JPH0510626B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing microcapsules for immunoanalysis with high sensitization efficiency. BACKGROUND ART A hemagglutination reaction has traditionally been used as a method for immunologically testing antigens or antibodies. Furthermore, a latex aggregation reaction using polystyrene latex as a carrier has already been put into practical use. However, these conventional methods have drawbacks such as easy non-specific aggregation, insufficient sensitivity, poor long-term storage, and a long time required for determination. Instead of such red blood cells or latex,
A method using microcapsules as a carrier was proposed. (Unexamined Japanese Patent Publication No. 55-94636, No. 57-19661, No.
57−19662, etc.). This method eliminates the above-mentioned drawbacks inherent to animal-derived carriers (red blood cells) and the disadvantages of synthetic carriers such as latex.If this microcapsule carrier method is applied, sensitivity can be increased, simple operation, and - Improved effects compared to conventional methods, such as reliable determination of off state, are being obtained, and new tests that are practically impossible with conventional methods are being developed. The present inventors continued to conduct various studies on the performance of microcapsules as carriers for immunoassays, and found that microcapsules with a specific combination of core material, wall material, and additives could be used for highly sensitive immunoassays. It has been found that microcapsules can be obtained that provide reagents. That is, in the present invention, an oily substance liquid consisting of a polyvalent isocyanate and an oily substance is added to an aqueous solution containing polyvinyl alcohol and polystyrene sulfonic acid, emulsified and dispersed in the coexistence of polyvinyl alcohol and polystyrene sulfonic acid, and then a water-soluble polyvalent amino acid is added. The present invention relates to a method for producing microcapsules for immunoanalysis, which comprises adding a compound to the emulsified dispersion and polymerizing it with the polyvalent isocyanate. The microcapsules of the present invention are manufactured by the following steps. (1) An oily substance liquid is prepared by dissolving an oil-soluble polyvalent isocyanate, isothiocyanate, or a prepolymer thereof (hereinafter referred to as isocyanate) in an oily substance that forms the core. (2) Aqueous solution containing both PVA and PSS (PVA
-Prepare a PSS aqueous solution). (3) Adding an oily substance liquid to the PVA-PSS aqueous solution,
Emulsifying and dispersing in the coexistence of PVA and PSS. (4) Add a polyvalent amino compound to the emulsified dispersion. (5) Perform thermal polymerization (about 60 to 90℃, usually about 70℃)
(heat for at least 1 hour, usually about 2 hours). Microencapsulation is completed through the above steps. In the combination of a polyvalent isocyanate and a polyvalent amino compound, in which the condensation polymerization reaction progresses slowly, step (4) is
process, but this is not desirable. PVA and PSS must always coexist when emulsifying and dispersing polyvalent isocyanate. For example, even if one of them is emulsified and dispersed and the other is added afterwards to form a coexistence system, the microcapsules of the present invention cannot be obtained. The microcapsules thus obtained exhibit very high detection sensitivity in agglutination reactions when used as microcapsule reagents sensitized with antigens or antibodies. The microcapsules of the present invention can be produced only under the above combination conditions, and if any one of them is missing, the desired microcapsules will not be obtained.
The reason is not necessarily clear, but PVA−
In the coexistence of PSS, polymerization at the interface is thought to be controlled extremely skillfully, and this seems to be due to the fact that the wall surface has many irregularities and therefore has a large surface area. PVA having a polymerization degree of about 200 to 1500 is usually used. If the degree of polymerization is too low, the emulsifying and dispersing power will be weak; if the degree of polymerization is too high, it will be difficult for antigens or antibodies to bind. It is desirable to use PVA in an amount of about 4 to 16% by weight based on the oily substance. There is no particular correlation with the degree of saponification as shown in microcapsules for pressure-sensitive paper (Japanese Patent Application Laid-Open No. 132631/1983), but in practical terms it is approximately 85%.
The above is good. A suitable PSS has a molecular weight of about 100,000 to 2 million, preferably 200,000 to 1 million, but practically 50
It is used with a degree of sulfonation ranging from 40 to 99%. The amount used is 10 to 100% by weight of PVA.
