JPS63230086A - Preformed chemical mediator immobilizing carrier - Google Patents

Abstract

PURPOSE:To obtain the titled carrier having excellent shelf stability and dispersion stability, by reacting the epoxy group of polymer particles containing epoxy group on the surface with a chain disulfide compound containing a specific functional group to introduce disulfide bond to the surface of the particles. CONSTITUTION:A latex comprising polymer particles containing epoxy group on the surface and having 0.03-3mum, preferably 0.04-1.5mum average particle diameter is reacted with a chain disulfide compound (e.g. cystamine or dithioglycolic acid) containing amino group or carboxyl group on the molecular ends to give an immobilizing carrier having preferably 1X10<-7>-1X10<-4>mol./m<2>, especially 1X10<-6>-1X10<-5>mol./m<2> disulfide bond introduced to the surface of the particles. The carrier can relatively readily immobilize a preformed chemical mediator containing disulfide bond or thiol group in the molecular by disulfide bond exchange reaction.

Description

[Detailed description of the invention] <Field of industrial application> The present invention relates to a carrier for immobilizing a physiologically active substance, and more specifically, a carrier capable of immobilizing a physiologically active substance having a disulfide bond or a thiol group in one molecule by a disulfide bond exchange reaction. The present invention relates to a carrier for immobilizing a physiologically active substance.

<Prior Art> In recent years, physiologically active substances such as enzymes and antibodies have been used in a variety of fields by taking advantage of their specific and selective biological reactions. Typical examples thereof include immobilized enzymes, which are formed by immobilizing enzymes on water-insoluble carriers, and immunological diagnostic reagents, which are formed by immobilizing immunoactive substances.

As described above, particles having functional groups such as carboxyl groups, amino groups, and hydroxyl groups introduced onto their surfaces have been known as carrier particles for immobilizing physiologically active substances such as enzymes and antibodies through covalent bonds.

As a method for immobilizing a physiologically active substance on such a carrier particle, 1. For example, after activating the carboxyl group on the surface of the carrier particle or in the physiologically active substance with carbodiimide, this is immobilized on the surface of the corresponding physiologically active substance or the carrier particle. A method in which a peptide bond is formed by reacting with an amine group, or a method in which a hydroxyl group on the surface of a carrier particle is converted into an imidocarbonal derivative with cyanogen bromide, and then this is reacted with an amino group possessed by a physiologically active substance. It has been known.

However, according to this method, the carboxyl group or amino group of the active substance participates in the immobilization reaction without any selectivity. The higher-order structure may be destroyed, resulting in a decrease in the activity of the immobilized physiologically active substance.

Furthermore, as another method for immobilizing enzymes and antibodies, a method using a disulfide bond exchange reaction is conventionally known. When using this method, the physiologically active substance is immobilized on the carrier only by the reaction of its disulfide bonds or thiol groups, so that the higher-order structures of enzymes and antigens as described above are not destroyed.

Conventionally, particle carriers having such a disulfide bond or a thiol group, which is a reduced form of this disulfide bond, on the particle surface have been used, for example.

7 Gallois derivative with the following group OOH 10CONHCH(CH2) 2 C0NHCHCH2
Activated thiol Sepharose 4B (7 Armasia Fine, manufactured by Chemicals Co., Ltd.) bound to SHC0NHCH2C0OH, Enzacryl polythiol (manufactured by Kotsuholite Laboratories), which has the following group -CH2CHCONHCHCHZ SHOOH bound to acrylamide del, glass. Those in which a substituent containing a thiol group is bonded to a pea are known. However, since the fine carrier particles described in h are mainly used as a filler for affinity chromatography, two particles of one particle do not have sufficient dispersion stability in an aqueous medium.

Therefore, it is difficult to use conventional carrier particles as described above in applications requiring monodispersion stability. For applications requiring such dispersion stability.

For example, after immobilizing antibodies on particles, they are mixed with a sample such as serum or urine to cause a two-antigen-antibody reaction.

