JPH0811764B2 - Method for producing cationic polyelectrolyte copolymer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial field of application> The cationic derivative of water-soluble linear polysaccharide-olefin monomer graft copolymer of the present invention is a cationic derivative of water-soluble linear polysaccharide having a hydroxyl group. The olefin monomer is graft-polymerized in water to produce a latex polymerization product useful as an immunoassay material. INDUSTRIAL APPLICABILITY The present invention suggests that a cationic derivative of a linear polysaccharide having a hydroxyl group and a water-soluble olefin monomer can be generally graft-polymerized in water to produce a latex polymerization product useful as an immunoassay material. It is a thing.

<Prior Art> Most conventional methods for producing a latex polymerization product as an immunoassay material are emulsion-polymerized in an aqueous solution in the presence of a surfactant, and a soapless product without a surfactant is used. Is desired. This is because the surfactant present in the aqueous solution affects the action as a latex diagnostic agent.

<Means for Solving Problems> The cationic derivative of the water-soluble linear polysaccharide-olefin monomer graft copolymer of the present invention is a cationic derivative of a water-soluble linear polysaccharide having a hydroxyl group and an olefin in water. Graft polymerization of a monomer by a suspension polymerization method or an emulsion polymerization method to produce a latex polymerization product useful as an immunoassay material, which is a cationic derivative of the water-soluble linear polysaccharide of the present invention-olphine monomer The polymer graft copolymer latex is used for the antibody-adsorbed latex diagnostic agent according to the claims, which is a latex diagnostic agent. Emulsion polymerization is a polymerization method in which an olefin monomer is suspended in an aqueous solution and usually emulsified using a surfactant or the like. To be more specific, hydrogen bonding, Coulomb force, charge transfer This is a polymerization method in which a polymer chain is polymerized and grows to form fine particles in an aqueous solution by interaction at an aqueous solvent interface by action, Van der Waals force, or the like. Usually, the polymerization product is present as a mixture of polymerized monomer and surfactant. This surfactant, which is considered to be an impurity, may interfere when used in the antibody-adsorbed latex diagnostic agent described in Reference Example which is a latex diagnostic agent, which is a problem.

<Effect of the Invention> The present invention is a product obtained by graft-polymerizing an olefin monomer in water to a cationic derivative of a water-soluble linear polysaccharide having a hydrogen group. Each bond is shared by chain transfer of olefin monomer double bond resulting from radical generation by abstraction of hydrogen atom of hydroxyl group (as proton by oxidation of initiator) of cationic derivative of water-soluble linear polysaccharide. It is clear that it is a bond. This is 4
Since it is carried out by using a valent cerium ion or the like as an initiator and no surfactant is used, it is very useful without being disturbed when it is used as an antibody-adsorbed latex diagnostic agent. The cationic derivative of water-soluble linear polysaccharide-olefin monomer graft copolymer latex of the present invention is intended to be used as an antibody-adsorbed latex diagnostic agent. That is, the objective is to graft-polymerize an olefin monomer in water to a water-soluble polymer having a hydroxyl group to produce a latex polymerization product useful as an immunoassay diagnostic material. On the other hand, such a graft copolymer which is said to be soap-free and grows at the interface between the solvent and the solute is a useful substance having a wide variety of applications. In particular, in addition to immunoassay materials, they are attracting attention as overmembranes and biomaterials. Focusing on its hydrophilicity, it can be applied to artificial kidney membranes, components, blood substitutes, and contact lenses.

<Detailed Description of Structure of Invention> Hereinafter, a cationic derivative of the linear polysaccharide of the present invention-
The olefin monomer graft copolymer will be described in detail. The cationic derivative of linear polysaccharide-olefin monomer graft copolymer is obtained through the following two steps.