A range of is appropriate. The polyvalent amino compound used as a component of the wall material in the microcapsules of the present invention is a water-soluble primary amino compound. For example, linear diamines (ethylene, tetramethylene, hexamethylene, octamethylene diamine, etc.), diethylenetriamine, triethylenetetramine, tetraethylpentamine, aromatic diamines (phenylene, xylene diamine, diaminobenzoic acid, aminophenylethylamine) ), heterocyclic diamines (diamino-pyridine, -triazole, -pyrimidine, -mercaptopyrimidine, etc.), basic amino acids (arginine, lysine, hydroxylysine, ornithine, citrulline, glutamine, asparagine, etc.), triamines (triamino-propane, -benzene, -pyrimidine, etc.), among which linear diamines and basic amino acids, particularly hexamethylene diamine and lysine, are preferred. Oily substances that serve as capsule core materials include natural mineral oils, animal oils, vegetable oils, and synthetic oils. Since the surface of these core substances is completely covered by the capsule wall, they do not seem to have a direct effect on antigens or antibodies, but it is preferable to avoid biochemically active substances. Examples of mineral oils include kerosene, naphtha, and paraffin oil; examples of animal oils include fish oil and lard oil. Examples of vegetable oils include peanut oil, linseed oil, soybean oil, castor oil and corn oil. Examples of synthetic oils include biphenyl compounds (e.g., isopropylbiphenyl, isoamylbiphenyl), terphenyl compounds, naphthalene compounds (e.g., diisopropylnaphthalene), and alkylated diphenylalkanes (e.g., 2,4-dimethyldiphenylmethane). , phthalic acid compounds (eg, diethyl phthalate, dibutyl phthalate, dioctyl phthalate), chlorinated paraffin, and the like.
A polyvalent isocyanate, a polyvalent isothiocyanate, or a prepolymer thereof refers to a compound having two or more isocyanate groups or isothiocyanate groups and is soluble in an oily substance. Specific examples include m-phenylene diisocyanate, p-phenylene diisocyanate, 2,6-tolylene diisocyanate, 2,4-tolylene diisocyanate, naphthalene-1,4-diisocyanate, diphenylmethane. -4,4'-diisocyanate,
3,3'-dimethoxy-4,4'-biphenyl diisocyanate, 3,3'-dimethyldiphenylmethane-
4,4'-diisocyanate, xylylene-1,4
-diisocyanate, xylylene-1,3-diisocyanate, 4,4'-diphenylpropane diisocyanate, trimethylene diisocyanate,
Hexamethylene diisocyanate, propylene-
1,2-diisocyanate, butylene-1,2-
Diisocyanate, ethyridine diisocyanate, cyclohexylene-1,2-diisocyanate, cyclohexylene-1,4-diisocyanate, p-phenylene diisothiocyanate, xylylene-1,4-diisothiocyanate diisocyanate or diisothiocyanate such as diisothiocyanate, ethyridine diisothiocyanate; 4,4′,4″-triphenylmethane triisocyanate, toluene-2,
4,6-triisocyanate, polymethylene polyphenyl triisocyanate, 1,1,1-tris [(3-isocyanato-4-methylphenyl)
triisocyanates such as carbamoyloxymethyl]propane; tetraisocyanates such as 4,4'-dimethyldiphenylmethane-2,2',5,5'-tetraisocyanate; addition of hexamethylene diisocyanate and hexanetriol thing,
Adduct of 2,4-tolylene diisocyanate and Brenz catechol, adduct of tolylene diisocyanate and hexanetriol, adduct of tolylene diisocyanate and trimethylolpropane, adduct of xylylene diisocyanate and trimethylolpropane , a polyisocyanate prepolymer such as an adduct of hexamethylene diisocyanate and trimethylolpropane; or any similar suitable polyisocyanate or polyisothiocyanate. It is also possible to use two or more of these in combination. The amount used is oil-based liquid
It is preferably about 0.1 to 20 parts, particularly about 1 to 10 parts, per 100 parts. Other conditions for microencapsulation that can be applied to the method for producing microcapsules of the present invention are described in detail in "Microcapsules" by Asashi Kondo, published by Nikkan Kogyo Shimbun (1971), JP-A-72346-1973, etc. has been done. Although the specific gravity of the microcapsules of the present invention can be freely changed by changing the core material,
Generally speaking, a value within the range of 0.85-1.25 is appropriate for use as an immunological test. The average size of microcapsules is 0.5 μm ~
It is desirable to select from the range of 20 μm, preferably from 1 μm to 10 μm. Since the microcapsules of the present invention exhibit extremely high sensitization efficiency, they are extremely useful as carriers for immunoassay reagents. Furthermore, since it provides a highly sensitive immunological test reagent with a very high S/N ratio, it is extremely advantageous in terms of manufacturing costs. The present invention will be explained in more detail with reference to Examples below. Example 1 Oil-soluble red fluorescent dye oleosol Red BB was added to a mixed oil (specific gravity approximately 1.14) of 8.4 g of diisopropylnaphthalene and 16.6 g of chlorinated paraffin (Toyoparax 150, Toyo Soda Co., Ltd., chlorination degree 50%).