Diagnostic test reagents that make a diagnosis based on the degree of aggregation.

After immobilizing the enzyme on the particles, it is dispersed in an aqueous medium,
Examples include an enzymatic reaction in which the reaction is performed with a substrate.

On the other hand, it is already known that a latex containing polymer particles with excellent dispersion stability can be obtained by a method such as selecting a monomer component of 2 m or more and carrying out emulsion copolymerization. However, it is difficult to directly produce polymer particles having a disulfide bond or a thiol group on one surface by a method such as emulsion polymerization because a suitable monomer is not found. In addition, even if disulfide bonds and thiol groups can be introduced into one polymer particle by subjecting the polymer particles obtained by emulsion polymerization etc. to a post-reaction, in general, the polymer particles obtained in this way are In many cases, the dispersion stability is significantly impaired and the long-term storage stability is poor. Furthermore, even if the polymer particles have excellent dispersion stability after introducing disulfide bonds or thiol groups, generally speaking, after immobilizing # elements, antibodies, etc.

The dispersion stability is often impaired. On the other hand.

Generally, polymer particles have thiol groups on their surface.

As a result of the oxidation of thiol groups during storage in an aqueous medium, the number of thiol groups decreases or even disappears over a long storage period, making it difficult to achieve the desired effect of introducing thiol groups. It disappears.

<Problems to be Solved by the Invention> The present invention was made to solve the above-mentioned problems in conventional carriers having disulfide bonds or thiol groups on the particle surface. Another object of the present invention is to provide a carrier for immobilizing a physiologically active substance that does not reduce or disappear in active groups even when stored in an aqueous medium for a long period of time and has excellent dispersion stability.

Means for Solving the Problems〉 Namely, 1. The carrier for immobilizing a physiologically active substance of the present invention is a latex containing polymer particles having an epoxy group on the surface and having an average particle size of 0.03 to 3 μm, and 1. It is characterized in that a diluted disulfide compound having a mino group or a carboxyl group is bonded to an epoxy group (h).

In the present invention, the polymer particles having an epoxy group on one surface are the chain disulfide compounds described below.

It can be bonded using the epoxy group on its surface, and has a thickness of 0.03 to 3 μm, preferably 0.04 to 1.0 μm.
It has an average particle size of 5 μm. If the particle size is too small, when a physiologically active substance is immobilized on the polymer particles and the reaction is carried out in an aqueous medium, recovery after one reaction becomes difficult. On the other hand, if the particle size is too large.

Since the particle surface area per unit volume of the polymer particles becomes small, the amount of physiologically active substances immobilized decreases, and dispersion into an aqueous medium becomes difficult, which is not preferable.

The above-mentioned polymer particles are obtained in a latex state and are usually prepared using an emulsion polymerization method. In other words, the polymer particles used in the present invention have an epoxy group on the surface of 7, and are not particularly limited as long as they satisfy the conditions described in 7. This is because the polymer particles obtained in this manner are preferable in terms of adjustment of the average particle diameter and ease of operation for introducing epoxy groups.

Such polymer particles are preferably emulsion copolymerized using glycidyl (meth)acrylate as a monomer in order to introduce epoxy groups.

However, this glycidyl (meth)7 acrylate generally has poor polymerization stability, so when a large amount is emulsion copolymerized, it easily aggregates during the polymerization reaction.

It has been considered difficult to obtain a carrier by emulsion copolymerization of a relatively large amount of glycidyl (meth)acrylate as in the present invention in order to immobilize a physiologically active substance. Therefore, in the present invention, when the above monomer is used, it is preferable to carry out emulsion copolymerization with a polyfunctional internal crosslinking monomer and (meth)acrylonitrile.

That is, by adopting such a composition, the epoxy group can be introduced into the surface of the t-polymer particle at high density while maintaining polymerization stability and dispersion stability in an aqueous medium.

This results in a preferable monomer composition. It is presumed that this is because the polyfunctional internal crosslinking monomer and (meth)acrylonitrile copolymerize effectively with glycidyl (meth)acrylate, creating water-insoluble polymer particles while increasing the internal cohesive force. Ru.

