JPS5958004A - Method for manufacturing microparticles for clinical testing - Google Patents

Abstract

PURPOSE:To obtain the titled particles capable of immobilizing an antigen, antibody, etc. to the surfaces of them firmly at low temperature, useful for clinical examination using immunochemical reactions, etc., by irradiating a system consisting of two or more specific monomers with ionizing irradiation at a lower temperature <= room temperature. CONSTITUTION:A system consisting of (A) a polymerizable monomer (e.g., crotonaldehyde, etc.) containing an aldehyde group and a polymerizable double bond and (B) a monomer (e.g., hydroxyethyl methacrylate, etc.) for a glassy vinyl polymer is irradiated with light rays or ionizing radiation in a state kept at room temperature --100 deg.C, and the monomer is polymerized, to give the desired particles having 0.5-20 micron particle diameters. The particles are washed and brought into contact with a physiologically active substance under a given condition so that an antigen, antibody, etc. is immobilized to them. EFFECT:Having high sensitivity, capable of detecting even a very small amount of a substance. Capable of producing particles having various kinds of properties.

Description

[Detailed description of the invention] The present invention relates to a method for producing single-print particles for clinical testing.

When measuring trace amounts of substances such as IgA, IgD, IgE, and IgG in body fluids in immunochemical clinical tests, a method using microparticles as a carrier is adopted.

For example, red blood cells are used as biological particles as a carrier, and the red blood cells are sensitized with antigens or antibodies, which are then reacted with the antigen or antibody in the test fluid to produce trace amounts of substances by immunochemical agglutination or agglutination inhibition reaction. There is a way to measure it. Also,
Instead of red blood cells, etc.

Recently, fine particles of synthetic resin latex and minerals such as bentonite and crystal have been used as non-biological particles.

Conventionally, most of the solid particulate phase materials used in immunochemical clinical tests do not have special binding functional groups on their surfaces.
The binding between the carrier and the antigen or antibody is physical adsorption that utilizes the simple adsorption properties of the antigen or antibody. Therefore, the binding force is weak and some bonds may be broken during the operation, making it difficult to measure in immunochemical clinical tests. This was causing an error.

Traditionally, latex, which has been widely used among non-biological particles, has a low monomer concentration and is mostly synthesized by emulsion polymerization using a catalyst under a high emulsifier concentration, and its particle size is 0. The distribution is wide, ranging from .01 to 2 microns, and the particle size is often less than 1 micron.The carrier particles used in normal immunochemical measurements preferably have a particle size of about 2 to 6 microns. However, there are many difficulties in synthesizing fine particles with a particle size of 2 to 3 microns by emulsion polymerization, such as the need for a long time or the need to set specific synthesis conditions.Furthermore, in the case of catalytic polymerization, There is a drawback that impurities such as catalysts are mixed into the particles, which deteriorates the physical properties of the particles.Also, in US Pat. Although the method of making microparticles is disclosed, the latex synthesized in this way is expected to be swollen with a solvent and not necessarily spherical in shape, and is different from latex synthesized by direct polymerization. It is thought that they have different properties.

As a result of research aimed at improving the shortcomings of the prior art, the present inventors have determined that the particle size can be improved by irradiating a system consisting of two types of specific monomers of μm with a low dose of light or ionizing radiation at a low temperature below room temperature. It has been discovered that solid particulates of 0.5 to 20 microns are produced.

Therefore, the main object of the present invention is to provide a method for producing particulate carriers for clinical testing having a particle size of 0.5 to 20 microns.

Further objects of the present invention are immunochemical reactions, receptor reactions,
Particle size 0 used for clinical tests using lectin reactions etc.
．． The object of the present invention is to provide a method for producing solid particulate carriers of 5 to 20 microns.

Other objects and advantages of the present invention will be highlighted below.

According to the present invention, a system consisting of a polymerizable monomer having an aldehyde group and a polymerizable double bond in the same molecule and one or more vitrifiable vinyl-based polymerized monomers is added at a low temperature below room temperature, preferably from 5 to - Fine particles for clinical and testing purposes with a particle size of 0.5 to 20 microns are produced by maintaining the temperature at -10 DC and irradiating the particles with a low dose of light or ionizing radiation to polymerize the polymerizable monomer.

One of the important features of the present invention is that crotonaldehyde 1 having an aldehyde group and a polymerizable double bond in the molecule, such as acrolein, methacrolein, and citral, is used as a polymerizable monomer.

