottom image of the agglutination tube based on the antigen-antibody reaction was visually observed to evaluate the performance of the dry reagent for agglutination reaction sensitized with heat-denatured human IgG. The results are shown in Table 1.

Comparative Examples 1 and 2 A dry reagent for agglutination reaction was prepared in the same manner as in Example 1 except that the dye and the surface treatment agent were changed as shown in Table 1, and the performance was evaluated. The results are shown in Table 1.

Comparative Examples 3 and 4 The procedure of Example 1 was repeated except that polystyrene latex talc shown in Table 1 was used instead of the water-insoluble inorganic particles. The results are shown in Table 1.

Examples 6, 7, 8 and Comparative Examples 5, 6 Human chorionic gonadotropin produced by rabbits (hereinafter referred to as HC
The purified HCG antibody obtained by purifying the antibody (abbreviated as G) by affinity chromatography was adjusted to PBS containing 2 mg / ml concentration, and then diluted by the multiple dilution method to prepare a diluted HCG antibody solution.

Then, a water-insoluble inorganic particle prepared by using the surface treatment agent described in Table 2 in the same manner as in Example 1
1 ml of PBS dilution and 1 ml of the above antibody dilution were added and sensitized at room temperature for 1 hour with stirring.

Then, centrifuge to remove the HCG antibody in the supernatant, and remove the precipitate with a small amount of PBS2 containing sucrose, bovine serum albumin, and glycine.
Redispersed in ml. The method for freeze-drying the suspension of aggregation-reactive particles sensitized with the HCG antibody was in accordance with the method of Example 1.

2 ml of PBS containing a small amount of normal rabbit serum was added to and dispersed in the dry reagent for agglutination reaction thus obtained.

The antigen-antibody reaction was performed using pool pregnant woman urine containing HCG at a concentration of 10 IU / ml. First, a pregnant woman urine diluted solution is prepared by using 10 times diluted PBS pregnant woman urine as a stock solution. In order to carry out the antigen-antibody reaction, prepare a microtiter plate and add 25 μl of pregnant woman urine dilution to each well. Then, 25 μl of the dispersion of the dry reagent for the agglutination reaction was added to each well.
After adding, shaking the microplate for 5 minutes,
It was left at room temperature. At this time, the agglutination reaction dry reagent was added dropwise to the well 5 minutes after dispersion. Then, the bottom image of the agglutination tube based on the antigen-antibody reaction was visually observed to evaluate the performance of the HCG antibody-sensitized dry reagent for agglutination reaction. The results are shown in Table 2.

Examples 9 to 13 and Comparative Examples 7 to 11 The water-insoluble inorganic particles of Example 2 in Table 1 were sensitized with heat-denatured human IgG in accordance with the method of Example 1 to prepare aggregation-reactive particles.

A dry reagent for agglutination reaction was obtained in the same manner as in Example 1 except that the agglutination-reactive particles were suspended in a suspension containing the additive having the composition shown in Table 3.

The performance of the antigen-antibody reaction of the dried reagent for agglutination reaction sensitized with the heat-denatured human IgG thus obtained is shown in Table 3.

For comparison, the performance of the agglutination reaction reagent obtained by similarly treating polystyrene is also shown in Table 3.

Example 14, Comparative Example 12 Water-insoluble inorganic particles of Example 3 in Table 1 were sensitized with heat-denatured human IgG according to the method of Example 1 to prepare aggregation-reactive particles, which were then lyophilized to be used for aggregation reaction. A dry reagent was obtained.

The performance of the antigen-antibody reaction of the dry reagent for agglutination reaction sensitized with this heat-denatured human IgG was examined by the following method according to the method of Example 1. That is, 2 ml of PBS containing a small amount of normal rabbit serum was added to a vial containing a dry reagent for agglutination reaction to disperse, and the standing time before adding this dispersion to each well containing the diluted serum of rheumatism patients was changed. The influence on the reagent performance was investigated.

The results are shown in Table 4. For comparison, a dry reagent for agglutination reaction produced using the water-insoluble inorganic particles of Comparative Example 2 in Table 1 was also examined in the same manner, and the results are also shown in Table 4.

Example 15 A dry reagent for agglutination reaction for a plate method for detecting hepatitis B virus antigen (hereinafter abbreviated as HBs) using the water-insoluble inorganic particles of Example 2 in Table 1 was prepared as follows.

A HBs antibody diluted solution was prepared by preparing PBS containing purified HBs antibody at a concentration of 2 mg / ml obtained by purifying the goat-produced HBs antibody by affinity chromatography and then diluting it by the multiple dilution method. Then, sensitization and lyophilization were carried out according to Example 1 to obtain a dry reagent for agglutination reaction, which was then dispersed in 4 ml of PBS.

The antigen-antibody reaction was performed by using human serum containing various concentrations of HBs in PBS.
After 20-fold dilution, 25 μ of this diluted solution and 25 μ of a dispersion of the above-mentioned dried reagent for agglutination reaction were mixed by stirring on a glass plate, and the strength of the agglutination image was measured after 3 minutes. The dry reagent for agglutination reaction was dropped on a glass plate 5 minutes after the dispersion. Table 5 shows the results of aggregation by the glass plate method.

As a comparative example, a dry reagent for agglutination reaction prepared using the inorganic particles of Comparative Example 6 in Table 2 was evaluated by the same method,
Poor dispersibility resulted in non-specific aggregation.

Examples 16, 17, and 18 To a glass flask equipped with a stirrer, 2800 cc of isopropyl alcohol (IPA) and 28 cc of water were added and kept at 10 ° C., and then Table 6
1020 ml of a 30 wt% IPA solution of each compound to be hydrolyzed shown in 2
The inorganic compound particles were obtained by dropwise addition at a rate of 2.5 ml / hour.
These particles were washed and purified in the same manner as in Example 1 and then surface-treated with γ-glycidpropyltrimethoxysilane. These water-insoluble inorganic particles were sensitized with heat-denatured human IgG in the same manner as in Example 1 and lyophilized to obtain a dry reagent for agglutination reaction, and the performance was evaluated by the microtiter method.
The results are also shown in Table 6.

Claims (1)

[Claims] 1. A water-insoluble inorganic compound having a contact angle with water of 80 degrees or less and having an average particle diameter of 0.1 to 10.0 μm.
A dry reagent for condensation reaction obtained by freeze-drying a suspension of agglutinating reactive particles in which biological material is sensitized on the surface of particles having a particle dispersion value of 80% or more.