In the above copolymer composition, it is preferable that glycidyl (meth)acrylate is in the range of 10 to 60% by weight in the monomer mixture. This is not preferable because the amount of groups decreases, and the amount of disulfide compounds bound and the amount of physiologically active substances immobilized, which will be described later, decreases.Furthermore, if an amount exceeding 60% by weight is emulsion copolymerized, polymerization stability deteriorates. During the polymerization reaction, a large amount of aggregates are produced.

As a polyfunctional internal crosslinking monomer, it has good copolymerizability,
Furthermore, polyhydric alcohol poly(meth)acrylate (hereinafter referred to as poly(meth)acrylate) has good monopolymerization stability.
It is preferable to use ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, diethylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, triethylene glycol diacrylate, and trimethylol. Propane trimethacrylate.

Trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, and the like are preferably used. Also, divinylbenzene? N.

N'-methylenebisacrylamide and the like can also be used as a polyfunctional internal crosslinking monomer. The internal crosslinking monomer is used in the monomer mixture in an amount of 1 to 304% by weight, preferably 42 to 2011% by weight. If the amount of the internal crosslinking monomer is too small, the addition effect will not be exhibited even in emulsion copolymerization, and if it is used in excess, the stability of the 111 polymerization may be impaired, the amount of aggregates may increase, or the resulting polymer may This is not preferred because the dispersion stability of the particles in an aqueous medium is impaired.

Emulsion copolymerization with the above polyfunctional internal crosslinking monomer (
meth)7crylonitrile in the monomer mixture from 1 to 60
It is preferable to use it within a range of % by weight; outside the # range, polymerization stability and dispersion stability may be impaired.

In forming polymer particles used in the present invention.

By emulsion copolymerizing each of the above monomers, it is possible to obtain polymer particles with excellent dispersion stability, etc., but it is necessary to adjust the glass transition point of the obtained polymer particles to a desired value according to the intended use. In the monomer mixture, radically polymerizable monomers other than the above-mentioned monomers are contained in an amount of 10 to 8811% by weight, preferably ti 20 to 78? may be copolymerized within a range of % by weight. Such monomers include vinyl acetate, (meth)acrylic acid alkyl ester, styrene, and methylstyrene.

The use of one or more monomers with a relatively high glass transition temperature such as vinyltoluene and (meth)acrylamide, especially monomers with a glass transition temperature higher than room temperature, is useful for rounding to prevent fusion between polymer particles. preferable. Of these,
Particularly suitable is Metak! An alkyl ester having 1 to 3 carbon atoms is used.

As mentioned above, when obtaining polymer particles having an average particle size Q of 03 to 3μ having an epoxy group on the surface by emulsion copolymerization, the emulsion copolymerization reaction can be carried out while keeping the pH around 7. desirable. This is because if the pH during one reaction is too acidic or alkaline, epoxy groups (glycidyl groups) tend to rA4iIl and water-soluble polymers are produced, and polymer particles obtained by cleavage of nixyl groups tend to This is because it does not have many epoxy groups on its surface, and the amount of bonding of the disulfide compound becomes insufficient.

The monomer composition as described above is conventionally I) in an aqueous medium.
Although emulsion copolymerization can be carried out by a known ordinary method, the carrier for immobilizing the physiologically active substance iX of the present invention may be mixed with an emulsifier by adsorption or the like.

For example, when used in a specific reaction determined by an agglutination phenomenon in the antigen-antibody reaction 1, the target antigen-antibody will aggregate even if there is no antibody in the sample.

In some cases, IF4 non-specific aggregation may occur. Therefore.

It is preferable not to use an emulsifier in the emulsion copolymerization, and especially when the monomer composition is as shown below, the single polymerization stability is good even in the absence of an emulsifier. However, depending on the type of physiologically active substance to be immobilized, the emulsifier may not have the effect of illumination III in its application.

