JPS58113754A - Production of artificial carrier - Google Patents

Abstract

PURPOSE:To obtain a carrier suited for serodiagnosis, etc. by contg. anionic or nonionic surfactants into a soln. contg. gelatin, water soluble polysaccharides, sodium polymetaphosphate as well as a hydrophilic org. solvent and water prior to adjusting of said soln. to a specific pH range. CONSTITUTION:Prior to adjusting of a soln. contg. gelatin, water soluble polysaccharides such as gum arabic, sodium polymetaphosphate (NaPO3)n, a hydrophilic org. solvent such as lower alcohol or aceton, and water to 2.5-6.0pH by adding acids under stirring at temp. higher than the gelling temp. of the gelatin, a soln. of anionic or nonionic surfactants is added thereto, then acids such as acetic acid soln. are added thereto to form gel particles. A particle dispersion is cooled and when the temp. thereof drops to <=10 deg.C, aldehyde crosslinking agents (glutaraldehyde, H2CO, etc.) are added to the soln. to obtain crosslinked gells. Thus, optional grain sizes can be provided to the carrier, and the homogeneous and stable carriers is obtained in good yields. The carrier bound with antigens or antibodies retains the initial characteristics without deterioration even when frozen abd preserved for a long period of time.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improvement in a method for producing a novel artificial carrier that can be widely used in medicine and the like. In fields such as clinical testing that utilize antigen and antibody reactions, antigens and antibodies are adsorbed or bonded to particles of a certain appropriate size as carriers, and the presence of the corresponding antibody or antigen is used as a carrier. This method of causing aggregation of the sensitized carrier 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 for serological diagnosis of various diseases.

The 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 particulate 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. In addition, among non-biological carriers, charcoal powder and kaolin are difficult to select particles of a certain size, and polystyrene latex is preferable as a reaction medium due to its natural aggregation, which causes non-specific aggregation in the neutral region. 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 fixed size, the particle size is fixed depending on the type of organism, and they can be of any size depending on the purpose. 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 blood cells and can cause non-specific agglutination reactions with antibodies. There is a possibility that a certain cross-reaction may occur and the desired agglutination reaction may be erroneously determined.

Another disadvantage is that 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 discovered that a solution containing gelatin, a water-soluble polysaccharide, and sodium polymetaphosphate was stirred using a mixture of water, alcohol, etc. as a solvent. We discovered that by precipitating particles by making one adjustment below, and insolubilizing these particles by treating them with an aldehyde-based crosslinking agent, an excellent carrier that overcomes all the drawbacks of the conventional ones can be obtained, and we have already filed a patent application for this content. did. In this method, a surfactant was added after the first adjustment to prevent agglomeration of the generated particles, but as a result of further research by the present inventors, it was found that the surfactant was added before the first adjustment. found that the yield of the carrier was greatly increased and, on this basis, proceeded to complete the present invention. , t 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 gelation temperature of gelatin is adjusted to pH 2 by adding an acid while stirring. ,5~6.0
In the method for producing an artificial carrier in which the carrier is made insolubilized by the action of an aldehyde crosslinking agent, an anionic or nonionic surfactant is added to the solution before the above adjustment. The present invention relates to a method for producing an artificial carrier characterized by the following.

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, caladanan, and gum arabic is particularly preferred.

Sodium polymetaphosphate is a substance represented by the chemical formula (NaPo5)n, such as sodium tetrametaphosphate and sodium hexametaphosphate.

Examples of hydrophilic organic solvents include lower alcohols, tt
f/tyl alcohol, ethyl alcohol, clobyl alcohol, etc., a7tone, etc. 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. Examples of coloring agents include Food Red No. 3, Rhodamine, Rose Bengal, /Meso-3R,yl? Red dyes such as Ludo S1 fuchsin, eosin, and dimetral red, or blue dyes such as crystal violet, toluidine blue and methidium/blue, and the like may be used. However, Reacti! - Reactive dyes such as red and direct blue are particularly suitable because the color will not fade if colored with reactive dyes. 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:
Gelatin: about 10.01 to 2, preferably 0.05 to 1
．． The water-soluble polysaccharide content is about 0.01 to 2 volumes, preferably about 0.05 to 1.0'j, and the hydrophilic organic solvent is about 4 to 25 volumes. /'J Sodium metaphosphate is preferably added in an amount of about 3 to 15% 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 0.005
-0.5 inch, but if a reactive dye is used, about 1 to 5 inch of gelatin drying is sufficient.

