JPS58113755A - Production of artificial carrier - Google Patents

Abstract

PURPOSE:To obtain a carrier which can be preserved for a long period of time by adjusting a soln. contg. gelatin, water soluble polysaccharides, sodium polymetaphosphate, as well as a hydrophilic org. solvent and water in a specific pH range by adding acids thereto to form particles then cooling the same to temp. lower than specific temp. and effecting crosslinking agents therewith. CONSTITUTION:Acids (acetic acid, etc.) are added to a soln. contg. gelatin, water soluble polysaccharides (gum arabic, etc.), sodium polymetaphosphate, and a hydrophilic org. solvent (alcohol, etc.) and water under stirring at temp. higher than the gelling temp. of the gelatin to adjust 2.5-6.0pH and to form a dispersion of gel particles. After the formation of the particles, the soln. is cooled and when the temp. thereof drops to <=10 deg.C, crosslinking agents (glutaraldehyde, CH3CHO, etc.) are added thereto to obtain crosslinked particles. The crosslinked particles are centrifugally separated and washed, whereby the carrier to be deposited thereon with antigens, etc. for serodiagnosis, etc. is obtained. This carrier is obtained in optional grain sizes according to production conditions, is good in yield, permits preservation for a long period of time and is usable anytime without liberation of antigens, etc. even when the carrier is bound with the antigens, etc. and is frozen and preserved.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improvement in the production method of a novel artificial carrier that can be widely used in medicine, etc. 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 presence of the corresponding antibodies or antigens induces sensitization. The method of causing aggregation of carriers is called indirect passive aggregation reaction. Since this indirect passive agglutination reaction can detect antibodies and antigens in a sample liquid with high sensitivity, it is widely used for serological diagnosis of various diseases.

Carriers used in this reaction include non-biological carriers such as polystyrene latex, kaolin, and charcoal powder, and biological particles such as animal red blood cells and bacterial cells. In general, non-biological particulate carriers have advantages such as being biologically stable and having no antigenic activity themselves; The drawback is that it is difficult for the body to be absorbed tightly. 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 polystyrene latex exhibits spontaneous aggregation, which is non-specific aggregation, in the neutral region, which is desirable as a reaction medium. On the other hand, although animal red blood cells and bacterial cells, which are biological carriers, have the advantage of having a fixed size, the size of the particles is fixed depending on the type of organism, and the size of the particles is determined according to the purpose. It is not possible to obtain particles of arbitrary 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, resulting in the desired agglutination. This may lead to erroneous reactions.

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 that does not have 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 through one adjustment, and insolubilizing these particles by treating them with an aldehyde-based crosslinking agent, we could obtain an excellent phase that eliminated all of the drawbacks of conventional methods, and we have already applied for a patent on this matter. . In this method, a surfactant was added after adjustment to prevent agglomeration of the generated particles, but as a result of further research by the present inventors, it was found that after the particles were generated by the addition of acid. It was discovered that by cooling quickly, carrier particles can be obtained without adding a surfactant at a good yield comparable to when a surfactant is added, and based on this finding, the present invention was completed. Ta. That is, in the present invention, a solution containing gelatin, a water-soluble polysaccharide, sodium polymetaphosphate, a hydrophilic organic solvent, and water and having a temperature equal to or higher than the gelatin temperature is adjusted to pH by adding an acid while stirring.
2.5 to 6.0, and then insolubilized by applying an aldehyde crosslinking agent, after particles are generated by adding the acid, the particle dispersion is cooled to 10 ° C. or less, The present invention relates to a method for producing an artificial carrier, characterized in that the crosslinking agent is allowed to act at a temperature of 10° C. or lower.

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, carrageenan, etc., with gum arabic being particularly preferred.

Polymetaphosphate puttrium has the chemical formula (Na PO3)n
Examples of such substances include sodium tetrametaphosphate and sodium hexametaphosphate.

As the hydrophilic organic solvent, lower alcohols, methyl alcohol, ethyl alcohol, flobyl alcohol, and acetone can be used.

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. As a coloring agent, food red No. 3,
Rhodamine, Rose Pen, Ponceau 3R1? Red dyes such as Ludo 81 fuchsin, eosin, and neutral red, or blue dyes such as crystal violet, toluino/blue and methylene blue, etc. can be used. However, reactive dyes such as Reactive Red and Direct Blue do not cause color fading, so reactive dyes are particularly suitable. It goes without saying that in addition to the coloring agent, various substances may be added depending on the purpose.