Needless to say, it may be added in that case.

In the emulsion copolymerization as described above, the concentration of the monomer component mixture in the aqueous medium is also related to the average size of the combined particles in the resulting dispersion.

Usually, it is in the range of 1 to 40g1% by weight.

As the polymerization initiator, a water bath radical polymerization initiator is used. Typically, persulfates such as potassium persulfate, sodium persulfate, and ammonium persulfate.

A redox polymerization initiator using these persulfates and a thiosulfate such as sodium thiosulfate, potassium thiosulfate, sodium hydrogen thiosulfite, etc., or a sulfite such as sodium sulfite, potassium sulfite, sodium hydrogen sulfite, etc. is preferable. The amount of these polymerization initiators used, but not limited thereto, is preferably in the range of 0.01 to 1% by weight based on the monomer mixture. Also the atmosphere of polymerization.

Although not particularly limited, an atmosphere of nine inert gases excluding oxygen is preferably used. Also, the combined temperature is ii.

Although not particularly limited, the temperature is usually in the range of 20 to 100°C, preferably 40 to 90°C.

As mentioned above, the polymer particles obtained as in the above 2 samples have an average particle diameter of 0.03 to 3 μm, and have excellent dispersion stability in an aqueous medium. ,
Adjusting the specific gravity of the linear combined particles to a range of 40.9 to 1.5 is preferable because it facilitates maintenance of dispersion stability in an aqueous medium, bonding reaction with disulfide compounds, and immobilization reaction of physiologically active substances. It is.

The carrier for immobilizing a physiologically active substance of the present invention can be obtained by bonding a dehydrogenated disulfide compound having an rH) group or a carboxyl group at the end of one molecule to the epoxy group present on the surface of the above polymer particles.

As such a chain disulfide compound.

For example, cystamine represented by the formula %, cystine represented by the formula, homocystine represented by the formula, penicillamine disulfide represented by the formula, penicillamine cysine disulfide represented by the formula, dithiodi Glycolic acid formula 1 HOOC-CH
Examples include dithiodipropionic acid represented by 2-CHz -8-8-CH2-CHx -COOH and dithiosalicylic acid represented by formula%.

In order to react the above-mentioned chain disulfide compound with an epoxy group, it is sufficient to simply add the disulfide compound to a latex containing polymer particles having an evaxide group on the surface, mix the mixture, and stir the mixture.

The reaction temperature at this time is usually 20 to 90°C, preferably 30°C.
The reaction is carried out at ~70°C and is completed in several hours to several days.

The carrier for immobilizing a physiologically active substance of the present invention is produced by reacting the epoxy group of a polymer particle having an epoxy group on one surface with a chain disulfide compound having a specific functional group to form a disulfide on the surface of the second particle. This method introduces a bond. The location of the disulfide bond introduced onto the surface can be arbitrarily selected depending on the amount of the physiologically active substance to be immobilized.

Considering the ease of immobilization and dispersion stability in an aqueous medium, disulfide bonds are
It is adjusted to be in the range of X 10-7 to I

The carrier for immobilizing a physiologically active substance of the present invention thus obtained has a chain disulfide bond on the surface of the combined particles, and the disulfide bond is exchanged with a physiologically active substance having a disulfide bond or a thiol group in one molecule. It can be immobilized by reaction. Furthermore, although the carrier of the present invention can be immobilized as it is by reacting with a physiologically active substance, the disulfide bond on the surface of the carrier can be reduced to a thiol group using an appropriate reducing agent, and the carrier can be used as a carrier having a thiol group. It can also be used for immobilizing physiologically active substances.

To reduce disulfide bonds, for example, but not limited to, one such as 2-mercaptoethylamine, 2-mercaptoethanol, dithioslidele, sodium borohydride, etc. may be added to the latex containing the carrier of the present invention. Add reducing agent, mix, let stand, and then centrifuge.

The supernatant liquid may be discarded, an aqueous medium may be added to the formed precipitate to re-disperse it, and then centrifugation and re-dispersion may be repeated.

