JPS6348021B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to 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 bound to particles of an appropriate size as carriers,
The method of causing agglutination of this sensitized carrier by the presence of the respective antibodies or antigens is called indirect passive agglutination. 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 to develop an excellent carrier free of these drawbacks, the present inventors succeeded in producing a novel carrier for immobilizing active proteins using gelatin, water-soluble polysaccharide, and metaphosphate as raw materials. The present invention was completed based on the discovery that this carrier is excellent in that it overcomes all the drawbacks of the conventional carrier. That is, the present invention contains gelatin, a water-soluble polysaccharide, and a metaphosphate ion, and the amino groups of gelatin are cross-linked with aldehyde, and the gelatin is dissolved in 0.15M phosphate buffered saline with a pH of 7.2 measured at 25°C. The electrophoretic mobility is between −1.3 and −0.5 μm/sec/V/cm,
The present invention relates to a carrier for immobilizing an active protein, which is characterized by being spherical, hydrophilic and insoluble particles with a particle size of 1 to 100 microns. First, the carrier of the present invention contains gelatin, a water-soluble polysaccharide, and metaphosphate ions as constituent components. Gelatin is a type of protein obtained from collagen. Among gelatins, acidic gelatin is particularly 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. Metaphosphate ions are anions that are formed when some cations of metaphosphate are separated, and the number of charges depends on the pH at which the carrier particles are formed, the type of metaphosphate used as a raw material, etc. Determined. Metaphosphate is a substance represented by the chemical formula: (MPO ₃ ) _o M: cation, n=3 to 6, and includes, for example, sodium trimetaphosphate and sodium hexametaphosphate. In the carrier particles, the amino groups of gelatin are crosslinked with aldehyde, and the water-soluble polysaccharide and metaphosphate ion are electrostatically bonded to gelatin. Although metaphosphate ions are contained inside the particles, they are thought to surround the surface in particular to form an electric double layer. Most of these carrier particles are water, and their composition is approximately 1 to 15% gelatin, approximately 0.5 to 8% water-soluble polysaccharide, and 0.05 to 3% metaphosphate ion.
%, and the water content is about 70-90%. When the carrier particles are colored, they contain a dye, and depending on the manufacturing method, they may also contain a surfactant, a hydrophilic organic solvent, and the like. The physical properties of the carrier particles of the present invention are shown below. (1) Electrophoretic mobility The electrophoretic mobility of the carrier particles of the present invention in 0.15M phosphate buffered saline (hereinafter abbreviated as PBS) with a pH of 7.2 at 25°C is -1.3 to -0.5μ.
m/sec/V/cm. (2) External shape: Spherical shape with a diameter of 1 to 100μ. (3) Color Colorless and opaque unless specifically colored. (4) Attitude towards water Hydrophilicity (5) Immobilization of active proteins The carrier particles of the present invention can be used to sensitize sheep red blood cells with antigens or antibodies, and can be used to sensitize sheep red blood cells with antigens or antibodies. Other active proteins such as can be immobilized. Examples of such sensitization methods include tannic acid, formalin,
Examples include methods using glutaraldehyde, pyruvitaldehyde, bis-diazotized benzidine, toluene-2,4-diisocyanate, and the like. In addition, the carrier particles of the present invention can be used to immobilize enzymes or antigens by a known method of immobilizing enzymes using amino groups, carboxyl groups, hydroxyl groups, etc.
Various active proteins such as antibodies can also be immobilized. Examples of such immobilization methods include diazo coupling method and acid azidization method. Such carrier particles of the present invention can be prepared by adding an acid to an aqueous solution containing gelatin, a water-soluble polysaccharide, and a metaphosphate while stirring to adjust the pH to 2.5 to 6.0, and then adding an aldehyde crosslinking agent. It can be produced by insolubilizing it by acting on it. In this case, the concentration of each of these substances in the solution before pH adjustment is about 0.01 to 2% of gelatin, preferably about 0.05 to 1.0%, and water-soluble polysaccharide.
It is about 0.01 to 2%, preferably about 0.05 to 1.0%. Metaphosphate is 3-15% of gelatin dry weight
It is preferable to include a certain degree. Each substance may be appropriately determined within these concentration ranges depending on the particle size and physical properties of the desired particles. Other ingredients such as a hydrophilic organic solvent, a surfactant, and a coloring agent are added as appropriate to the solution before pH adjustment. Hydrophilic organic solvents can improve the physical properties of the particles produced, particularly their dispersibility and hardness. Examples of hydrophilic organic solvents that can be used include lower alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol, and acetone, and the preferred concentration is about 4 to 25% by volume. The purpose of the surfactant is to prevent agglomeration of the generated particles and maintain dispersion, and an anionic or nonionic surfactant is preferable. 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. In the case of anionic surfactants, the concentration in the solution before pH adjustment is
Agglomeration prevention effects can be obtained at about 0.001 to 0.01%, and in the case of nonionic surfactants, about 0.01 to 0.1%.