The concentration of each of these substances in the solution before adjustment is as follows:
Zelab 10.01 to 2 inches, preferably 0.05 to 1
．． The volume of the water-soluble polysaccharide is about 0.01 to 2 fi, preferably about 0.05 to t, ol, and the volume of the hydrophilic organic solvent is about 4 to 25. /IJ Sodium metaphosphate should preferably be contained in an amount of about 3 to 15 g of the dry weight of gelatin. 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 colorant, it is usually 0005 to 0.
．． The amount is about 5%, but if a reactive dye is used, about 1 to 5% of the dry weight of gelatin is sufficient.

The process of preparing such a solution is not in question;
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.

On the other hand, when gelatin is below its isoelectric point, it reacts with water-soluble polysaccharides and becomes cloudy, so acidic gelatin is used.

If there is, it is a good idea to add an alkali to raise the - level of the solution to at least that level. 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 the acid addition begins.

The temperature of the solution must be above the gelatin's gelatinization 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 particularly preferably about 35 to 50°C.

Next, add acid to this solution while stirring and add -2,5 to 6
．． Adjust to 0. This step is where particles are generated. In order to form uniform particles, it is preferable to continue heating at 35 to 50°C and drop the acid while stirring moderately. The optimum pH within the range of pH 2.5 to 6.0 varies depending on the composition of the raw material solution and the intended particle size, and is therefore preferably determined by conducting experiments. For example, when the obtained particles are used as an antigen-sensitizing carrier, the square size is about 2 to 10μ.

It is best to set the particle size to 4.0 to 5.
It is in the range of 5. The acid used in this tone IIK is not particularly limited and may be an inorganic acid or an organic acid, but it is preferably as mild as possible, for example acetic acid is preferred.

The particles produced in this step do not disappear even if the temperature of the system is lowered below the gelatinization temperature, so there is no equilibrium relationship with the mother liquor. Furthermore, although it depends on the mixing ratio of gelatin and water-soluble polysaccharide, the particles are almost always positively charged, and polymetaphosphate ions are oriented on their surfaces, forming a so-called electric double layer. . This also promotes stable dispersion of particles.

The present invention is characterized in that after adding an acid to generate particles, the particle generation liquid is cooled to 10° C. or lower, thereby eliminating the need for adding a surfactant. It is preferable that the cooling be carried out immediately after the addition of the acid is completed. If the mixture is left at room temperature after addition, the particles will collide with each other and begin to aggregate.

The temperature is 10°C or lower, preferably 5°C or lower. After particle generation, the particles are insolubilized with an aldehyde crosslinking agent, and during this time it is necessary to maintain the particle dispersion at a temperature of 10° C. or lower. However, it is sufficient to maintain this temperature until insolubilization has been achieved to the extent that particles can be easily redispersed.

The amount of aldehyde crosslinking agent added is 0.00% of the dry weight of gelatin.
The amount is about 1 to 200 mm, and after addition, it is left to stand for about one night to allow the crosslinking reaction to occur sufficiently. Examples of crosslinking agents include glutaraldehyde, formaldehyde, glyoxal,
Examples include crotonaldehyde, acrolein, acetaldehyde, etc., with glutaraldehyde being particularly preferred.

After treatment with an aldehyde-based crosslinking agent, the particles are collected by centrifugation or the like and washed. Washing may be carried out 2 to 3 times using an aqueous solution of an anionic surfactant of about 0.001 to 0.01 inch or an aqueous solution of a nonionic surfactant of about 0.01 to 0.1 inch.

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 gel/acid buffer, so formalin treatment is performed under conditions that fix red blood cells.

This treatment prevents swelling and provides a carrier that can withstand long-term storage due to the sterilizing effect of formalin.

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 previously thought to be insufficient in quantity, as a carrier for indirect passive agglutination reactions, and is chemically and physically homogeneous and stable, 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 any particle size. Further, the present invention, as compared to the prior invention, eliminates the need for a surfactant by cooling the particles after they are generated until they are insolubilized. Therefore, the present invention is particularly effective when the carrier is used in applications where the presence of a surfactant is undesirable, for example.

Examples and usage examples of carriers are shown below. In this specification, unless otherwise specified, % represents weight %.

Example 1 Gelatin 4F, whose isoelectric point is P) (9), was dissolved in warm water at 40°C to a concentration of 10 (1+/!), and adjusted to P) (9) using a 10T sodium hydroxide solution. 4f of gum arabic was dissolved in water to a concentration of 100 m/g, and the insoluble matter was heated to 40°C on the r side edge.