Oleosol Red BB (Sumitomo Chemical CI 26105) 0.25g
was dissolved. To the resulting solution was added 1,1,1-tris[(3-isocyanato-4-methylphenyl)carbamoyloxymethyl]propane (Burnock D
-750, manufactured by Dainippon Ink Co., Ltd.) dissolved in 2 g of methyl ethyl ketone was mixed. This oily substance liquid was mixed with 2 g of polyvinyl alcohol, PVA-105 (manufactured by Kuraray Co., Ltd., saponification degree 98%, polymerization degree 500) and a partial sodium salt of polystyrene sulfonic acid (manufactured by Procter-Gamble Co., Ltd., VERSA, TL-500,
(average molecular weight: 500,000) was added to a solution of 75 ml of water, and stirred and emulsified to prepare oil droplets with an average size of about 5 μm. Add a solution of 2.6 g of hexamethylene diamine dissolved in 25 ml of water, and
After diluting with 75 ml, the mixture was reacted at 70°C for 2 hours to perform microencapsulation. After the microcapsules were produced, they were centrifuged and washed with physiological saline to remove unreacted residues, and then dispersed in physiological saline so that the microcapsule particle concentration was 10%. Comparative microcapsule A: Polyvinyl alcohol, PVA-117 (manufactured by Kuraray Co., Ltd., instead of polyvinyl alcohol, PVA-105)
Microcapsules A were prepared under the same conditions as in Example 1, except that the degree of saponification was 98% and the degree of polymerization was 1700. Comparative Microcapsule B: Microcapsule B was prepared under the same conditions as in Example 1 except that 3 g of polyvinyl alcohol, PVA-105, was used without using polystyrene sulfonic acid. Comparative Microcapsule C: Microcapsule C was prepared under the same conditions as in Example 1 except that 2 g of polystyrene sulfonic acid was used without using polyvinyl alcohol or PVA-105. Using the above four types of microcapsules, microcapsule reagents were prepared by the following operations, and their sensitivities were compared. Preparation of microcapsule reagent and evaluation thereof Reagent A using microcapsules of the present invention 1.5 g of the microcapsules of the present invention prepared in Example 1 were taken out and diluted and dispersed in 8.5 ml of physiological saline. Next, 10 ml of a 25% glutaraldehyde aqueous solution diluted 100 times with physiological saline was added to the diluted microcapsule solution and reacted at 37°C for 45 minutes. After the reaction was completed, the mixture was washed by centrifugation and redispersed in 10 ml of physiological saline. Aliquot 2 ml of this, add 2 ml of Affinity chromatography-purified anti-human IgG antibody (sheep immune, manufactured by Wako Pure Chemical Industries, Ltd., 1% solution) diluted 200 times with physiological saline, and incubate at 37°C for 120 minutes. did. Then 0.15M containing 0.2% glycine
3 with phosphate buffered saline (PBS, PH=7.2)
After centrifugation and washing, it was dispersed in PBS containing 3% bovine serum albumin (BSA) to prepare a human IgG detection reagent. Comparative microcapsule reagents A, B, and C Comparative microcapsules A, B, and C were each treated with glutaraldehyde in the same manner as in the process, and Afinitei chromatographic purified anti-human
A subsequent reaction was performed using a 50-fold diluted solution of IgG antibody under the same conditions as in the step to obtain comparative reagents A, B, and C. Evaluation of reagents. An antigen-antibody reaction was performed using the microtiter method using the reagent A prepared in the following procedure. The tube in which obvious agglutination was observed was considered positive, and the highest dilution factor of the serum showing positive was determined.