I. Preparation of Cationic Derivative of Linear Polysaccharide The formula of the unit of the cationic derivative of linear polysaccharide is [C ₆ H ₇ O ₂ (OH) _3-a・ (OX) _a ] x ・ H ₂ O (1) [wherein X _{is, - (CH 2) nR 1} (R 1 is -NH _2, -N (C
_{H 3) 2, -N (C} 2 H 5) 2, -N + (C 2 H 5) 3, -N + (C
_{2 H 5) 2 (C 2} H 5) N (C 2 H 5) 2, -C 6 H 4 · NH 2, -CO · C 6
A group selected from the group consisting of H ₄ · NH ₂ , n = 1 to 3), or —COR ₂ (R ₂ is —CH ₂ · NH ₂ or —C ₆ H ₄ · NH ₂ ).
Or _{-CH 2 CH (OH) · CH} 2 R 3 (R 3 is -NH _2, -N (C
_{H 3) 2, -N (C} 2 H 5) 2, -N + (C 2 H 5) groups selected from the group consisting of _3), or _{-NH · CH 2 · CH 3,} a is 0 <a <3
Positive number, x is an integer of 50,000 ≧ x ≧ 5], and (1)
It is shown by the formula. In the above formula (1), the hydroxyl group of the cationic derivative of this linear polysaccharide is partially substituted with an acid group such as carboxymethyl group or sulfate group by an ether bond, or partially substituted with an alkyl group. It may have a cationic functional group represented by X. Usually, the cationic derivatives of these linear polysaccharides are obtained by the Schotten-Baumann reaction in an alkaline solution of the hydroxyl group of the linear polysaccharide and the chlorine compound of the above cationic substituent represented by XC1. The linear polysaccharides referred to here include those that can be industrially produced by fermentation such as dextran and pullulan. As the olefin compound which can be graft-polymerized, a compound represented by the following general formula is considered.

[Wherein R ₄ , R ₅ and R ₆ are each selected from a hydrogen atom or CH ₃ , and R ₇ is (Wherein R ₈ is a hydrogen atom, a C _{1 to} C ₁₂ alkyl group, a cyclohexyl group, a C _{1 to} C ₄ hydroxyalkyl group, a C _{1 to} C ₈ aminoalkyl group, and a C _{1 to} C ₈ dialkylaminoalkyl group. Group, glycidyl group, tetrahydrofuran group, C _{1 to} C ₄ lower alkyl-substituted tetrahydrofuran group, benzyl group, and (—CH ₂ CH ₂ —O—) yCH ₂ CH ₂ OH group (where y is a positive number from 1 to 10) , -N (R ₉ ) ₂ (R ₉ is a hydrogen atom or a C ₁ -C ₄ alkyl group, two R ₉ may be the same or different)), (R ₁₀ represents a C ₁ -C ₈ alkyl group), a phenyl group, a pyridine group, a tolyl group, or a pyrrolidone group].

Specifically, α, β-unsaturated acid alkyl esters such as acrylic acid and methacrylic acid, lower alkyl-substituted cyclohexyl esters such as cyclohexyl ester, 2-hydroxyethyl ester, 2-hydroxypropyl ester, and 2-hydroxy. Butyl ester;
Acrylamide, methacrylamide, acrylamide,
Acrylic - or methacrylic - dimethyl amide, the alpha, aminoalkyl esters of C ₁ -C ₃ of β- unsaturated acids,
C ₁ -C ₃ dialkylaminoalkyl ester, glycidyl ester, tetrahydrofurfuryl ester, benzyl ester, polyethylene glycol monoesters;
Α, such as acrylonitrile and methacrylonitrile,
Nitrile group of β-unsaturated acid; vinyl alcohol, methyl vinyl alcohol, dimethyl vinyl alcohol; vinyl alcohol such as vinyl acetate, vinyl propionate, vinyl butyrate and its C ₁ -C ₃ alkyl ester of methyl-substituted vinyl alcohol; styrene , Vinyltoluene; vinylpyrrolidone; vinylmethylpyrrolidone and the like.

II. Preparation of graft copolymer The reaction is usually carried out in an aqueous solution. That is, the above olefin monomer is added to an aqueous solution of a cationic derivative of a polysaccharide, and an initiator is added to react. As the initiator, tetravalent cerium salt, tetravalent manganese salt, ferric salt-hydrogen peroxide are usually used, but in addition, potassium persulfate (KPS), azobisisobutylnitrile (AIBN), benzoyl peroxide. Radical initiators such as (BPO) are also used. The reaction temperature can be widely selected from normal temperature to 80 ° C. If necessary, the reaction is continued by replacing with nitrogen. Each bond is a covalent bond due to chain transfer of an olefin monomer double bond, which is caused by generation of a radical by abstracting as a proton of a hydrogen atom by oxidation of a hydroxyl group of a cationic derivative of a water-soluble polysaccharide.

