JPS58113756A - Production of artificial carrier - Google Patents

Abstract

PURPOSE:To obtain a carrier for serodiagnosis, etc. which can be preserved for a long period of time and has excellent characteristics by contg. anionic or nonionic surfactants in an aq. soln. of gelatin, water soluble polysaccharides and sodium polymetaphosphate prior to controlling of said soln. to a specific pH range. CONSTITUTION:An aq. soln. of gelatin, water soluble polysaccharides such as gum arabic and sodium polymetaphosphosphate (in other words, only water is used, without making combination use of org. solvents) is maintained at temp. higher than the gelling temp. of the gelatin and a soln. of anionic or nonionic surfactants are added thereto; thereafter a soln. of acetic acid, etc. is dropped to the soln. under stirring to adjust 2.5-6.0pH and to form particles. According to the conditions such as pH and compsns. of raw materials, grain sizes can be controlled to desired values. After the formation of the particles, the soln. is cooled and when the temp. drops to <=10 deg.C, aldehyde crosslinking agents are added to the soln. to insolubilize the same. The particles are centrifugally sepa rated and washed, whereby the carrier is obtained. This carrier is obtainable in good yields and with uniform properties, and is preservable for a long period of time. The carrier bound with antigens or antibodies is stable without liberation of the antigens, etc. even when preserved by freeze drying.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improvement in a method for producing a novel artificial carrier, which can be widely used in various applications. 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 carriers are sensitized by the presence of the corresponding antibodies or antigens. The method of causing aggregation 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.

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. 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 particle size is fixed depending on the type of organism, and it depends on 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, which cause a cross-reaction, a non-specific agglutination reaction, with antibodies. This may cause errors in the agglutination reaction.

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.

The present inventors conducted various studies to develop excellent carriers free from these drawbacks, and as a result, gelatin, water-soluble polysaccharides, and polymethalin i4. ．． If particles are precipitated by adjusting a solution containing sodium and using a mixture of water, alcohol, etc. as a solvent under stirring, and the particles are insolubilized by treatment with an aldehyde-based crosslinking agent, the drawbacks of the conventional method can be overcome. We have discovered that an excellent carrier that completely eliminates these problems can be obtained, and have already filed four patent applications on this subject. 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 content as well. However, in both methods, a surfactant was added after the initial adjustment to prevent agglomeration of the generated particles. The present invention was made based on the fact that in a method in which the solvent is only water and no hydrophilic organic solvent is used, the yield of the carrier increases if a surfactant is added in advance before any adjustment.

That is, in the present invention, an aqueous solution containing gelatin, a water-soluble polysaccharide, and sodium polymetaphosphate and having a temperature equal to or higher than the gluing temperature of gelatin is adjusted to pH by adding acid while stirring.
2.5 to 6.0, and then treated with an aldehyde crosslinking agent to make it insolubilized.
The present invention relates to a method for producing an artificial carrier by adding an anionic or nonionic surfactant to the aqueous solution prior to the above adjustment.

As the gelatin used in the present invention, commercially available gelatin may generally be used as is. Among commercially available products, liquid 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.

Sodium polymetaphosphate is a substance represented by the chemical formula (NaPO3)n, such as sodium tetrametaphosphate and sodium hexametaphosphate.

Alternatively, if the carrier is to be colored, a coloring agent may be added to the solution prior to 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, for example, Food Red No. 3,
Red pigments such as rhodamine, rose bengal, Pontno 3R, yN Ru-81 fuchsin, eosin, and neutral red, or crystal/metal iolet,
toluidine blue and methi. A blue dye such as Renblue can be used. However, reactive dyes such as Active Red and Direct Blue do not cause color fading, so reactive dyes are particularly preferred.

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 is about 0.01 to 2, preferably 005 to 1.
The content of the water-soluble polysaccharide is about 0.01 to 2%, preferably about 0.05 to 1.0%. It is preferable that sodium polymetaphosphate is contained in an amount of about 3 to 15 times 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 colorants, usually 0
．． 0.005 to 0.5 es, but if a reactive dye is used, about 1 to 5 es of gelaten dry weight is sufficient.