This was taken as the antibody titer. 1% human IgG (Miles) 1% solution
Normal goat serum was diluted 20,000 times with PBS containing BSA, and normal goat serum was diluted 10 times with PBS containing 1% BSA as a negative control. Place 25μ of each into each tube hole of the V-bottom microplate.
A dilution series was prepared by diluting the samples at 2-fold intervals using PBS containing 1% BSA. next. 25μ of the reagent A prepared in step A was collected with a dropper and dropped into the tube hole of the test solution dilution row of the microplate. The microplate was shaken for 5 minutes to advance the antigen-antibody reaction. After standing at room temperature for 3 hours, the aggregation image at the bottom of the microplate tube was observed, and the antibody titers as shown in Table 1 were obtained. ．． Comparative microcapsule reagents A to C obtained in 1.
Antibody titers as shown in the table were obtained.

[Table] Reagent A, in which the microcapsules of the present invention are sensitized with anti-human IgG antibodies, is 800% lower than comparative reagent A.
It was found that the antibody titer was twice as high. In addition, the non-specific agglutination property measured by the negative control was lower than that of comparative reagents B and C, and a reagent with an extremely high signal-to-noise ratio was obtained. The reagent obtained using the microcapsules of the present invention provides a sufficiently high antibody titer at a lower antibody concentration than the comparative reagent, and is therefore extremely advantageous in terms of production cost. Anti-human that has not been purified by Affinity chromatography instead of anti-human IgG antibody purified by Affinity chromatography
The reaction was carried out under the same conditions as in the step except that IgG antibody (manufactured by Miles) was diluted 20 times with physiological saline to obtain anti-human IgG detection reagent B. According to the procedure, the antigen-antibody reaction was carried out using a microtiter method to determine the antibody titer, and the results shown in Table 2 were obtained.

[Table] It has been found that the microcapsules of the present invention give sufficiently high antibody titers even when commercially available antibodies are directly immobilized thereon. Regarding the microcapsules of Comparative Examples A to C. Although the same conditions as above were performed, no agglutination images were observed in the human IgG dilution series. In the step, the diluted microcapsules were incubated at 37°C for 90 minutes using a 100-fold dilution of Affinitei chromatography-purified anti-human IgG antibody without being treated with glutaraldehyde to cause an adsorption reaction. Next, centrifugal washing with PBS was repeated three times, and human IgG was dispersed in PBS containing 3% BSA.
Detection reagent C was obtained. Following the inoculation, the antibody titer was determined by the microtiter method. The results obtained are shown in Table 3.

[Table] Sufficiently high antibody titers were obtained even when antibodies were directly physically adsorbed onto the microcapsules of the present invention without using a crosslinking agent such as glutaraldehyde. Example 2 Microcapsules were prepared under the same conditions as in Example 1, except that 1.2 g of DL-lysine hydrochloride was dissolved in 25 ml of water instead of hexamethylene diamine, and the solution was neutralized with a caustic soda solution. 1.5 g of the microcapsules prepared in this example were taken out and diluted and dispersed in 8.5 ml of physiological saline. Then 1-ethyl-3-(3-dimethylaminopropyl)
Physiological saline containing 0.2% by weight of carbodiimide hydrochloride
10ml was added to the diluted microcapsules and reacted at 37°C for 45 minutes. After the reaction was completed, the mixture was washed by centrifugation and redispersed in 10 ml of physiological saline. Add the following to this 2ml. Sensitized microcapsules obtained by reacting Affinitei chromatographically purified anti-human IgG antibodies under
It was used as an IgG detection reagent. According to the procedure, the antibody titer was determined by the microtiter method. The results obtained are shown in Table 4.

[Table] The reagent conjugated with antibodies to the microcapsules of the present invention using lysine as a component for forming the microcapsule wall gave extremely high antibody titers. Example 3 1.5 g of the microcapsules prepared in Example 1 were taken and diluted and dispersed in 8.5 ml of physiological saline.