In this reaction, the product occurs in latex. This latex polymer is generally insoluble in water, alcohol, or an organic solvent such as acetone or tetrahydrofuran, but can be easily formed into a film by a casting method or the like. Alternatively, an excessively insoluble solvent such as alcohol is added to obtain a precipitate, and then a molded product can be easily formed by a hot press method or the like.

For the above purpose, the ratio of the backbone polymer to the graft polymer or the degree of polymerization in the graft polymer can be selected variously according to the purpose. In the graft polymerization, the polymerization rate is determined by the graft rate (%). This is defined by graft ratio (%) = (amount of monomer graft-polymerized / amount of trunk polymer in graft copolymer) × 100.

In the present invention, the olefin compound is formed as a graft chain, and it is considered appropriate that the graft ratio is in the range of 2% to 5000%. Although the present invention repeatedly states that it is a product obtained by graft-polymerizing an olefin monomer in water into a cationic derivative of a water-soluble polysaccharide having a hydroxyl group, the structure of the resulting copolymer chain is dextran or the like. A cationic derivative of a soluble linear polysaccharide is used as a trunk polymer, and its hydroxyl group serves as a binding site by abstracting a hydrogen atom, and an olefin compound serves as a graft chain. Each connection is
As mentioned above, it is obvious that the bond is a covalent bond due to chain transfer of an olefin monomer double bond due to radical generation by abstraction of a proton of a hydroxyl group of a water-soluble polysaccharide cationic derivative.

Example 1 2 g of DEAE (diethylaminoethyl) dextran hydrochloride having a nitrogen content of 5% and having dextran having an average molecular weight Mw of 500,000 as a matrix was dissolved in 50 ml of water, and then 5 ml of methanol and 30 ml of methyl methacrylate (MMA) were added, and the mixture was thoroughly added. Reaction solution,
After the air in the reaction vessel is replaced with nitrogen gas, 190 mg of ceric ammonium nitrate nitrate dissolved in 15 ml of 0.1N nitric acid is added with thorough stirring to start the reaction. Reaction is 3
A latex is formed at 0 ° C. for 1 hour. At the end of the reaction, 3 ml of a 1% hydroquinone solution was used as a terminating agent.
Then, the reaction solution was poured into 3 times the amount of methanol to obtain a precipitate.

The precipitate was washed thoroughly with hot water, centrifuged, dried under reduced pressure at 50 ° C, and then the dried product was placed in a Soxhlet extractor.
Acetone extraction was carried out for a period of time to obtain 1.5 g of DEAE (diethylaminoethyl) dextran-MMA copolymer hydrochloride.

Nitrogen content 2.0% Graft ratio 25% vs DEAE-dextran yield 30% This is insoluble in water which is a good solvent for DEAE dextran hydrochloride and acetone which is a good solvent for polymethylmethacrylate. FIG. 1 is an infrared absorption spectrum of this product. Absorption of the carbonyl group, which is not found in DEAE-dextran hydrochloride, is observed near 1730 cm ^-1 .

Example 2 Nitrogen content 4 based on pullulan having an average molecular weight of Mw 200,000 4
% DEAE (diethylaminoethyl) -pullulan hydrochloride 4 g
Was dissolved in 80 ml of water, then 10 ml of methanol and 35 ml of styrene monomer were added, and the air in the reaction solution and the reaction vessel was sufficiently replaced with nitrogen gas.
Cerium ammonium nitrate nitrate 20 dissolved in
Add 0 mg to start the reaction.

The reaction is carried out at room temperature for 1 hour to form a latex. At the end of the reaction, 3 ml of a 1% hydroquinone solution was used as a terminating agent.

Subsequent purification and drying steps were performed as in Example 1 and DEAE
6 g of hydrochloride of (diethylaminoethyl) pullulan-styrene copolymer was obtained.