The present invention is characterized in that such a solution further contains an anionic or nonionic surfactant.

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. In the case of anionic surfactants, the concentration in the solution of tone Buzen is 0.001 to 0.0.
Approximately 1%, 0.01 to 0 for nonionic surfactants
Agglomeration prevention effect can be obtained with about 1 inch. The process for preparing such solutions is not critical; for example, each may be dissolved in hot water and then mixed, or each may be dissolved together. However, since water-soluble polysaccharides often contain small amounts of insoluble components, it is preferable to dissolve them separately and add them. On the other hand, when gelatin is below its isoelectric point, it reacts with water-soluble polysaccharides and becomes cloudy, 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 gluing temperature of gelatin. 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 in the range of 2.5 to 60 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 this case, the optimum value of - is in the range of 40 to 5.5. The acid used for this adjustment is not particularly limited and may be either an inorganic acid or an organic acid, but preferably one that is as mild as possible, such as acetic acid.

The particles produced in this step do not disappear even when the temperature of the system is lowered below the gluing temperature of gelatin, so there is no equilibrium relationship with the mother liquor. Also, although it depends on the mixing ratio of gelatin and water-soluble polysaccharide, in most cases the particles are positively charged, and polymetaphosphate ions are oriented on the surface, forming a so-called electric double layer. There is. 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. 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 g of the dry weight of gelatin, and after addition, the mixture 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-based 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 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 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 particle-sized red blood cells.
It is. In addition, the present invention greatly simplifies the manufacturing equipment and facilitates operation by not using such organic solvents in contrast to the prior invention that contains alcohol or the like as a solvent. For the prior invention in which the surfactant is not used, the yield is improved by changing the timing of adding the surfactant.

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

Example 1 Gelatin 4F'i having an isoelectric point of pH 9 was dissolved in warm water at 40° C. to a concentration of 100 at, and the pH was adjusted to 9 with a 10-ton sodium hydroxide solution. Gum arabic 49 was dissolved in water to make 100 ml, and after separating insoluble matter tF, it was heated to 40°C.

Mix 59 ml of the gelatin solution obtained in this way with 501 ml of gum arabic solution + 11 ml of gum arabic solution, and add 40 ml of this mixed solution.
Added to 300 tug of distilled water at °C. Add to this 1.5 m of 10% sodium hexametaphosphate solution, 8 ml of 1 i% HoIJ oxyethylene phenyl ether (Emuldan A-60, registered trademark of Kao Soap Co., Ltd.) solution.
, and ILs Direct Blue Solution 61 were added.

The solution was kept at 40° C., and while stirring, 10 volumes of thiacetic acid solution were added dropwise to adjust the temperature to 48° C., and particles were generated.

The particle dispersion was ice-cooled to 5°C, 1.31 g of gel tar aldehyde was added, and the mixture was kept overnight at the same temperature as after stirring. The particle dispersion was then centrifuged at 2 ooo rpm for 10 minutes to collect the particles as pellets.

The particles were suspended in 0.02 volume of Emuldan A-60 solution and centrifuged. The washing operation was repeated three times, and then dispersed in 4 volume i% formalin solution so that the particle concentration was 5 °C. It was left undisturbed for one week.

The carrier particles obtained in this example were 6.2II, and the 75
The thickness was in the range of 3-6μ.

Example 2 Dodge KI of 8 ml of emaldan A-60 solution of 1 volume
Same as Example 1 except that 2 ml of % alkyl sulfomaleic acid (Thymol Ep1 Kao Soap (registered trademark)) solution was used, and 6 ml of 1% Reactive Red was used for 6 ml of 1% direct pull solution. Carrier particles were prepared as follows. However, it was adjusted by dropping acetic acid...
was 46. The carrier particles thus obtained had a pH of 11 and a particle size of 75 μm in the range of 3 to 6μ.

