JPH05306113A - Artificial particle - Google Patents

Abstract

PURPOSE:To obtain artificial particles having a specified range of average particle size which are useful for agglutination immunoassay by mixing silica, gelatine, water-soluble polysaccharide and methaphosphate in a specified weight ratio. CONSTITUTION:Silica, gelatine, water-soluble polysaccharide (e.g. gum arabic) and methaphosphate (e.g. sodium trimethaphosphate) are mixed to obtain artificial particles having 0.5-10mum average particle size. The content of the mixture of gelatine, water-soluble polysaccharide and methaphosphate is 0.02-50mg/g based on silica. The production method of these particles is as follows. An aq. soln. of gelatine and aq. soln. of water-soluble polysaccharide are mixed with an aq. soln. of hydrophilic org. solvent while stirring, and further an aq. soln. of methaphosphate is added thereto. The mixed soln. is controlled to pH 5.5 to 8, and then a soln. containing silica is further added. The mixed soln. is controlled to pH 4 to 6.5 and then cooled.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to particles obtained by mixing silica, gelatin, a water-soluble polysaccharide and a metaphosphate, wherein gelatin, water-soluble polysaccharide and metaphosphate are contained in the particles. To the silica mixture,
The present invention relates to artificial particles having an average particle size of 0.02 mg / g to 50 mg / g and an average particle size of the obtained particles of 0.5 μm to 10 μm. This particle can be used for an agglutination immunoassay by binding, for example, an antigen or an antibody.

[0002]

2. Description of the Related Art Conventionally, a diagnostic method based on an agglutination immunoassay is known as one of immunodiagnostic methods. The method is carried out by using particles to which an immunologically active substance such as an antigen or an antibody is bound. These particles include polystyrene latex, kaolin, charcoal powder, animal red blood cells,
At present, bacterial cells are known and used.
These particles are easy to obtain and can be obtained in large quantities, but they are not suitable for rapid measurement, it is difficult to obtain particles with an arbitrary fixed particle size, the properties differ from one production lot to another, spontaneous aggregation and non-aggregation Since it has drawbacks such as being likely to cause specific aggregation, attempts have been made to improve it, but these drawbacks have not been overcome yet. Therefore, particles composed of a water-soluble polysaccharide, gelatin, sodium metaphosphate and an aldehyde crosslinking agent (Japanese Examined Patent Publication Nos. 63-29223 and 63-63).
No. 48021) and the like were found.

[0004]

However, although this particle can solve some of the drawbacks of the conventional particles such as the fact that a large amount can be obtained with an arbitrary particle size, agglutination immunoassay can be carried out rapidly. I couldn't handle that.

[0005]

As a result of research on new artificial particles, the present inventors found that particles obtained by mixing silica, gelatin, water-soluble polysaccharides and metaphosphates had the conventional drawbacks. The present invention has been completed by finding that it can be improved and can be applied to a rapid agglutination immunoassay. The present invention is an artificial particle obtained by mixing silica, gelatin, a water-soluble polysaccharide and a metaphosphate.

The particles of the present invention comprise silica, a water-soluble polysaccharide,
It can be obtained by mixing gelatin and metaphosphate. The mixture of water-soluble polysaccharide, gelatin and metaphosphate is 0.02 mg / g to silica.
50 mg / g, preferably 1 mg / g to 10 mg
/ G. The amount of this mixture is 0.02 with respect to silica.
When it is less than mg / g, the amount of antigen or antibody that can be bound to the surface is small when used for agglutination immunoassay, and the sensitivity of the measurement cannot be sufficiently increased, and when it is more than 50 mg / g for silica. In addition, it is not preferable because particles having a uniform size cannot be obtained during the production of particles.

The particles obtained by mixing have an average particle diameter of 0.5 .mu.m-by adopting the operation method described below.
It can be 10 μm. When used for agglutination immunoassay, 1 μm to 5 μm is preferable.