Nitrogen content 1.14% Graft ratio 350% vs DEAE pullulan yield 4
3% Example 3 4 g of AE (aminoethyl) dextran hydrochloride having a nitrogen content of 5% and having dextran having an average molecular weight Mw of 40,000 as a matrix was treated with water.
Dissolve in 90 ml, then add 5 ml of methanol and 20 ml of butyl methacrylate, sufficiently replace the air in the reaction solution and reaction vessel with nitrogen gas, and then with stirring well, add 15 ml of 0.1N nitric acid.
Cerium ammonium nitrate nitrate 50 dissolved in
Add mg to start the reaction. The reaction is carried out at room temperature for 30 minutes to form a latex. Upon completion of the reaction, 3 ml of a hydroquinone 1% solution was used as a terminating agent. Subsequent purification and drying steps were performed in the same manner as in Example 1 to obtain 6 g of hydrochloride of AE (aminoethyl) dextran-butyl methacrylate copolymer.

Nitrogen content 2.0% Graft ratio 300% to AE-dextran yield 30% This is insoluble in water, which is a good solvent for AE dextran hydrochloride, and in acetone, which is a good solvent for polybutyl methacrylate.

Example 4 Nitrogen content 3 with pullulan having an average molecular weight Mw of 30,000 as a matrix
% HPTMA (2-hydroxypropyltrimethylammonium) -pullulan hydrochloride 4 g was dissolved in 100 ml of water, then 30 ml of methyl acrylate monomer was added, and the reaction solution and air in the reaction vessel were sufficiently replaced with nitrogen gas. Then, while stirring well, 200 mg of ceric ammonium nitrate nitrate dissolved in 20 ml of 0.1N nitric acid is added to start the reaction. The reaction is carried out at room temperature for 1 hour to form a latex. At the end of the reaction, 4 ml of a 1% solution of hydroquinone was used as a terminator. The subsequent purification and drying steps were performed in the same manner as in Example 1, and HP
2 g of hydrochloride of TMA (2-hydroxypropyltrimethylammonium) pullulan-methyl acrylate copolymer was obtained.

Nitrogen content 2% Grafting rate 150% HPTMA pullulan yield 33% Reference Example 1 After carrying out the same reaction as in Example 1, the reaction completed solution of the latex was not poured into methanol, but the reaction was carried out in water to cause unreacted reaction. The substance and the initiator were removed to obtain a DEAE dextran-MMA copolymer latex mixed with a homopolymer.

This is useful as a reagent for detecting rheumatoid factor and shows the test results.

Test Method A 2% solution of this latex was mixed with an equal volume of antibody, ie glycine-to give 20 μg / mg latex.
Nacl buffer-gamma globulin (GB) (0.05mol / 1, PH8.
2) diluted with) was added drop by drop with stirring,
After stirring for 2 hours at room temperature (if necessary, centrifuge at 4000 rpm for 30 minutes and repeat resuspension with GB) Finally, store at 4 ° C as a 1% suspension in GB with 0.5% BSA (bovine albumin). .

One drop of the above-mentioned sensitized latex liquid of the present invention was dropped onto a wiped glass plate with a capillary tube, and rheumatoid factor-positive serum diluted to 1/20 with GB in advance with a capillary tube.
When the glass plate was swirled and shaken, the presence or absence of aggregation was visually judged after about 3 minutes, and an aggregate was observed. Similar operation known 1/20
No aggregates were observed when performed with rheumatoid factor-negative serum diluted to 1%.

Reference Example 2 After the same reaction as in Example 2, the reaction-completed solution of the latex was not poured into methanol, but the solution was allowed to disintegrate in water to remove unreacted materials and the initiator, and the homopolymer was mixed. A DEAE pullulan-styrene copolymer latex was obtained.

This was useful as a reagent for detecting a lithium-like factor.

As in Reference Example 1, a 2% solution of this latex was mixed with an equal amount of the antibody, that is, glycine-containing 20 μg / mg latex.
Nacl buffer-γ globulin (GB) (0.05mol / 1, PH8.
The latex solution diluted in 2) and sensitized was visually confirmed to have aggregates in the rheumatoid-like factor-positive serum by the procedure of Reference Example 1, but aggregates were observed in the rheumatoid-like factor-negative serum. I couldn't see it.

Reference Example 3 After the same reaction as in Example 3, the reaction-completed solution of the latex was not poured into methanol, and unreacted substances and the initiator were removed by performing the folding in water to mix the homopolymer. AE (aminoethyl) dextran-bryl methacrylate copolymer latex was obtained.