Mix 50 ffiz of the above gelatin solution and 5 g of gum arabic solution (it/!), add 300 ml of 30 volume thiethyl alcohol solution, 1.5 ml of 1'0% sodium hexametaphosphate solution, and 6 g of 1 titanium lactate solution. was added, stirred thoroughly, and the whole was heated to 40°C.Next, 10 volumes of acetic acid solution was added dropwise to the mixture while stirring to adjust the pH to 5.0.

::・.

Immediately place the particle dispersion produced by one adjustment in a water bath for 50 minutes.
It was cooled to 0.degree. C. and 1.32 g of glutaraldehyde was added.

After stirring well, the mixture was kept at 5° C. overnight. Then, this particle dispersion was centrifuged for 10 minutes using 2QQQrpn, and the particles were collected as red.
The washing procedure of turbidity and centrifugation was repeated three times in a 5% alkylsulfomaleic acid (Demol E, Kao Soap Co., Ltd. registered trademark) solution, and then dispersed in a 4 volume t% formalin solution.
It was left standing at 5°C for one week.

The yield of the carrier particles thus obtained was 6.42, and 75 particles of the obtained particles were in the range of 3 to 6μ. was prepared.

That is, first, instead of 4 parts of gum arabic, 12 parts of calcined methylcellulose, and 1 part of 1 part of Direct Blue solution instead of 5 ml of 1 part of Acty Red solution.
．． 5 ml was used. Also, the amounts of gelatin, 10% hexametaphosphoric acid solution, and glutaraldehyde were reduced to 1/414.

Also, the end of acetic acid addition was set to 4.6.

The yield of carrier particles thus obtained was 4.5f, and 95% of the particles obtained were in the range of 0.8-1.5μ.

Example 3 Carrier particles were prepared in the same manner as in Example 1 except for the following points.

That is, the gelatin solution was changed from 5omt to 40ml,
Then, the gum arabic solution was made up from 50ffi/ to 6Qml. Then, from 1.6 ml to 1.2 ml of 10% sodium hexametaphosphate solution, add 1 tiliacte 4
Bray Dro 1R1 1% Direct Blue 4.811I
K.

Then, the glutaraldehyde was changed by 1.3F and 1.0PK respectively. Moreover, the end of acetic acid addition was set at 4.6.

The yield of carrier particles obtained in this example was 7.8f, and 90% of the particles obtained were in the range of 1 to 2μ.

Example 4 Carrier particles were prepared in the same manner as in Example 1 except for the following points.

That is, the gelatin solution was changed from 50 liters to 6011,
The gum arabic solution was changed from 50+u/ to 4Qntl. Then add 10% sodium hexametaphosphate solution to 1.6
The amount of glutaraldehyde was changed from 5 ml to 7.2 ml of 1-tidirect blue, and from 132 to 1.8 g of glutaraldehyde. In addition, the completion of acetic acid addition was set at 4.8.

The yield of carrier particles obtained in this example was 9.6 mm, and 75 particles of the obtained particles were in the range of 3 to 6 microns.

Usage Example 1 The carrier particles obtained in Example 1 were dispersed in phosphate buffered saline (hereinafter abbreviated as PBS) of pH 7.2 to a concentration of 1%, and the 5d was dispersed at 5 ppm. of PBS 5 yrl, pH 7.2, containing tannic acid.

The mixed solution was heated at 37°C for 15 minutes, centrifuged, thoroughly washed with physiological saline, and then dispersed in 85 ml of pH 7.2 to give a concentration of 0 and 1 iKff streptokinase. P) to be 32 UAI
(Dissolved in 7,2 PBS.

5 ml of the tannic acid-treated particle dispersion and 5 ml of the streptokinase solution were mixed and heated at 37° C. for 30 minutes. Thereafter, the particles were centrifuged, washed three times with physiological saline, dispersed in a dispersion medium to a concentration of 5%, and freeze-dried.

The titer of the Strezotokinabi-immobilized particles thus obtained was determined by a microplate method. The results are shown in the table below.

Patent applicant: Fuji Organ Pharmaceutical Co., Ltd. Agent Patent Attorney 1) Masahiro Naka

Claims (1)

[Claims] gelate/, water-soluble polysaccharide, polymetaphosphate,
A solution containing a hydrophilic organic solvent and water and having a temperature higher than the gelatin gelatin temperature is heated by adding an acid while stirring.
In the method for manufacturing an artificial carrier in which H2 is adjusted to 5 to 6.0 and insolubilized by applying an aldehyde crosslinking agent after sonication, particles are generated by the addition of the acid, and then the particle dispersion is cooled to below IOC, 1. A method for producing an artificial carrier, characterized in that the crosslinking agent is applied at a temperature of 10° C. or lower.