By cooling the solution, aggregation can be prevented at lower concentrations. If the carrier is to be colored, it is preferable to add a coloring agent 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. Coloring agents include, for example, 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. 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. When adding colorants, usually
The amount is 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. 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 falls below the gelling temperature, an aldehyde-based crosslinking agent is added to insolubilize the particles. The amount of this 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 the aldehyde crosslinking agent, the particles are collected by centrifugation or the like, and washed if necessary. The washing may be carried out two or three times using water containing the same surfactant at the same concentration as the surfactant 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 has performance equivalent to that of animal red blood cells, which has been considered to be the best carrier for indirect passive agglutination reactions, and is chemically and physically homogeneous and stable, free of antigenic activity, and free from antigenic activity. It has many advantages over animal red blood cells, such as the ability to easily and inexpensively mass-produce particles with a particle size of Examples of manufacturing and usage of the carrier are shown below. In this specification, % represents weight % unless otherwise specified. Production 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 using a 10% sodium hydroxide solution. 4 g of gum arabic was dissolved in water to make 100 ml, and after removing insoluble materials, the mixture was heated to 40°C. 50 ml of the gelatin solution thus obtained and 50 ml of the gum arabic solution were mixed, and this mixed solution was poured into 300 ml of a 30% by volume methyl alcohol solution previously heated to 40° C., and the mixture was thoroughly stirred. to this
1.6ml of 10% sodium hexametaphosphate solution, 1
% alkyl sulfomaleic acid (trade name Demol
E _p , 2 ml of Kao Soap Co., Ltd. product solution, and 1%
6 ml of Direct Blue solution was added and stirred thoroughly. Next, while maintaining the temperature at 40°C, a 10% by volume acetic acid solution was added dropwise to adjust the pH to 4.8 to generate particles. After cooling the particle dispersion obtained by this pH adjustment to 5°C, 1.3 g of glutaraldehyde was added.
was added, stirred well, and left at this temperature overnight. The particle dispersion was then centrifuged at 2000 rpm for 10 minutes to collect the particles as pellets. A washing operation in which the particles were suspended in a 0.005% Demol _Ep solution and centrifuged was repeated three times, then dispersed in a 4% by volume formalin solution and left at 5°C for one week. The carrier particles obtained in this example weighed 7.7g;
75% were in the range of 3-6μ. The electrophoretic mobility of this carrier particle was measured. The device is Laser Zee System 3000 manufactured by Pen Ken Inc. in the United States.
The measurements were carried out at 25°C using a microcomputer (system 3000).
As for the measurement method, this particle is 0.15M with a pH of 7.2.
Suspend in PBS and add 1 ml of it to a diameter of 1 mm and a length of
Place in a 20 mm cylindrical electrophoresis cell, per 1 m
The migration speed was measured by applying a voltage gradient of 1048V.
As a result, the electrophoretic mobility of this particle is −0.969±
It was 0.009μm/sec/V/cm. On the other hand, when we measured the electrophoretic mobility of uncolored particles produced under the same conditions, we found that -0.972±0.012μm/sec/V/
It was cm. Production Example 2 Carrier particles were prepared in the same manner as Production Example 1 except for the following points. That is, first, 1 g of carboxymethyl cellulose was used in place of 4 g of gum arabic, and a 30 volume % ethyl alcohol solution was used in place of the 30 volume % methyl alcohol solution. Also, the amounts of 10% gelatin sodium hexametaphosphate solution, 1% Demol _Ep 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 3.8 g, 90% of which were in the 1-2 micron range. Production Example 3 Carrier particles were prepared in the same manner as Production 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
1.6ml to 1.2ml, 1% Direct Blue solution from 6ml to 4.8ml, and glutaraldehyde.
The amount was changed from 1.3g to 1.0g. In addition, the pH at the end of acetic acid addition was set at 4.6. The carrier particles obtained in this example weighed 6.4g;
90% were in the range of 1-2μ. The electrophoretic mobility of this carrier particle was measured in the same manner as in Production Example 1: -0.939±0.059μm/sec/V/cm
It was hot. Production Example 4 Carrier particles were prepared in the same manner as Production Example 1 except for the following points. That is, the gelatin solution was changed from 50 ml to 60 ml, and the gum arabic solution was changed from 50 ml to 40 ml. Then add 1.6% 10% sodium hexametaphosphate solution
ml to 2ml, add 6ml of 1% Direct Blue solution
to 7.2 ml, and 1.3 g of glutaraldehyde.