These polymerizable monomers used in the present invention are also vinyl monomers, so when these monomers are polymerized by the usual radiation polymerization method, bulk polymerization proceeds and undesirable substances are produced, which is inconsistent with the purpose of the present invention. A homogeneous, amorphous polymer is precipitated. For this reason, the present inventors searched and researched irradiation polymerization conditions, and found that
It was discovered that it is possible to obtain the desired solid fine particles by irradiating the polymer with a low dose of light or ionizing radiation at low temperatures to allow the polymerization reaction to proceed slowly and grow the polymer into particles.

Since the academic group used in the present invention has aldehyde groups, the surface of the fine particles obtained by radiation polymerization is covered with aldehyde groups. Therefore, when biologically active substances such as antigens and antibodies are immobilized on the microparticles of the present invention, it is only necessary to wash the microparticles with water and then bring them into contact with the biologically active substances under certain conditions. An immobilized product in which the substance is firmly fixed is produced. Incidentally, in order to immobilize the biologically active substance on the fine particles, it is also possible to add the biologically active substance during the irradiation polymerization reaction and immobilize it simultaneously with the polymerization. That is, in the present invention, since the polymerization reaction is carried out at low temperature and low dose, biologically active substances are present in the polymerization reaction process.
However, there is no fear that they will become inactive.

The term "bioactive substance" used in the present invention refers to antigens,
A component with some biological function (referred to as a biocatalyst or bioactive substance) such as an antibody, complement, cell, enzyme, bacterial cell, or organelle.

Conventional methods of immobilizing biologically active substances on particulate latex have disadvantages, such as the fact that the biologically active substances tend to desorb from the surface of the latex because non-specific adsorption to the latex is utilized. However, since the surface of the microparticles of the present invention is covered with functional groups called aldehyde groups that have a high ability to bind to proteins, they strongly bind to trace amounts of biologically active substances, especially antigens and antibodies. Therefore, immunochemical measurements using particle carriers have high sensitivity and are characterized by the ability to detect trace amounts of substances in body fluids.

The only feature of the invention is the use of vitrifiable vinyl polymerizable monomers as the polymerizable monomers. The vitrifying polymerizable monomer is a polymerizable monomer that does not crystallize at low temperatures but becomes supercooled and does not lose its polymerizability. The polymerization of vitrifying polymerizable monomers in a supercooled state can be called solid-phase polymerization in an amorphous state, and the polymerizability is high in low-temperature overhead, so it is useful for immobilizing physiologically active substances. It is an extremely useful tool for low-temperature polymerization applications.

Normally, physiologically active substances are thermally unstable, so
In order to avoid deactivating the cow, it is ideal to fix it at a lower temperature. Radiation polymerization is a method that can effectively carry out polymerization at low temperatures, and considering that it is the vitrification 1'lE vinyl polymerization 1 biomonocarbon group that has high polymerizability at low temperatures by radiation. It can be said that one of the important features of the present invention is the use of the vitrifying polymerizable monomer 'lfW. By the way, the vitrified 1'lE polymerizable monomer has a hydrogen-bonding functional group with moderate strength in the molecule, a bulky or extremely asymmetric substituent, and a low free energy of rotation such as an ether bond. One of the following general formulas that contains bonds, etc.
a, CH2-Cx-C-0(CH2)nOR11 where X is H or a methyl group, R1 is H or CH2=CX
-C- where X is H or a methyl group, and n is an integer from 4 to 1o; where -QX is H or butyl
0-.

-CH-CH20-, also CH2-CH○-11 CH3CH3 and m is an integer from 1 to 6; where X is H or a methyl group; d, CH2=CX-C-0-R3-0-R11 here where X is H or a methyl group, R3 is a straight or branched alkylene group having 1 to 10 carbon atoms, and R4 is a vinyl or alkyl group having 1 to 10 carbon atoms; where R5 is an alkane (C, -05) ~Iruiribi.

Alkylene (C, -C5) amino group, R6 and R7 are each H, an alkyl group having 1 to 5 carbon atoms.

alkylamino group having 1 to 5 carbon atoms, 1 to 5
hydroxylalkyl group having 4 carbon atoms.

