JPS6332150B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved method for producing a novel artificial carrier that can be widely used for sensitizing antigens and antibodies, immobilizing enzymes, and the like. In fields such as clinical tests that utilize antigen and antibody reactions, antigens or antibodies are adsorbed or bonded to particles of an appropriate size as carriers, and the particles are sensitized by the presence of the corresponding antibodies or antigens. The method of causing aggregation of carriers is called indirect passive agglutination reaction. Since this indirect passive agglutination reaction can detect antibodies and antigens in a sample liquid with high sensitivity, it is widely used in serological and hematological diagnosis of various diseases. Carriers used in this reaction include non-biological particles such as polystyrene latex, kaolin, and charcoal powder, and biological particles such as animal red blood cells and bacterial cells. In general, non-biological particle carriers have advantages such as being chemically stable and having no antigenic activity themselves, but have the disadvantage that antigens or antibodies are difficult to adsorb closely. For example, when freeze-drying for storage, antigens and antibodies are released from the carrier. For this reason, it is unavoidable to store it in a liquid in a cool, dark place, but as a result, it cannot be stored for a long period of time. Furthermore, among non-biological carriers, it is difficult to select particles of a certain size for charcoal powder and kaolin, and for polystyrene latex, natural aggregation occurs due to non-specific aggregation in the neutral region, which is desirable as a reaction medium. There is a risk of causing On the other hand, although animal red blood cells and bacterial cells, which are biological carriers, have the advantage of having a constant size, the particle size is fixed depending on the type of organism, and can be adjusted to suit the purpose. It is not possible to obtain particles of size . For example, animal red blood cells are the most readily available carrier with a constant size, but they have unique antigens on the surface of the blood cells, and cross-react with antibodies, which is a non-specific agglutination reaction. This may cause errors in the agglutination reaction. Furthermore, the biological, chemical, and physical properties of red blood cells vary among individual animals, making it difficult to obtain blood cells of constant quality. As a result of various studies in order to develop an excellent carrier free of these drawbacks, the present inventors prepared a solution containing gelatin, a water-soluble polysaccharide, and sodium polymetaphosphate in a mixture of water, alcohol, etc. as a solvent under stirring. We discovered that by precipitating particles by adjusting the pH with water and insolubilizing these particles by treating them with an aldehyde-based crosslinking agent, an excellent carrier that overcomes all of the drawbacks of conventional methods can be obtained.We have already patented this content. An application was filed (Japanese Patent Application Laid-Open No. 153658/1983). Later, they discovered that an artificial carrier could be obtained in good yield without using a hydrophilic organic solvent such as alcohol, and filed a patent application for this patent (Japanese Patent Laid-Open No. 160465/1983).
However, in both methods, a surfactant was added to prevent the particles produced from agglomerating. As a result of further research, the present inventors found that even when the solvent is water only and no hydrophilic organic solvent is used, if particles are formed by adding an acid and then cooled quickly, the surfactant The inventors have discovered that carrier particles can be obtained at a good yield comparable to when a surfactant is added without the addition of a surfactant, and based on this finding, the present invention has been completed. That is, the present invention provides gelatin, a water-soluble polysaccharide,
Production of an artificial carrier by adding acid to an aqueous solution containing sodium polymetaphosphate and having a temperature higher than the gelling temperature of gelatin, adjusting the pH to 2.5 to 6.0 with stirring, and then insolubilizing it by applying an aldehyde crosslinking agent. A method for producing an artificial carrier, characterized in that after particles are generated by the addition of the acid, the particle dispersion is cooled to 10°C or lower, and the crosslinking agent is allowed to act at a temperature of 10°C or lower. It is something. As the gelatin used in the present invention, commercially available gelatin may generally be used as is. Among commercially available products, acidic gelatin is preferred. Water-soluble polysaccharides can be used as thickeners or thickeners, and also include derivatives and salts of polysaccharides. Examples include gum arabic, carboxymethyl cellulose, sodium alginate, agar,
Examples include carrageenan, and gum albia is particularly preferred. Sodium polymetaphosphate has the chemical formula ( _NaPO3 )
It is a substance represented by _o , such as sodium tetrametaphosphate and sodium hexametaphosphate. Alternatively, if the carrier is to be colored, a coloring agent may be added to the solution before particle formation. An example where coloring is required is when the product of the present invention is used as a carrier for an indirect passive aggregation reaction. That is, since the product of the present invention is normally colorless and opaque, by coloring it, the agglomerated image can be easily determined. Examples of coloring agents include red pigments such as Food Red No. 3, Rhodamine, Rose Bengal, Ponceau 3R, Bordeaux S, Fuchsin, Eosin, and Neutral Red, or blue pigments such as Crystal Violet, Toluidine Blue, and Methylene Blue. can be used. However, if colored with reactive dyes such as Reactive Red and Direct Blue, the color will not fade, so in addition to the coloring agent for which reactive dyes are particularly suitable, various substances can be added depending on the purpose. Needless to say, this is a good thing. The concentration of each of these substances in the solution before pH adjustment is about 0.01 to 2% gelatin, preferably
About 0.05-1.0%, and water-soluble polysaccharides 0.01-2%
degree, preferably about 0.05 to 1.0%. Sodium polymetaphosphate is 0.5 of the dry weight of gelatin.