The present invention is characterized in that an anionic or nonionic surfactant is further added to such a solution before adjustment.

Examples of anionic surfactants include alkyl sulfosuccinic acids, alkyl sulfomaleic acids, Nispur alkyl sulfates, polyoxyethylene alkyl ether sulfates, and examples of nonionic surfactants include polyoxyethylene fatty acids. Ester, polyoxy, Chetyle/alkyl ether, t? Examples include lJ oquin ethylene alkylphenyl ether and polyethylene glycol fatty acid ester. 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. The concentration in the liquid before voice adjustment is 0.001 for anionic surfactants.
~0.01%, and 0.01% for nonionic surfactants.
Agglomeration prevention effect can be obtained at about 0.01 to 0.1 inch. Agglomeration can be prevented at lower concentrations by cooling the solution.

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 the 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 at temperatures below its isoelectric point, producing cloudiness, so when using acidic gelatin, it is best to add an alkali to raise the solution's temperature to at least around that point. . However, even after opacity has occurred, it can be eliminated by adding alkali. In any case, the solution is K before starting the acid addition.
must be kept in a cloudy state.

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. 35-50 U to form uniform particles
It is best to continue heating and add the acid dropwise while stirring moderately. The optimum value 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, so it is best to determine it by conducting experiments in advance. 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 that case, the optimum − is 4.0 to 10 μm.
It is in the range of 5.5. The acid to be used is not particularly limited and may be an inorganic acid or an organic acid, but it is preferably as mild as possible, and for example, acetic acid is preferred.

The particles produced in this process do not disappear even if the temperature of the system is lowered below the delification temperature of gelatin, 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 the female particles.

After adding the acid, it is preferable to quickly cool the particle dispersion to prevent the formed particles from agglomerating. And the liquid temperature is 10
When the temperature drops below 0.degree. C., an aldehyde crosslinking agent is added to insolubilize the particles. The amount of this crosslinking agent added is about 0.1 to 200 inches of the dry weight of gelatin, and after addition, the mixture is left to stand overnight 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 is done with water containing the same surfactant at the same concentration as the one used for particle dispersion.
Just go around.

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 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 antigens or antibodies, animal red blood cells are used as carriers, which have the same performance as the best single red blood cells, and are chemically and physically homogeneous and stable. It has many advantages over animal red blood cells, such as no antigenic activity and the ability to easily and inexpensively mass-produce particles of any particle size. In addition, the present invention greatly increases the yield of the carrier by changing the timing of addition of the surfactant as compared to the prior invention.

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

Example 1 4 tons of gelatin with an isoelectric point of pH 9 was added to 40°C hot water.
The solution was dissolved to a pH of 0.001% and adjusted to pH 9 using 10% sodium hydroxide solution. 4 tons of gum arabic was dissolved in water to a concentration of 100 m/g, and after removing insoluble matter, the solution was heated to 40°C.

59ml of the gelatin solution obtained in this way and 50ml of the gum arabic solution were mixed, and this mixed solution was heated to 40°C in advance to form a 30 volume methyl alcohol solution.
0.0 II/ and stirred well. 10 for this
Sodium thihexametaphosphate solution 1.6II7! ,1
Thialkyl sulfomaleic acid (trade name Demol E) solution 2I1111 and 1T direct blue solution 5 m
1 was added and stirred well.

Next, while maintaining the temperature at 40°C, 10 volumes of acetic acid' solution was added dropwise to adjust the p) to 14.8 to form particles.

The particle dispersion obtained by this adjustment was cooled with water to 5°C.
Then, 1.32 g of geltaraldehyde was added, and after stirring well, the mixture was allowed to stand overnight at the same temperature. The particle dispersion was then centrifuged at 200 rpm for 10 minutes to remove the particles.
It was collected as a lettuce. A washing operation in which the particles were suspended in a 0.005% Demol E solution and centrifuged was repeated three times, then dispersed in 4 volumes of thiformin solution and left at 5° C. for one week.

The carrier particles obtained in this example were 7.7? And 75m of 1,000
was in the range of 3 to 6μ.

Example 2 Carrier particles were prepared by diluting 30 volumes of methyl alcohol solution. However, the value of - prepared by dropping acetic acid was 449. The carrier particles thus obtained were 7.8 tons.
, and 70% of them were in the range of 7 to 11μ.