The artificial particles of the present invention contain silica. Silica can be produced as monodisperse silica particles by hydrolyzing an alkyl silicate in water or an organic solvent that is miscible with water under alkaline conditions (edited by the Chemical Society of Japan, Experimental Chemistry Course, Vol. 18, 330). ~ 3
(See page 31). As the alkyl silicate, commercially available methyl silicate, ethyl silicate (ethyl orthosilicate) and the like can be used. The size of silica produced can be adjusted by the amount of alkali added during production.

The mixture used for producing the particles of the present invention is a water-soluble polysaccharide, gelatin and metaphosphate. Examples of the water-soluble polysaccharides include gum arabic, carboxymethyl cellulose, sodium alginate, agar, carrageenan, etc., but gum arabic is preferably used because stable particles can be obtained with good dispersibility.

As the gelatin, usually commercially available gelatin can be used, but in order to control the charge on the surface of the obtained particles and make the particles uniform, it is preferable to use acid-treated gelatin having an isoelectric point of 8-9. .. Examples of the metaphosphate include sodium trimetaphosphate, sodium tetrametaphosphate, sodium hexametaphosphate, potassium trimetaphosphate, potassium tetrametaphosphate, potassium hexametaphosphate, etc. Among them, sodium hexametaphosphate is used. Preferably.

The mixture added to the silica during the production of the artificial particles can be prepared in a hydrophilic organic solvent. The ratio of each component and the solvent at that time is 0.05% to 2.0% of the water-soluble polysaccharide, preferably 0.1% to 1.0%, and gelatin 0.05% to 2. 0%, preferably 0.1% to 1.0%, hydrophilic organic solvent 4% to 2
5%, metaphosphate is 0.5% to dry weight of gelatin
It can be used at 20% (volume% for organic solvent only, weight% for others), and can be changed within this range as necessary. Examples of the hydrophilic organic solvent include alcohols such as methyl alcohol, ethyl alcohol and propyl alcohol, and ketones such as acetone and methyl ethyl ketone. Among these alcohols, methyl alcohol and ethyl alcohol are preferably used.

For the production of artificial particles, a gelatin aqueous solution and a water-soluble polysaccharide aqueous solution are mixed in a hydrophilic organic solvent aqueous solution with stirring, and a metaphosphate aqueous solution is further added to the mixture to give an acid having a pH of 5.5 to 8.0. The pH is adjusted to 6.0 to 7.0 to give a mixture solution. Further, a silica-containing solution was added to this mixed solution and stirred, and then the pH was adjusted to 4.0 with an acid.
To 6.5, preferably pH is adjusted to 4.5 to 5.5, and the particles can be obtained by cooling. Each aqueous solution used in the production of the mixture solution can be heated to 28 to 60 ° C., preferably 38 to 40 ° C. before use. The gelatin aqueous solution is preferably a uniform solution obtained by adjusting the pH to around 9 with an alkaline aqueous solution, and the water-soluble polysaccharide aqueous solution is preferably dissolved in water and then removed when impurities are present. The acid used for adjusting the pH in the production can be preferably an organic acid, especially acetic acid, since the particle size can be finely adjusted.

When the artificial particles composed of silica, water-soluble polysaccharides, gelatin and metaphosphate of the present invention are used as a reagent for agglutination immunoassay, they can be further colored and allowed to act with an aldehyde compound. The obtained artificial particles are treated in the same manner as in the case where animal red blood cells are used (Boyden method; J. Exp. Med., 93 , 107-120).
(1950) etc., an immunologically active substance, such as an antigen or an antibody, is bound thereto, and then used as a reagent for agglutination immunoassay.

[0014]

EXAMPLES The present invention will be described in more detail with reference to the following examples and test examples. (Example 1) 1400 ml of ethanol, 520 ml of distilled water, 71 ml of 25 wt% ammonia water and 6 ml of 20 mM sodium hydroxide aqueous solution were added to the flask, and the mixture was stirred at room temperature (20 to 25 ° C) for 5 minutes, and then ethyl orthosilicate ( 500 ml of Wako Pure Chemical Industries, Ltd. was added dropwise at a rate of 375 ml / hour, and silica particles (average particle diameter 3.04μ)
m) made. The silica particles thus obtained were repeatedly washed with ethanol by centrifugation to obtain purified silica particles. The particle size of the silica particles can be adjusted by the concentration of the sodium hydroxide aqueous solution as shown in Table 1.