It is preferable to contain about 20%. Each substance may be appropriately determined within these concentration ranges depending on the particle size and physical properties of the desired particles. When adding a coloring agent, the amount is usually about 0.005 to 0.5%, but if a reactive dye is used, about 1 to 5% of the dry weight of gelatin is sufficient. The process for preparing such a solution is not critical; for example, each may be dissolved in warm water and then mixed, or each may be dissolved together. However, in order to facilitate the dissolution of each substance, it is best to add a hydrophilic organic solvent later, and since water-soluble polysaccharides often contain small amounts of insoluble components, they must be dissolved separately. It is good to add it. On the other hand, gelatin reacts with water-soluble polysaccharides and becomes cloudy at a pH below its isoelectric point, so when using acidic gelatin, it is advisable to add an alkali to raise the pH of the solution to at least around that point. However, even after this cloudiness occurs, it can be eliminated by adding an alkali. In any case, the solution must be free from cloudiness before starting the addition of acid. The temperature of the solution must be above the gelatin temperature. This temperature varies depending on the concentration of gelatin, etc., but is usually about 25 to 30°C. From the viewpoint of good particle formation, the temperature is preferably about 35 to 50°C. Next, add acid to this solution while stirring to make the pH
Adjust to 2.5-6.0. This step is where particles are generated. In order to form uniform particles, it is best to continue heating to 35-50°C and drop the acid while stirring moderately. The optimum PH in the range of PH2.5 to 6.0 varies depending on the composition of the raw material solution and the target particle size, so it is best to determine it in advance by conducting experiments. For example, when the obtained particles are used as an antigen-sensitizing carrier, the particle size is preferably about 2 to 10 μm, and in this case, the optimum pH is in the range of 4.0 to 5.5. The acid used for this pH adjustment is not particularly limited and may be an inorganic acid or an organic acid, but it is preferable to use one that is as mild as possible, and for example, acetic acid is preferable. The particles produced in this step do not disappear even if the temperature of the system is lowered below the gelatin temperature, so there is no equilibrium relationship with the mother liquor. In addition, most of the particles are negatively charged, and the cations in the solution are oriented on their surfaces, forming a so-called electric double layer. This also promotes stable dispersion of particles. In the present invention, after adding an acid to generate particles, the particle generation liquid is cooled to a temperature below the gelling temperature,
The feature is that this makes it unnecessary to add a surfactant. Cooling is preferably carried out quickly after the addition of acid is completed. If the mixture is left at room temperature after addition, the particles will collide with each other and agglomeration will begin. The temperature is below the gelation temperature, preferably below 5°C. After particle generation, the particles are insolubilized using an aldehyde crosslinking agent, but during this time it is necessary to maintain the particle dispersion at a temperature below the gelling temperature.
However, it is sufficient to maintain this temperature until insolubilization progresses to the extent that particles can be easily redispersed. The amount of aldehyde crosslinking agent added is 0.1 to 200 of the dry weight of gelatin.
%, and after addition, it is left to stand for about one night to allow the crosslinking reaction to occur sufficiently. Examples of the crosslinking agent include glutaraldehyde, formaldehyde, glyoxal, crotonaldehyde, acrolein, and acetaldehyde, with glutaraldehyde being particularly preferred. After treatment with an aldehyde crosslinking agent, the particles are collected by centrifugation or the like and washed. Cleaning is done with an anionic surfactant aqueous solution of about 0.001-0.01% or 0.01-0.01%.
2 using a 0.1% nonionic surfactant aqueous solution.
You only need to do this 3 times. The carrier thus obtained may be used for various purposes, but if crosslinking is insufficient, it may swell in a salt solution. Therefore, when used for such purposes, it is recommended to treat with an aldehyde crosslinking agent to prevent swelling. For example, when sensitizing an antigen, it is carried out in a phosphate buffer, so formalin treatment is performed under conditions that fix red blood cells. This treatment prevents swelling and, due to the sterilizing effect of formalin, provides a carrier that can be stored for a long period of time. The carrier of the present invention can immobilize a wide variety of antigens, antibodies, enzymes, and the like. For example, when sensitizing an antigen or antibody, a conventional method using animal red blood cells as a carrier may be used. The carrier of the present invention has performance equivalent to that of animal red blood cells, which was conventionally considered to be the best carrier for indirect passive agglutination reactions, and is chemically and physically homogeneous and stable, has no antigenic activity, and has no antigenic activity. It has many advantages over animal red blood cells, such as the ability to easily and inexpensively mass-produce particles of a certain size. The present invention eliminates the use of organic solvents and surfactants in contrast to the prior invention, and the present invention makes it possible to easily and conveniently produce an artificial carrier having such excellent properties. Can be done. By eliminating the need for organic solvents, the production equipment is simplified and operations are facilitated, and the carrier obtained by this method can also be used, for example, in applications where the presence of surfactants is undesirable. Examples and usage examples of carriers are shown below. In addition,
In this specification, unless otherwise specified, % is % by weight
It represents. Example 1 4 g of gelatin having an isoelectric point of PH9 was dissolved in 40°C warm water to a total volume of 100 ml, and the pH was adjusted to 9 with a 10% sodium hydroxide solution. 4g gum arabic
was dissolved in water to a total volume of 100 ml, and after removing insoluble matter, the mixture was heated to 40°C. 50 ml of gelatin solution and 50 ml of gum arabic solution were mixed, and to this were added 300 ml of distilled water at 40°C, 1.6 ml of 10% sodium hexametaphosphate solution, and 6 ml of 1% Direct Blue solution. This solution was then kept at 40°C and stirred.