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

That is, first, carboxymethylcellulose 111i was used in a wafer of gum arabic 4II, and 5 ml of a 1% Demol Ep solution Q was used in 8 ml of a 1-volume emuldan A-60 solution. Then, regarding the amount of addition, gelatin, 10 thihexametaphosphate sodium solution,
Both the 1% Direct Blue solution and glutaraldehyde were reduced to one quarter. Also, - is also 4.8 to 4.
I made it 6. The carrier particles thus obtained were 4.81
Yes, that 95 inch was in the range of 08 to 1.5μ.

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

That is, gelatin solution'r: 50 ml to 40t
nl and add gum arabic solution from 5Qml to 60rrr.
Changed to l. Then, from 1.6 ml to 1.21 nl in 10 thiexametaphosphate solution, 1 volume of Chemarg 7A-60 solution 8 rug was added to 1 tidemole Ep.
6 ml of 1% Direct Blue in a 2M solution 4.
8 ml and glutaraldehyde'i 1.3
# to 1. I changed each to Og. Further, the pH at the end of the dropwise addition of acetic acid was set to 4.2.

The carrier particles thus obtained weighed 9.6 g, 90% of which were in the 1-2 micron range.

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 ml, and the gum arabic solution was changed from 50 ml to 40 ml. Then, from 1.6 ml to 2 ml of 10% sodium hexametaphosphate solution, 1 volume of Chemarun A-60 solution ii 2 ml of % Demol Ep solution, 1 ml of TiDirect Blue 6 #It f to 7.2 ml, Then, the amount of glutaraldehyde was changed from 1.3g to 1.81I. Further, the end p)l of acetic acid dropwise addition was set to 4.6.

The carrier particles thus obtained had a particle diameter of 10.4.9, 75% of which were in the range of 3-6μ.

Usage Example 1 The carrier particles obtained in Example 1 were dispersed in phosphate buffered saline (hereinafter abbreviated as PBS) with a pH of 7.2 to a concentration of 5 ppm, and 20 ml of the suspension was mixed with 5 ppm of tannin. It was mixed with 820 ml of pH 7.2 containing acid.

The mixture was heated at 37°C for 15 minutes, centrifuged, thoroughly washed with physiological saline, and then dispersed in PBS with a pH of 6.4 to a concentration of 5%, and the total volume was 20mA! Closed. On the other hand, pH6
, 4, the syphilis pathogen Treponema mother dam suspended in PBS was destroyed by ultrasonication and used as an antigen solution.

Tannic acid treated particle dispersion liquid 20rrtl and antigen solution 20r
ul and heated at 37° C. for 40 minutes. The antigen-sensitized particles thus obtained were thoroughly washed with PBS of pH 6.4, dispersed in 19 ml of dispersion medium so that the particle size was 5 cm, 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 using sheep red blood cells as a carrier (
The results of a similar measurement using Fuji Organ Pharmaceutical Co., Ltd.) are also shown.

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 dispersed in 85 ml of pH 7.2 so that the particle concentration was 1%.

Highly purified streptokinase was dissolved in 85 ml of pH 7.2 to a concentration of 3211, and this solution was mixed with the particle dispersion.

The mixture was heated at 37° C. for 30 minutes, and the particles were centrifuged and thoroughly washed with saline. Disperse the particles in a dispersion medium to a concentration of 5%, freeze-dry and
Stored at °C.

When the titer of the streptokinase-immobilized particles thus obtained was determined by the microplate method, the results shown in the table below were obtained.

Patent applicant: Fuji Organ Pharmaceutical Co., Ltd. Representative Patent Attorney Masahiro Tanaka

Claims (1)

[Claims] An aqueous solution containing gelatin, water, soluble polysaccharides, and sodium polymetaphosphate, whose temperature is above the gelatin dalification temperature, is adjusted to a pH of 2.5 to 60 by adding acid while stirring, and then adding an aldehyde crosslinking agent. A method for producing an artificial carrier in which the aqueous solution is made insolubilized by the action of an anionic or nonionic surfactant prior to the above-mentioned adjustment. 0