This was useful as a reagent for detecting rheumatoid factor.

As in Reference Example 1, a 2% solution of this latex was mixed with an equal amount of the antibody, that is, glycine-containing 20 μg / mg latex.
Nacl buffer-γ globulin (GB) (0.05mol / 1, PH8.
The latex liquid diluted and sensitized in 2) was visually confirmed by the procedure of Example 2 for the presence or absence of aggregates in the rheumatoid factor-positive serum, but aggregates were observed. I couldn't see it.

Reference Example 4 After carrying out the same reaction as in Example 4, the unreacted material and the initiator were removed by performing folding in water to remove homopolymer, and HPTMA (2-hydroxypropyltrimethylammonium) pullulan-acrylic acid mixed with a homopolymer. A methyl copolymer latex was obtained.

This was useful as a reagent for detecting rheumatoid factor.

As in Reference Example 1, a 2% solution of this latex was mixed with an equal amount of the antibody [that is, glycine-Nacl buffer γ globulin (GB) (0.05) to obtain a 20 μg / mg latex).
The latex solution diluted with (mol / 1, PH8.2) and sensitized was subjected to the procedure of Reference Example 1 to visually confirm the presence or absence of aggregation in the rheumatoid factor positive serum. No aggregates were found in the negative serum.

[Brief description of drawings]

FIG. 1 shows DEAE-dextran-of Example 1 of the present invention.
FIG. 3 is an infrared absorption spectrum diagram of the hydrochloride salt of the MMA copolymer.

Claims (1)

[Claims] 1. A compound represented by the general formula [C ₆ H ₇ O ₂ (OH) _3-a · (OX) _a ] x · H ₂ O (1) [wherein X is — (CH ₂ ) nR ₁ (R ₁ is ^{_{-NH 3 +, -NH + (CH}} 3)
^{_{2, -NH + (C 2 H}} 5) 2, -N + (CH 2) 2 CH 2 CH (OH) CH 3,
^{_{-N + (C 2 H 5)}} 2 (C 2 H 5) N (C 2 H 5) 2, -N + (C 2 H 5) 2 C
_{H 2 CH (OH) CH 3} , -N + (C 2 H 5) 3, -C 6 H 4 · NH 3 +, -CO
A group selected from the group consisting of C ₆ H ₄ · NH ₃ ⁺ , n = 1 to 3), or —COR ₂ (R ₂ is —CH ₂ · NH ₃ ⁺ or —C ₆ H ₄ · NH ₃
⁺ ) Or --CH ₂ CH (OH) ・ CH ₂ R ₃ (R ₃ is --NH ₃ ⁺ , --NH ⁺ (CH
^{_{3) 2, -NH + (C}} 2 H 5) 2, -N + (C 2 H 5) groups selected from the group consisting of ₃₎ or _{-NH · CH 2 · CH 3,} a is 0 <a <3 Is a positive number, x is an integer of 50,000 ≧ x ≧ 5], and is a trunk polymer composed of a cationic derivative of a water-soluble linear polysaccharide, [Wherein R ₄ , R ₅ and R ₆ are each selected from a hydrogen atom or CH ₃ , and R ₇ is (R ₈ is a hydrogen atom, a C _{1 to} C ₁₂ alkyl group, a cyclohexyl group, a C _{1 to} C ₄ lower alkyl-substituted cyclohexyl group, a C _{1 to} C ₈ hydroxyalkyl group, and a C _{1 to} C ₈ amino group. Alkyl group, C ₁ -C ₈ dialkylaminoalkyl group, glycidyl group, tetrahydrofuran group, C ₁ -C ₄ lower alkyl-substituted tetrahydrofuran group, benzyl group and (-CH
_{2 CH 2 -O-) yCH 2 CH} 2 OH group (y is 1 to 10 a positive _{number), - N (R 9)} 2 (R 9 is hydrogen atom or an alkyl group of C ₁ -C _4, two R ₉ may be the same or different)), (R ₁₀ represents a C _{1 to} C ₈ alkyl group), a phenyl group, a pyridine group, a tolyl group, a pyrrolidone group, and a group selected from the group consisting of a C _{1 to} C ₄ lower alkyl-substituted pyrrolidone group] A method for producing a graft copolymer, which comprises graft-polymerizing a compound.