Table 1 −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Silica particles Sodium hydroxide concentration (mM) Silica particle size ( μm) −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Silica particles A 10 1.64 Silica particles B 15 2.64 Silica particles C 20 3.04 --------------------------------------.

(Example 2) Gelatin 5 having an isoelectric point of 9
g was dissolved in warm water at 40 ° C. so as to be 100 ml.
5 g of gum arabic was dissolved in water to 100 ml, impurities were filtered off, and the mixture was heated to 40 ° C. 40 ml of the aqueous gelatin solution thus obtained and the aqueous solution of gum arabic 4
0 ml was mixed, and this mixture was preheated to 40 ° C. 4
It was poured into 504 ml of a 4% by volume aqueous ethanol solution and stirred well. 20 ml of a 10% aqueous solution of sodium hexametaphosphate (manufactured by Wako Pure Chemical Industries, Ltd.), 20 ml of an aqueous solution of 10% by weight of alkylsulfomaleic acid (trade name: Demol EP, registered trademark of Kao Soap Co., Ltd.), and a dye of 0.5% by weight 40 ml of New Fuchsin (Tokyo Kasei) aqueous solution
After the addition of 10% by volume of acetic acid aqueous solution, the pH was adjusted to 6.
It was set to 0. Next, 400 ml of a 25% by volume aqueous solution of the silica particles (silica particles A, B or C) obtained in Example 1 was added, and after stirring well, a 10% by volume aqueous acetic acid solution was added dropwise to adjust the pH to 4.9, Colored silica, gelatin,
Particles containing gum arabic and sodium hexametaphosphate were obtained.

The particle dispersion obtained by this pH adjustment was ice-cooled to 5 ° C., 8 ml of a 25% by volume glutaraldehyde aqueous solution was added, and after stirring well, the mixture was allowed to stand at this temperature for two nights to insolubilize the particles. did. The particle dispersion was then centrifuged at 2000 rpm for 5 minutes to collect the particles as pellets. The washing operation of suspending these particles in a 0.01% demol EP solution and centrifuging was repeated three times, and then dispersed in a 4% by volume aqueous formalin solution, and allowed to stand at 5 ° C. for 1 week to give colored artificial particles A. , B or C were obtained.

(Example 3) Gelatin 5 having an isoelectric point of 9
g was dissolved in warm water at 40 ° C. so as to be 100 ml.
5 g of gum arabic was dissolved in 100 ml of water, impurities were filtered off, and the mixture was heated to 40 ° C. 0.5 ml of the aqueous gelatin solution thus obtained and 0.5 ml of the aqueous solution of gum arabic were mixed, and the mixture was preheated at 40 ° C.
It was poured into 272 ml of a 41% by volume ethanol solution which had been warmed up and stirred well. To this, 2.0 ml of 10% sodium hexametaphosphate aqueous solution, 20 ml of 10% by weight demole EP aqueous solution and 10% by volume aqueous acetic acid solution were added dropwise to adjust the pH to 6.0. Next, 200 ml of 25% by volume of the aqueous solution of the artificial particles (particles A, B and C) obtained in Example 2 was added, and after stirring well, a 10% by volume acetic acid aqueous solution was added dropwise to adjust the pH to 4.9. The obtained particle dispersion liquid was ice-cooled to 5 ° C., 2.0 ml of a 25% by volume aqueous glutaraldehyde solution was added, and the mixture was well stirred and left at this temperature for two nights to insolubilize the particles. Further, the particles were recovered by the same operation as in Example 2. Table 2 shows the particle sizes of the obtained artificial particles A, B or C. The average particle diameter can be determined by measuring with a Coulter Counter TAII (manufactured by Nikkaki Co., Ltd.). For comparison, the measurement results of sheep red blood cells and gelatin particles are shown.

