JPS6332149B2 - - 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, 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 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, 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, 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 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 after pH adjustment to prevent agglomeration of the generated particles. The present invention was made based on the fact that in a method in which only water is used as a solvent and no hydrophilic organic solvent is used, the yield of the carrier can be increased by adding a surfactant in advance before adjusting the pH. That is, the present invention provides gelatin, a water-soluble polysaccharide,
An artificial carrier is prepared by adding an acid to an aqueous solution containing sodium polymetaphosphate and having a temperature higher than gelatin gelling temperature to adjust the pH to 2.5 to 6.0 with stirring, and then insolubilizing it by applying an aldehyde crosslinking agent. The present invention relates to a method for producing an artificial carrier, characterized in that the aqueous solution contains an anionic or nonionic surfactant before the pH adjustment. 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 arabic 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 colorants 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 Villette, Toluidine Blue, and Methylene Blue. 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 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 present invention is characterized in that an anionic or nonionic surfactant is further added to such a solution before pH adjustment. Examples of anionic surfactants include alkyl sulfosuccinic acids, alkyl sulfomaleic acids, alkyl sulfates, polyoxyethylene alkyl ether sulfates, and examples of nonionic surfactants include polyoxyethylene fatty acid esters. , polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyethylene glycol fatty acid ester, and the like. Surfactants are added for the purpose of preventing particle aggregation, but cationic surfactants cannot prevent particle aggregation and are therefore outside the scope of the present invention. Regarding the concentration in the solution before pH adjustment, an aggregation prevention effect can be obtained at a concentration of about 0.001 to 0.01% in the case of anionic surfactants and about 0.01 to 0.1% in the case of nonionic surfactants. By cooling the solution, aggregation can be prevented at lower concentrations. 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, since water-soluble polysaccharides often contain small amounts of insoluble components, it is better to dissolve them separately and add them. On the other hand, gelatin has a temperature below its isoelectric point.
At pH, it reacts with water-soluble polysaccharides and becomes cloudy, so when using acidic gelatin, it is best to add an alkali to raise the pH of the solution to at least around 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 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. After adding the acid, it is preferable to quickly cool the particle dispersion to prevent the formed particles from agglomerating. Then, when the liquid temperature becomes below the gelation temperature, preferably below 10°C, an aldehyde crosslinking agent is added to insolubilize the particles. The amount of this crosslinking agent added is about 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 the aldehyde crosslinking agent, the particles are collected by centrifugation or the like, and washed if necessary. Washing may be carried out two or three times with water containing the same surfactant at the same concentration as that used for particle dispersion. 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 greatly simplifies the manufacturing equipment and facilitates operation by not using such organic solvents as opposed to the prior invention which contains alcohol etc. as a solvent. The yield was improved by changing the timing of addition of the surfactant in the prior invention, which is not used for this purpose. 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 make 100 ml, and the pH was adjusted to 9 with 10% sodium hydroxide solution. 4g of gum arabic was dissolved in water to make 100ml, and after separating insoluble matter, the mixture was heated to 40°C. Mix 50 ml of the gelatin solution obtained in this way and 50 ml of gum arabic solution, and heat the mixture at 40°C.
was added to 300ml of distilled water. To this was added 1.6 ml of 10% sodium hexametaphosphate solution and 1% by volume polyoxyethylene phenyl ether (Emulgen A-
60, Kao Soap Co., Ltd. registered trademark) solution 8 ml, and 1%
6 ml of Direct Blue solution was added. 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 suspended in a 0.02 volume % Emulgen A-60 solution and centrifuged. The washing procedure was repeated three times, and then the particle concentration was adjusted to 5%.
It was dispersed in a volume% formalin solution and allowed to stand at 5°C for one week. The carrier particles obtained in this example weighed 6.2 g, of which 75
% was in the range of 3-6μ. Example 2 1% alkyl sulfomaleic acid (Demol Ep,
Using 2 ml of Kao Soap Co., Ltd. (registered trademark) solution, and
Carrier particles were prepared in the same manner as in Example 1, except that 6 ml of 1% Reactive Bread was used instead of 6 ml of 1% Direct Blue solution. However, the pH adjusted by dropping acetic acid was 4.6. The carrier particles thus obtained weighed 11 g, 75% of which were in the 3-6μ range. Example 3 Carrier particles were prepared in the same manner as in Example 1 except for the following points. That is, first, 1 g of carboxymethyl cellulose was used instead of 4 g of gum arabic, and 0.5 ml of 1% Demol Ep solution was used instead of 8 ml of 1% by volume Emulgen A-60 solution. Also, the amounts of gelatin, 10% sodium hexametaphosphate solution, 1% Direct Blue solution, and glutaraldehyde were all reduced to one quarter. Also, the pH was increased from 4.8 to 4.6. The carrier particles thus obtained weighed 4.8 g, of which 95%
It was in the range of 0.8 to 1.5μ. 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 40ml,
The gum arabic solution was changed from 50ml to 60ml. Then, 10% sodium hexametaphosphate solution
1.6 ml or 1.2 ml, 8 ml of 1% by volume Emulgen A-60 solution was changed to 2 ml of 1% Demol Ep solution, 6 ml of 1% Direct Blue was changed to 4.8 ml, and glutaraldehyde was changed to 1.3 g or 1.0 g. In addition, the pH at the end of acetic acid addition was set at 4.2. The carrier particles thus obtained weighed 9.6 g, and 90% of them were in the range of 1 to 2 microns. Example 5 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
The volume was changed from 1.6 ml to 2 ml, 1% by volume Emulgen A-60 solution was changed to 2 ml of 1% Demol Ep solution, 6 ml of 1% Direct Blue was changed to 7.2 ml, and glutaraldehyde was changed from 1.3 g to 1.8 g. Furthermore, the pH at the end of acetic acid addition was set at 4.6. The carrier particles thus obtained weighed 10.4 g, 75% of which were in the 3-6μ range. Usage Example 1 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. The mixture was heated at 37°C for 15 minutes, centrifuged, thoroughly washed with physiological saline, and adjusted to a pH of 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 Organ 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] Use Example 2 The carrier particles obtained in Example 1 were treated with tannic acid in the same manner as in Use Example 1. Then, the tannic acid-treated particles were heated to a pH of 7.2 so that the particle concentration was 1%.
Dispersed in 5 ml of PBS. Highly purified streptokinase at 32V/
ml of PBS at pH 7.2, and this solution was mixed with the particle dispersion liquid. The mixture was heated at 37°C for 30 minutes, and the particles were centrifuged and thoroughly washed with physiological saline. The particles were dispersed in a dispersion medium to a concentration of 5%, freeze-dried, and stored at 4°C. When the titer of the streptokinase-immobilized particles thus obtained was determined by a microplate method, the results shown in the table below were obtained.

【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 gelling temperature of gelatin is stirred and an acid is added thereto.
In an artificial carrier production method in which the pH is adjusted to 2.5 to 6.0 and then an aldehyde crosslinking agent is applied to make it insolubilized, an anionic or nonionic surfactant is added to the aqueous solution before the pH adjustment. A method for producing an artificial carrier characterized by containing the carrier.