<Effects of the Invention> As described above, according to the carrier for immobilizing physiologically active substances of the present invention, physiologically active substances such as enzymes and various proteins having disulfide bonds or thiol groups can be relatively easily shared by disulfide bond exchange reaction. It can be immobilized by bonding, and the resulting immobilization carrier also has excellent dispersion stability in an aqueous medium.

In addition, when the epoxy group for bonding with the chain disulfide compound is introduced by emulsion copolymerization of a specific monofood, the epoxy group can be uniformly and densely deposited on the particle surface of the carrier for immobilizing a physiologically active substance. Since it can be introduced, it is also possible to increase the amount of physiologically active substances immobilized.

<Examples> The present invention will be specifically described below with reference to Examples, but the present invention is not limited to these and various applications are possible without departing from the technical idea of the present invention.

Example 1 Glycidyl methacrylate 18. li+, 26 g of methyl methacrylate, 4 g of triethylene glycol dimethacrylate, and 12 g of acrylonitrile in 340 g of distilled water.
In addition to I, an aqueous polymerization initiator solution prepared by dissolving 0.3 g of potassium persulfate in 10 p of water was added under a nitrogen stream to initiate emulsion polymerization at a temperature of 70°C.

Polymerization was carried out by stirring for 6 hours while maintaining the system pH 1 at 7.0, resulting in a solid content of approximately 15% by weight and an average particle size of 0.45.
A latex containing polymer particles having epoxy groups on the surface of μm was obtained. The polymerization reaction was carried out very stably, and the amount of aggregates was about 0.605% by weight.

After repeating the operation of centrifuging the obtained latex and washing the precipitated polymer particles with distilled water three times,
It was redispersed in distilled water so that the solid content concentration was 15% by weight.

(b) 10% of the latex 40d containing the polymer particles obtained in the above and the bonding of the disulfide compound.
ii Add 40 d of cystamine aqueous solution.

The reaction was carried out at 40° C. for 24 hours with stirring. Next, in order to remove all unreacted cystamine present in the aqueous phase,
After centrifuging and washing 5 times with distilled water, 0.01 m
It was redispersed in oJ/l borate buffer (pH 7,0).

The amount of disulfide bonds bonded to the surface of the obtained polymer particles was cleaved with sodium borohydride, completely reduced to thiol groups, and then quantified with 4゜4'-dithiodipyridine. 6.7X10 mol/rr
? Met.

latex (solid content concentration 15 @ volume %) to 5Al.

2-Mercaptoethylamine water ml (1 mo! Otsu no 20
The mixture was stirred and reacted at 25° C. for 3 hours.

Next, 0.01 moJ/J borate buffer (1) H7, O
) and centrifugal washing 5 times, and redispersed in the same buffer solution as in section h so that the solid content concentration was 51%.

β-D-galactosidase (4m9/
ld) Jml fU, and allow the disulfide bond exchange reaction to occur at 5°C for 12 hours with stirring. Then, after washing three times by centrifugation, 0.01 mad/
β-
D-galactosidase immobilized carrier obtained 1. The amount of β-D-galactosidase immobilized was 36 m per 111 carrier particles.
g, and the activity yield was 64%. Incidentally, the activity yield is defined as the ratio of actual activity to the theoretical amount of enzyme activity. In the present invention, NAD
The actual enzyme activity was determined by measuring the absorbance at 340 nmKh of NADHe produced by the reaction in the presence of NAD and galactose dehydrogenase, and the amount of free enzyme having the same activity was divided by the amount of immobilized enzyme.

On the other hand, after storing the latex containing the carrier for immobilizing a physiologically active substance obtained in section h (b) at 25°C, J:
β-D-galactosidase was immobilized in the same manner as described above.

The relative activity of the immobilized enzyme i and the enzyme activity per unit weight polymer particle at θ month of storage is taken as 100%.
Shown in the table.