Allyl or vinyl group; f, CH2=CX-C-R3-0-R8-0-R4
Here, X, R3 and R4 are j defined in -, R
8 is the same as R3, R3 and R8 are the same and each different; g+ CH2-CX-C-0-R3-C-R4111 0 where X, R3 and R4 are as defined above; 1],
CH2=GX CORG OR 111 00 where X, R3 and R4 are as defined above; i,
0H2=CX-C-0-R, 11 where X is as defined above and R1□ is tolyl, tolyl, xylyl, phenyl, furfuryl.

Naphthyl, phthalyl, cyclohexyl, cyclophenyl, cycloheptyl, cyclobutyl, pyridyl or ro-oxinepyrrolidinyl group; j, R9-C-(CH2-0-C-CX=CH2)
31 where X is a group defined in ``2'' and R9 is an ethyl or prokyl group; or 0H2-CX-C-0-0-R, o-0-C-
CX=CH21110 where X is as defined above and R3° is isopropylene, imbutylene, a branched alkylene group having 1 to 5 carbon atoms, HH1-G- or -C-10HR ,, where R11 is as defined above.

Specific examples of the vitrifiable vinyl-based polymerizable monomers used in the present invention include;
0xypropyl acrylate, human 90xypropyl methacrylate, diethylene glycol methacrylate,
Diethylene glycol acrylate, triethylene glycol methacrylate-4, ToIJ ethylene glycol acrylate, tetraethylene glycol acrylate, tetraethylene glycol methacrylate, polyethylene glycol methacrylate, polyethylene glycol acrylate, diethylene glycol dimethacrylate,
Diethylene glycol diacrylate, methoxydiethylene glycol dimethacrylate, methoxydiethylene glycol diacrylate, methoxytetraethylene glycol dimethacrylate, neopentyl glycol dimethacrylate, hexanediol monomethacrylate.

glycidyl methacrylate, glycidyl acrylate,
Examples include heptanediol monomethacrylate, butanediol monomethacrylate, ethylene glycol diacrylate, and propylene glycol dimethacrylate.

An advantage of the production method of the present invention is that fine particles with various properties can be produced by copolymerizing the two types of polymerizable monomers mentioned above.

For example, particles with excellent strength can be produced by using a hydrophobic monomer as a glass vinyl polymerization monomer, and hydrophilic monomers such as hydroxyethyl methacrylate and acrylic acid By using F heavily, it is possible to synthesize certain particles in a short time and also to control the number of aldehyde groups on the particle surface.

When carrying out the present invention, water or an organic solvent can be used as a solvent in addition to the above-mentioned polymerization injection method, and particles can be produced by irradiation polymerization in the solvent. For example, when polymerization is carried out in an aqueous solution, a water-soluble polymer such as polyvinyl alcohol or a surfactant such as sodium dodecyl sulfate may be used as a particle generation stabilizer.

The reaction temperature employed when carrying out the present invention is below room temperature, preferably in the range of 5 to -100C, and the amount of gland irradiated is in the range of 104 to 106H. When the combination reaction of the present invention is carried out at a temperature of 00 or more, it is carried out with stirring, but if the temperature is below OC, the reaction solution containing the polymerizable mono-111 is cooled and frozen before irradiation, and the reaction solution is irradiated in that state. Since particles can be synthesized by combining 'f and t, the operation is extremely simple.

Another advantage of the present invention is that uniform particles with a particle size of 1 to 2 microns can be synthesized by irradiation polymerization with the reaction solution cooled and solidified. This was made possible by allowing the polymerization reaction to proceed.

The solid microparticles of the present invention can be used as carriers not only for general immune reactions but also for clinical measurements that utilize all binding reactions such as receptor reactions and lectin reactions. It can be used for separation and purification of substances.

Hereinafter, the structure and effects of the present invention will be specifically clarified in Examples, but these Examples are only one aspect of the present invention.
This is not intended to limit the invention in any way.

Example 1 1 part of a monomer mixture consisting of 0.7 parts of acrolein and 0.3 parts of hydroxyethyl methacrylate and 9 parts of a 1% polyvinyl alcohol aqueous solution were mixed in a container and cooled to 1 t-78 tl'. In this first stage, gamma rays emitted from cobalt-60 were irradiated for 1 hour at a rate of 1 x 1 OR/l'#. Particles taken out after irradiation: 1.
Fine particles with an acid diameter of 4 microns were obtained.

Example 2 The particles obtained in Example 1 were purified with 1% P using a centrifuge.
It was washed three times with BS buffer and dried after washing. Gamma of anti-human placental gonato-9 tropin rabbit serum obtained by immunizing rabbits with human placental gonato-9 tropin (HCG)
The globulin fraction was diluted in a glycine buffer at a ratio of 0.1%, and 10 rng of the particles of Example 1 were added thereto for 6 hours at room temperature.
After standing for 0 minutes, the particles were separated using a centrifuge.