I changed it from 1.8g to 1.8g. Furthermore, the pH at the end of acetic acid addition was set at 4.8. The yield of carrier particles obtained in this example was 8.6 g, and 75% of the particles obtained were in the range of 3 to 6 μm. The electrophoretic mobility of this carrier particle was measured in the same manner as in Production Example 1: -0.996±0.040μm/sec/V/cm
It was hot. Production Example 5 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 using a 10% sodium hydroxide solution. 4 g of gum arabic was dissolved in water to a total volume of 400 ml, and after separating insoluble matter, the mixture was heated to 40°C. Mix 50 ml of the above gelatin solution and 50 ml of gum arabic solution, add 300 ml of 30% by volume ethyl alcohol solution, 1.6 ml of 10% sodium hexametaphosphate solution, and 6 ml of 1% reactive bread solution, stir well, and mix thoroughly. was heated to 40°C. Next, a 10% by volume acetic acid solution was added dropwise to the mixture while stirring to adjust the pH to 5.0. The particle dispersion produced by pH adjustment was immediately cooled to 5°C in an ice bath, and glutaraldehyde 1.3
g was added. After stirring well, the mixture was allowed to stand overnight at 5°C. Then this particle dispersion was heated at 2000 rpm.
The particles were collected as a pellet by centrifugation for 10 minutes. The particles were suspended in a 0.005% Demol _Ep solution and centrifuged. The washing procedure was repeated three times, and then
It was dispersed in a volume% formalin solution and allowed to stand at 5°C for one week. The yield of carrier particles thus obtained was 6.4 g, and 75% of the particles obtained were in the range of 3-6μ. Production Example 6 4 g of gelatin with 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 materials, 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%
was in the range of 2 to 3.2μ. Usage Example 1 The carrier particles obtained in Production Example 1 were added to a phosphate buffered saline solution (hereinafter abbreviated as PBS) with a pH of 7.2 containing tannic acid at the concentration shown in the table below to a particle concentration of 2.5. % and heated at 37°C for 15 minutes. The particles were centrifuged and thoroughly washed with saline. The syphilis pathogen Treponema pallidum (hereinafter referred to as
Abbreviated as TP. ) The bacterial cells were suspended in PBS at pH 6.4 and destroyed using ultrasound to obtain a TP antigen solution. The tannic acid-treated particles were dispersed in PBS at a concentration of 5%, and this dispersion was mixed in equal volumes with the TP antigen solution diluted at the multiples shown in the table below. 37% of the mixture
The particles were sensitized to the TP antigen by warming at ℃ for 40 minutes. The TP antigen-sensitized particles were centrifuged, thoroughly washed with PBS of pH 6.4, and dispersed in a dispersion medium to a concentration of 5%. When the thus obtained TP antigen-sensitized particle dispersion was reacted with syphilis-positive serum using a microplate method, the results shown in the table below were obtained. As a control, the results obtained when sheep red blood cells were used as a carrier are also shown in the table below.

[Table] Usage Example 2 The carrier particles obtained in Production Example 1 were mixed with tannic acid.
The particles were dispersed in PBS containing 10 ppm at pH 7.2 to a particle concentration of 2.5%, and heated at 37°C for 15 minutes. The particles were centrifuged, thoroughly washed with physiological saline, and dispersed in PBS at pH 7.2 to a concentration of 1%. On the other hand, highly purified streptokinase was used at pH 7.2.
It was dissolved in PBS to a concentration of 128 U/ml. 4 ml of the tannic acid-treated particle dispersion and 4 ml of the streptokinase solution were mixed and heated at 37° C. for 30 minutes. Thereafter, the particles were centrifuged, washed with PBS at pH 7.2, and dispersed in gelatin buffer at pH 7.5 to a particle concentration of 1%. The gelatin buffer solution was prepared by dissolving 5 g of gelatin, 10 g of sodium chloride, and 13.6 g of potassium phosphate in 1 ml. The streptokinase activity of this particle dispersion is approximately
Since it was 60 U, approximately 12% of the enzyme in the solution was immobilized on the particles. The enzyme activity was measured as follows. That is, 0.1 ml of human plasma and 0.05 ml of thrombin solution were added to 0.25 ml of the enzyme solution or particle dispersion, and the mixture was heated at 37° C. for 30 minutes. Thrombin solution is
This is 500 units of thrombin diluted 80 times with borate buffer. Enzyme activity was expressed as the highest dilution of streptokinase that inhibited linear coagulation.

Claims (1)

[Claims] 1 Contains gelatin, water-soluble polysaccharides and metaphosphate ions, and the amino groups of gelatin are cross-linked with aldehyde, with a pH of 7.2 measured at 25℃.
The electrophoretic mobility in 0.15M phosphate buffered saline is -1.3 to -0.5μm/sec/V/cm, and the particle size is 1 to
A carrier for immobilizing active proteins characterized by being spherical, hydrophilic and insoluble particles of 100μ.