10ml of physiological saline containing 1% by weight of n-propylamine
were mixed and incubated at 37°C for 45 minutes. After centrifugation and washing, the mixture was dispersed in 10 ml of physiological saline, and 10 ml of a 25% glutaraldehyde aqueous solution diluted 100 times with physiological saline was added thereto, followed by reaction at 37° C. for 45 minutes. After the reaction was completed, the mixture was washed by centrifugation and redispersed in 10 ml of physiological saline. Take 2 ml of this and react with Affinity chromatography purified anti-human IgG antibody in the same manner as in the step.
It was used as an IgG detection reagent. According to the procedure, the antibody titer was determined by the microtiter method. The results obtained are shown in Table 5.

[Table] The reagent in which an amine compound was adsorbed to the microcapsules of the present invention and an antibody was bound using a crosslinking agent such as glutaraldehyde had a slightly stronger nonspecific aggregation property, but gave an extremely high antibody titer. In addition, linear amines (n-propylamine and n
-butylamine), diamines (hexamethylene diamine and octamethylene diamine) and basic amino acids (lysine, arginine, hydroxylysine). All of the obtained reagents gave extremely high antibody titers. Reference Example Leptospira autumnalis strain A was grown in Kortov's medium (containing 10% normal rabbit serum), and the culture solution after 6 to 10 days of culture was centrifuged at 9000 RPM for 20 minutes at 5°C. Dilute the sediment with physiological saline
After washing twice, it was redispersed in physiological saline and crushed for 10 minutes using a 20kHz sonicator (manufactured by Otake Seisakusho). This is centrifuged at 12,000 R.PM, and the precipitate is diluted with physiological saline to 10 times the original amount to prepare the antigen solution.
On the other hand, the supernatant was further centrifuged at 50,000 R.PM for 1 hour, and the supernatant was adjusted to have an optical density of 0.2 at a wavelength of 280 nm using a spectrophotometer, and used as an antigen solution. 1.5 g of the microcapsules prepared in Example 1 were separated and diluted and dispersed in 8.5 ml of physiological saline. To this dispersion, add 10 ml of a 25% glutaraldehyde aqueous solution diluted 100 times with physiological saline, and
It was allowed to react for a minute. Then, the mixture was washed by centrifugation and redispersed in 10 ml of physiological saline. Take 2 ml of this, add 2 ml each of the above antigen solutions A and B, and heat at 37℃.
Incubated for 120 minutes. Next, perform centrifugation washing three times with PBS containing 0.2% glycine, then 3% BSA
By dispersing it in PBS containing Leptosvira autumnalis A disease detection reagents A and B were obtained. Rabbits were hyperimmunized with Leptospira autumnalis strain A to prepare antiserum. The Kortov medium culture of the autumn plague strain A was centrifuged, and the precipitated bacterial bodies were suspended in saline water. 4~
Rabbits were injected subcutaneously twice at 5-day intervals, followed by an additional 4-5
Nine intravenous injections are given at daily intervals. Seven to eight weeks had passed since the first subcutaneous injection, and after confirming that the mice had a predetermined antibody titer, whole blood was collected and antiserum was prepared. According to the procedure, the antibody titer was determined using a 100-fold dilution of the above antiserum using Leptospirosis A disease detection reagents (a) and (b) using the microtiter method. The results obtained are shown in Table 6.

[Table] Separate water-insoluble antigens and water-soluble antigens,
When the microcapsules of the present invention were sensitized to each antibody separately, reagents capable of detecting each antibody could be prepared.

Claims (1)

[Claims] 1 Add an oily substance liquid consisting of a polyvalent isocyanate and an oily substance to an aqueous solution containing polyvinyl alcohol and polystyrene sulfonic acid, emulsify and disperse in the coexistence of polyvinyl alcohol and polystyrene sulfonic acid, and then add the water-soluble polyvalent amino compound to the above-mentioned solution. A method for producing microcapsules for immunoanalysis, which comprises polymerizing the polyvalent isocyanate in addition to an emulsified dispersion.