JPH02168163A - Carrier for immobilizing hydrophobic substance and method for immobilizing hydrophobic substance employing such carrier - Google Patents

Abstract

PURPOSE:To immobilize hydrophobic substance even in the existence of a substance weakening the hydrophobic mutual action by forming a carrier of an inactive carrier into which a cationic functional group is introduced or which absorbs a compound having a cationic functional group. CONSTITUTION:For the inactive carrier, a carrier with the hydrophobic surface, for example, latex is used. For the cationic functional group, quaternary amino group and its salt are used. In order to introduce the cationic functional group into the carrier, for example, a compound containing a cationic functional group is arranged to be physically attracted to the surface of the carrier. For the compound, particularly, for the low molecular compound, dodecilamine or the other kinds of amine is used, and for the high molecular compound, a cationic polymer including a hydrophobic part is used. The hydrophobic part is composed of alkyl group or others. For the cationic polymer, a polymer containing fourth-class amino group represented by the formula is most preferable. In the formula, R1-R10 represent a hydrogen or alkyl group, Y1 and Y2 represent an alkyl or alkylene group which may contain S, N or O.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a carrier for immobilizing a hydrophobic substance and a method for immobilizing a hydrophobic substance using the carrier. For more details 1
The present invention provides an immobilization carrier that can effectively immobilize a hydrophobic substance even in the presence of a substance that weakens hydrophobic interactions, such as a surfactant, and a method for immobilizing a hydrophobic substance using the same. Regarding.

(Prior art) Antigens and antibodies are immobilized on carriers for the purpose of measuring physiologically active substances using antigen-antibody reactions.

Immobilized enzymes have been prepared by immobilizing enzymes on carriers. Immobilization of such substances.

Generally, this is done using hydrophobic interactions. For example, latex, plastic beads.

Immobilization is achieved by directly contacting the substance to be immobilized with a hydrophobic carrier such as a plastic plate in a buffer, physiological saline or purified water.

However, in the conventional direct immobilization method using physical adsorption, only hydrophobic interactions are used, so substances that weaken hydrophobic interactions, such as surfactants, are present in the reaction system during adsorption and immobilization. If it contains, immobilization is not possible. Therefore.

This direct immobilization method by physical adsorption can be used for extraction and/or
Also, it cannot be used to immobilize hydrophobic substances such as membrane proteins and lipids that require surfactants for stabilization.

This problem can be solved if the substance to be immobilized is immobilized by a covalent bond. However, it is difficult to control the reaction for introducing covalent bonds, and in some cases, the activity of the immobilized substance may be significantly impaired.

Patent Publication No. 63-501902 discloses that latex is treated with polycationic polyamino acids (polylysine or polyarginine) or methylated serum albumin to physically adsorb or chemically bond, and then calciolipin (syphilis antibody and It has been disclosed that ionically adsorbing a phospholipid (which exhibits a specifically reactive antigenic action) is carried out. The calciolibin-bound latex thus prepared is used for the diagnosis of syphilis. However, the above polylysine and polyarginine are homopolymers of hydrophilic amino acids and exhibit hydrophilicity. Therefore, even if hydrophobic materials such as latex, plastic beads, plasti, and cuprate are treated with polylysine or polyarginine,
These amino acid homopolymers cannot be effectively introduced. Also.

The ε-amino group of lysine is a primary amino group, and the pH is 11.
Above this, dissociation is almost suppressed and it no longer has a charge. Therefore, polylysine can be applied to substances that can be immobilized at a pH below 91111, such as calciolipin, but there are restrictions in that it cannot be applied to physiologically active substances that need to be immobilized at pH 11 or higher. Therefore, other physiologically active substances such as proteins may be inappropriate. Furthermore, the above-mentioned polylysine and polyarginine are polymers of amino acids, that is, polypeptides, and are not suitable for adsorbing and immobilizing proteins, which are also polypeptides, because the bonding force between them is weak. In addition, polylysine is expensive and toxic to living organisms.

Care must be taken when handling.

In this way, in the above-mentioned conventional substance immobilization method.

A large amount of physiologically active substance is required for immobilization due to high efficiency; the amount of physiologically active substance that can be immobilized per unit area is small; a wide ρ region cannot be immobilized; in the presence of a surfactant There were problems such as: it could not be fixed in place;

(Problems to be Solved by the Invention) The present invention is intended to solve the above-mentioned conventional problems, and its purpose is to solve the above-mentioned problems in the prior art. An object of the present invention is to provide an immobilization carrier capable of immobilizing hydrophobic substances such as physiologically active substances.

Another object of the present invention is to provide an immobilization carrier that can effectively immobilize hydrophobic substances that cannot be immobilized by mere physical adsorption, or even if immobilized, the amount is insufficient. be.