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

That is, first, 1 ton of caldeximable cellulose was used instead of 4 tons of gum arabic, and 30 volumes of ethyl alcohol solution was used instead of 30 volumes of methyl alcohol solution. Also, regarding the amount added, gelatin,
The 10% sodium hexametaphosphate solution, 1thidemol E, solution, 1es Direct Blue solution, and glutaraldehyde were all reduced to one quarter. In addition, - was also increased from 4.8 to 4.6.

The carrier particles thus obtained were 3.8t, 90% of which were in the range of 1-2μ.

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 increased from 50 ml to 40 ml,
The gum arabic solution was changed from 501 nl to 60 ml.

Then, 1.0 sodium thihexametaphosphate solution was added from 5 ysl to 1.2 ml, 1 thidirect pull solution was added from 6 ysl to 4.8 ml, and glutaraldehyde was added from 1.32 to 1.2 ml. I changed each OyK.

Moreover, the end of acetic acid addition was set at 4.6.

The carrier particles obtained in this example had a particle size of 6.42, and 90 particles were in the range of 1 to 2μ.

Example 5 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 ml to 60 d, and the gum arabic solution was changed from 50 ml to 40 rnl.

Then add l of 10% sodium hexametaphosphate solution.
, the amount of glutaraldehyde was changed from 1.32 to 1.82. The completion of acetic acid addition was set at 4.8.

The yield of carrier particles obtained in this example was 8.6f, and 75 particles of the obtained particles were in the range of 3 to 6μ.

Use Example 1 The carrier particles obtained in Example 1 were mixed with phosphate buffered saline (pH 7.2) containing tannic acid at the concentration shown in the table below.
Hereinafter, it will be abbreviated as PBS. ) and heated at 37° C. for 15 minutes. The particles were centrifuged and thoroughly washed with saline.

Bacterial cells of the syphilis pathogen Treponema ballidum (hereinafter abbreviated as TP) were suspended in PBS (P)(6,4), and the cells were destroyed using ultrasound and TP anti-antigen coloring was performed.

The tannic acid-treated particles were dispersed in PBS at a concentration of 5%, and this dispersion was mixed in equal volumes with the TPP stock solution diluted in the multiples shown in the table below. The mixture was heated at 37° C. for 40 minutes to sensitize the particles to the TP antigen.

The TPP original sensitized particles were centrifuged, thoroughly washed with -6.4 PBS, and dispersed in a dispersion medium to a concentration of 5-.

When the TPP original sensitized particle dispersion thus obtained was reacted with syphilis-positive serum by the micrograte method, the results shown in the table below were obtained.

As a control, the results obtained when human red blood cells were used as a carrier are also shown in the table below.

Use Example 2 The carrier particles obtained in Example 1 were dispersed in -7.2 PBS containing 10 ppm of tannic acid to a particle concentration of 2.5 cm, and heated at 37°C for 15 minutes.

The particles were centrifuged, thoroughly washed with physiological saline, and dispersed in PBS, pH 7.2, to a uniform volume. On the other hand, highly purified streptokinase was added to PBS at pH 7.2.
It was dissolved to give K 128 UAI.

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, pH 7.2, and dispersed in gelatin buffer, pH 7.5, to a particle concentration of 1%. In addition, the gelatin buffer solution is gelatin 5
1. Sodium chloride 101, and potassium phosphate 13
．． 61 to 1! It is dissolved in

Since the streptokinase activity of this particle dispersion was about 60 U, this means that about 12 units of the enzyme in the solution were immobilized on the particles.

The enzyme activity was measured as follows. That is,
0.05 ml of human plasma Q, l tnl and thrombin solution to 0.25 ml of enzyme solution or particle dispersion
1 was added and heated at 37°C for 30 minutes. The thrombin solution is 500 units of thrombin diluted 80 times with borate buffer. Enzyme activity was expressed as the highest dilution of streptokinase that inhibited the line apparatus.

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

Claims (1)

[Claims] Zentino, water-soluble polysaccharide, sodium polymetaphosphate,
A solution containing a hydrophilic organic solvent and water and having a temperature equal to or higher than the gelatinization temperature is heated by adding an acid while stirring.
In a method for producing a human carrier, the carrier is adjusted to H2,5 to 6o, and then treated with an aldehyde crosslinking agent to make it insolubilized.
A method for producing an artificial carrier, characterized in that an anionic or nonionic surfactant is added to the solution before the above adjustment.