This sensitizing particle? HCC, suspended in glycine buffer,
It was used as a projection reagent. This sensitizing particle is 0. Significant aggregation occurred in the presence of HCG at IIU/ne.

Example: 6 parts of a monomer mixture consisting of 0.5 part of Toral and 0.5 part of triethylene glycol diacrylate and 7 parts of a 1% polyvinyl alcohol aqueous solution were placed in a container,
r emitted from cobalt-60 while stirring at 4C
+r4M was irradiated for 2 hours at a storage rate of 1×106 R/hr. Next, in this container, add anti-human IgG Rabbit-IgG, 1M Na HG O3 buffer (8.94
) K,! z kashita ii (5111ν〃+e) to 1
me was added, and irradiation polymerization was carried out again under the same conditions for 60 minutes to obtain a particulate composition. The particle size of the particles obtained was 2-5 microns. The particles were washed with 0.01 M PBS buffer and the final sensitized particles were 2 x 108'f/r.
Take out ne and add it to human IgG 0.5 m9
/lne and left at 37C for 1 hour, and the state of aggregation was examined. As a result, significant aggregation of particles was observed.

Claims (1)

[Claims] 1. Polymerization having an aldehyde group and a polymerizable double bond in the same molecule; A system consisting of a 41 trimonomer and a vitrifiable vinyl polymeric monomer was maintained at room temperature to -100CK. A method for producing microparticles for clinical testing having a particle size of 0.5 to 20 microns, which comprises exposing them to light or ionizing radiation for 9 hours. 2. A system consisting of a polymerization monomer (1) having an aldehyde group and a polymerized Ellll type bond in the same molecule, a vitrifiable vinyl-based polymerization monomer (6), and a desired biologically active substance is heated at room temperature to - A method for producing microparticles for clinical testing with a particle size of 0.5 to 20 microns sensitized with the biologically active substance, which comprises irradiating with light or T41 radiation while maintaining the temperature at 10 DC. 6. The method according to claim 1 or 2, wherein the vitrifiable vinyl polymerizable monomer is one or more selected from the group consisting of the following general formula. a, CH2=CX-C-0(CH2)nOR11 where X is H or methyl group, R is H or CH2-CX] -C- (where X is H or methyl group), and an integer of 10; b, CH=CX-C-0-(R2)m-G-CX=
CH2II Il 0 where X is H or a methyl group, R2 is -CH2CH20-
. -CH-OH20- or -CH2-CHO-. CH3CH3 and m is an integer of 1 to 3; where X is H or a methyl group; a, 0H-CX-G-〇-R3-〇-R41 where X is H or a methyl group; a straight-chain or branched alkylene group having carbon atoms, and R4 is a vinyl or alkyl group having 1 to 10 carbon atoms; where R5 is an alkane (C1-C5)-yl-iribi. Alkylene (C, -C5) amino group, R6 and R7 each an alkyl group having 891 to 5 carbon atoms. alkylamino group having 1 to 5 carbon atoms, 1 to 5
hibiroxylalkyl group having 4 carbon atoms. Allyl or vinyl group; f, CH2CX, -C-R3-0-R8-0-R
. 1 where X, R and R4 are defined above and R8 is R
3, R3 and R8 are the same and each different; g, cu=cx-c-o-R-c-R4111 0 where x, R3 and R4 are as defined above; h,
CH=CX-C-0-R3-C-0-R41 00 where X, R and f (4 as defined above: i,
cH2=cx-c-o-R,. 1 Here, X is defined as one; ..., and RII is benzyl, tolyl, quinryl, phenyl, furfuryl, naphthyl, phthalyl, noclohexyl, cyclophenyl,
Cycloheptyl, cyclobutyl, pyridyl or ro-oxopyrrolidinyl group;
-○-C-CX=CH2) 31 where X is as defined above and R9 is an ethyl or pro-oyl group; or, CH=CX-C-0-0-Rlo-0-C- C
X=CH21110 as defined herein or above and Rlo is isopropylene, imbutylene, a branched alkylene group having 1 to 5 carbon atoms, HH1 where RII is as defined above. 4. Irradiation;? ,IL! ,! The method according to claim 1 or 2II'li, wherein the radius is 104 to 106R.