As is clear from Table 1, in the carrier for immobilizing physiologically active substances of the present invention, the disulfide bonds present on the particle surface function effectively for immobilizing enzymes even after storage for 4 months. .

Comparative Example 1 The reaction was carried out under the same conditions as in Example 1 except that the same amount of 2-mercaptoethylamine hydrochloride as a thiol compound was used instead of cystamine as the disulfide compound bonded in (b) of Example 1. and β-D
- A galactosidase immobilized carrier was obtained. The amount of thiol groups present on the surface of the immobilization carrier particles before immobilization is 3.9X
It was 10 mol/rrl.

Immobilized amount of β-D-galactosidase immobilized carrier and enzyme activity t per unit weight polymer particle at 0th month of storage
- Before storage in the same manner as in Example 1, the relative activity was set as 100%.
It was measured after storage and the results are shown in Table 2.

Table 2 As is clear from Table 2, in carriers for immobilizing physiologically active substances that have thiol compounds bonded to them instead of disulfide compounds, the thiol groups are oxidized during storage and the number of thiol groups necessary for immobilization decreases. Therefore, it was found that the amount of immobilization decreased with the passage of storage time, and the enzyme activity per unit weight of polymer particles also decreased.

That is, the disulfide bonds present in the carrier for immobilizing a physiologically active substance of the present invention can be used for the immobilization reaction as is or after being reduced to a thiol group, but it is necessary to preserve the disulfide bonds as they are until immobilization. This suggests something.

Example 2 Glycidyl methacrylate 18p, styrene 15y1
13 g of methyl methacrylate, 2 I of divinylbenzene and 12 Y of acrylonitrile were added to 340 g of distilled water, and emulsion copolymerization was carried out under the same conditions as in Example 1.
A latex containing polymer particles having an epoxy group on the surface of duckweed having a solid content concentration of about 15% by weight and an average grain size of 0.47μ was obtained. The polymerization reaction was carried out very stably, and the amount of aggregates was about 0.05% by weight.

The obtained latex was centrifuged and the precipitated 1 coalesced particles f were washed with distilled water, which was repeated three times, and then t! ! Shape separation concentration 5i! It was redispersed in distilled water so that the amount was %.

40ml of latex containing ft-M combined particles
40 ml of an aqueous solution of dithiodiglycolic acid containing % by weight of KLo was added, and the mixture was reacted at 40° C. for 48 hours with stirring. Next, in order to remove unreacted cystamine present in the aqueous phase, the mixture was centrifuged and washed five times with distilled water.
0.01 mol/l borate buffer (pH 7,0
) was redispersed.

The amount of disulfide bonds present on the surface of the obtained polymer particles was 1.9 x 10 mol/r! ? Met.

(e) Immobilization of enzyme on carrier β-D-galactosidase was immobilized on the obtained latex containing the carrier for immobilizing a physiologically active substance of the present invention in the same manner as in Example 1. 27 per gram of particles
The activity yield was 68%.

On the other hand, when the latex containing the carrier for immobilizing a physiologically active substance obtained in (b) above was stored at 25°C, 41
: β-D-galactosidase was immobilized in the same manner as described above. Table 3 shows the fixed amount of nine enzymes and the relative activity (enzyme activity per unit weight of polymer particles) f: 100% after 0 months of storage.

Table 3

Claims (3)

[Claims] (1) Average particle size 0.03 to 3 with epoxy groups on the surface
A carrier for immobilizing a physiologically active substance, characterized in that a chain disulfide compound having an amino group or a carboxyl group at the molecular end is bonded to the above-mentioned epoxy group to a latex containing μm-sized polymer particles. (2) the polymer particles are glycidyl (meth)acrylate;
The carrier for immobilizing a physiologically active substance according to claim 1, which is obtained by emulsion copolymerizing a monomer mixture containing a polyfunctional internal crosslinking monomer and (meth)acrylonitrile. (3) The carrier for immobilizing a physiologically active substance according to claim 2, wherein the polyfunctional internal crosslinking monomer is a poly(meth)acrylate of a polyhydric alcohol.