[0019]

Examination of the mixing ratio of artificial particles composed of a mixture of gelatin, gum arabic, sodium hexametaphosphate and silica particles and the presence or absence of spontaneous aggregation, which is one of the properties of the particles for agglutination immunoassay, depending on the concentration of each gelatin. I went. Table 3 shows the examination results of the mixing ratio of the mixture with respect to the silica particles and the spontaneous aggregation. The amount of the mixture can be determined from the amount of the mixture added to the reaction vessel, and the amount of the mixture decreased from the solution after the reaction by adding silica.

Table 3 ------------------ Gelatin Concentration Mixture for Silica Particles of Artificial Particles Ratio of (%) (mg / g) Spontaneous aggregation −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− 0. 80 20.6 Yes 0.40 9.0 Yes 0.20 4.0 No 0.10 1.3 No 0.05 0.1 No ----------------------- −−−−−−−−−−−−−−−−−−−

(Test Example) Rheumatoid Factor Measurement Test The artificial particles A, B or C obtained in Example 3 were used at a concentration of 5%.
To a pH 7.2 of phosphate buffered saline (hereinafter referred to as PBS), and 20 ml thereof was mixed with 20 ml of PBS (pH 7.2) containing 5 ppm of tannic acid. After heating this mixture at 37 ° C for 10 minutes, centrifuge it and wash it thoroughly with physiological saline.
It was dispersed in BS (pH 6.4) to make the total amount 20 ml.
On the other hand, a heat-denatured rabbit polymer IgG fraction suspended in PBS of pH 6.4 was used as a sensitizing solution.

20 ml of the tannic acid-treated particle dispersion liquid and 20 ml of the sensitizing liquid were mixed and heated at 37 ° C. for 60 minutes. The sensitized artificial particles thus obtained were mixed with PB of pH 6.4.
Thoroughly wash with S, 19ml so that the particle concentration becomes 5%
And was lyophilized. Distilled water was added to this lyophilized product to restore it to the same volume as before lyophilization, and each serum was measured by passive agglutination reaction by the microtiter method. Table 4 shows the time (judgment time) required to obtain the measurement result by performing measurement using the antigen-bound artificial particle produced from the sensitized artificial particle A, B or C obtained in Example 3. For comparison, sensitized gelatin particles (product name: Cellodia (registered trademark) RA; manufactured by Fujirebio Co., Ltd.),
Table 4 shows the determination times for sensitized sheep erythrocytes produced by binding heat-denatured rabbit polymer IgG fractions to sheep erythrocytes by a conventional method using tannic acid.

[0024]

Further, the titer of the positive serum and the negative serum was measured by a passive agglutination reaction by the microtiter method using each particle. The results are shown in Table 5. Table 5 ------------------------- Serum Serum Dilution Ratio (x40) Sample Particles --- −−−−−−−−−−−−−−−−−−−−−−− 1 2 4 8 16 32 32 64 128 256 −−−−−−−−−−−−−−−−−− −−−−−−−−−−−−−−−−−−− Positive artificial particles C + + + + + + + + + − Positive gelatin particles + + + + + + + + + + − Positive sheep red blood cells + + + + + + + + − −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Negative Artificial Particle C −−−− − − − − − Negative gelatin particles − − − − − − − − − Negative sheep red blood cell − − − − − − − − − −−−−−−−−−−−−−−−−−−−−−− −−−−−−−−−−−−− - +: Positive decision -: negative decision

[0026]

The present invention is a new artificial particle obtained by mixing silica, gelatin, a water-soluble polysaccharide and a metaphosphate. For example, when an antigen or antibody is bound to these particles and used as a reagent for agglutination immunoassay, 15
The result can be obtained in minutes, and quick measurement is possible.
Further, accurate measurement can be performed without causing nonspecific aggregation.

Claims (2)

[Claims] 1. Particles obtained by mixing silica, gelatin, a water-soluble polysaccharide and a metaphosphate,
The mixture of gelatin, water-soluble polysaccharides and metaphosphate in the particles is 0.02 mg / g to silica.
50 mg / g, and the obtained particles have an average particle diameter of 0.5 μm to 10 μm. 2. The artificial particle according to claim 1, wherein the water-soluble polysaccharide is gum arabic.