A 10% by volume acetic acid solution was added dropwise to adjust the pH to 4.8 to generate particles. The particle dispersion was ice-cooled to 5° C., 1.3 g of glutaraldehyde was added thereto, and after thorough stirring, it was allowed to stand at this temperature overnight. Then this particle dispersion
The particles were collected as a pellet by centrifugation at 2000 rpm for 10 minutes. The particles were dispersed in a 4% by volume formalin solution so that the particle concentration was 5%, and the mixture was heated at 5°C.
It was left undisturbed for one week. The particles obtained in this example weighed 7.5 g, and 75% of them were in the range of 2 to 3.2 microns. Example 2 Carrier particles were prepared in the same manner as in Example 1 except for the following points. That is, instead of 4 g of gum arabic, use 1 g of carboxymethyl cellulose, gelatin, 10
% sodium hexametaphosphate solution, 1% Direct Blue solution, and glutaraldehyde were all reduced to one quarter. Also, the pH is 4.8
I changed it from 4.2. The carrier particles thus obtained weighed 5.6 g, 90% of which were in the 1-2 micron range. Example 3 Carrier particles were prepared in the same manner as in Example 1 except for the following points. That is, increase the gelatin solution from 50ml to 40ml,
The gum arabic solution was changed from 50ml to 60ml. Then, 10% sodium hexametaphosphate solution
From 1.6ml to 1.2ml, add 6ml of 1% Direct Blue.
to 4.8ml, and glutaraldehyde from 1.3g.
Each was changed to 1.0g. Also, the pH at the end of acetic acid addition is
was set to 4.2. The carrier particles thus obtained weighed 10.4 g, 90% of which were in the 1-2 micron range. Example 4 Carrier particles were prepared in the same manner as in Example 1 except for the following points. That is, increase the gelatin solution from 50ml to 60ml,
The gum arabic solution was changed from 50ml to 40ml. Then, 10% sodium hexametaphosphate solution
From 1.6ml to 2ml, add 6ml of 1% Direct Blue.
to 7.2ml, and glutaraldehyde from 1.3g.
I changed it to 1.8g each. Also, the pH at the end of acetic acid addition is
was set at 4.7. The carrier particles thus obtained weighed 11.2 g, 70% of which were in the 6-9μ range. Usage example The carrier particles obtained in Example 1 were added to phosphate buffered saline (hereinafter referred to as
Abbreviated as PBS. ) and 20ml of it to 5ppm
of PBS containing tannic acid at pH 7.2. After warming the mixture at 37℃ for 15 minutes, centrifuge it, wash it thoroughly with physiological saline, and adjust the pH to 5%.
6.4 in PBS to make the total volume 20 ml. On the other hand, PH
The syphilis pathogen Treponema pallidum suspended in PBS in 6.4 was destroyed by sonication and used as an antigen solution. 20 ml of the tannic acid-treated particle dispersion and 20 ml of the antigen solution were mixed and heated at 37° C. for 40 minutes. The antigen-sensitized particles thus obtained were thoroughly washed with PBS of PH6.4,
The particles were dispersed in 19 ml of dispersion medium to a particle concentration of 5% and freeze-dried. Distilled water was added to this freeze-dried product to reconstitute it to the same volume as before freeze-drying, and the titer of the syphilis-positive serum was measured using the microtiter plate method. The results are shown in the table below. In addition, a commercially available TPHA kit (manufactured by Fuji Zoshihin Pharmaceutical Co., Ltd.) using sheep red blood cells as a carrier is available.
The results of similar measurements using the same method are also shown. 【table】

Claims (1)

[Claims] 1. An aqueous solution containing gelatin, a water-soluble polysaccharide, and sodium polymetaphosphate and having a temperature equal to or higher than the gelation temperature of gelatin is mixed with an acid while stirring.
In an artificial carrier production method in which the pH is adjusted to 2.5 to 6.0 and then an aldehyde-based crosslinking agent is applied to make it insolubilized, particles are generated by the addition of the acid and then the particle dispersion is cooled to below the gelation temperature. A method for producing an artificial carrier, characterized in that the crosslinking agent is allowed to act at a temperature below the gelation temperature.