Still another object of the present invention is to provide a method for efficiently immobilizing hydrophobic substances such as physiologically active substances using the immobilization carrier having the above-mentioned excellent properties.

(Means for Solving the Problems) A cationic functional group is introduced into the carrier for immobilizing a hydrophobic substance of the present invention. Alternatively, it is an inert carrier that has adsorbed a compound having a cationic functional group, thereby achieving the above object.

The method for immobilizing a hydrophobic substance of the present invention includes bringing a hydrophobic substance into contact with the carrier for immobilizing a hydrophobic substance and physically adsorbing the hydrophobic substance on the surface of the carrier. The above objective is achieved.

The inert carrier used in the present invention has a hydrophobic surface. Alternatively, any inert carrier with a partially hydrophobic surface can be utilized.

Examples include materials made of synthetic polymer compounds such as latex, plastic beads, and plastic plates; organic materials such as red blood cells treated with tannic acid; and inorganic materials such as silica.

In particular, latex, which can be mass-produced with industrially stable quality, or plastic molded products such as plastic beads and plastic plates are preferably used. The cationic functional groups introduced or adsorbed onto such an inert carrier include primary, secondary, tertiary or quaternary amino groups and salts thereof. Quaternary amino groups and salts thereof having a wide pH range that can act as cations are preferably used. Such a cationic functional group or a compound containing the same may partially contain an anion as long as it does not interfere with the immobilization of the hydrophobic substance.

Methods for introducing the above-mentioned cationic functional groups into the carrier include: (1) a method in which a monomer containing a cationic functional group is simultaneously reacted when synthesizing the plastic used as the carrier to prepare a carrier having the group; A method in which a cationic functional group is bonded to the surface of the carrier by chemical modification after the carrier is formed; and ■ A compound containing a cationic functional group (e.g., amines or cationic polymer) is physically adsorbed onto the carrier surface. There is a way to do it. Method (2) includes a method in which, for example, a heptamer having an amino group is simultaneously polymerized during one polymerization (for example, to prepare nylon or a derivative thereof).

Method (1) includes, for example, a method in which a stepless material such as silica is used as an inert carrier and the OH groups of the silica are chemically modified. As a compound used in method (2), as a low molecular weight compound.

Various amines such as dodecylamine and hexadecyltrimethylamine are used, and a cationic polymer containing a hydrophobic moiety is used as the high molecular weight compound. The hydrophobic portion of the polymer consists of alkyl groups, alkylene groups, phenyl groups, and the like. The number of carbon atoms in the alkyl group and alkylene group is suitably within the range of 1-15.

If the number of carbon atoms is larger than this, the polymer becomes less soluble in water and the carrier becomes difficult to treat. The molecular weight of the polymer is suitably 1,000 to 400,000. If the molecular weight is smaller than this, the hydrophobic interaction with the carrier will not be sufficient and it will not be effectively adsorbed onto the carrier.

At molecular weights above 400.000, the viscosity of the polymer becomes high and the support becomes difficult to process.

Examples of such polymers include polyallylamine (having an I-class amino group; produced by Nittobo Co., Ltd.), polyethyleneimine (having a secondary amine group), polydiallyldimethylammonium chloride, and polyamine sulfone (having a quaternary amino group). ), nucleic acids, etc. For example, a polymer having a quaternary amino group represented by the primary formula (I) is suitable: where R+""R+. is hydrogen or an alkyl group.

So and Vt are an alkyl group or an alkylene group, and Elv and Y2 may contain S, N or O.

In particular, polyamine sulfone represented by the primary structural formula (n) (Nittobo, PAS-A-5, average molecular weight 2.000~
5.000) is preferable: (Hereinafter in the margin) The substance to be immobilized on the carrier of the present invention is a substance that has a hydrophobic part and has a negative charge under the immobilization conditions. Such substances include physiologically active substances such as proteins, lipids, and the like. In particular, substances that require surfactants for extraction, stabilization, etc.1 For example, viral antigens, surface antigens of bacterial cells such as Treponema pallidum, a+
Various antigens, membrane proteins, receptors,
Enzymes and the like can be effectively immobilized by the carrier and method of the present invention.

As a surfactant used for the extraction and stabilization of surface antigens, membrane proteins, etc. of the above-mentioned bacterial cells.

Nonionic, amphoteric and cationic surfactants can be used, but anionic surfactants are not suitable. Examples of nonionic surfactants include 1, for example, Triton X,
Tween 20. Tween 80. Octyl glucoside, octyl thioglucoside, heptyl thioglucoside,
MEGA-8 (Octanoyl-N-methylglucamide)
e).

MEGA-9 (nonanoyl-N-methylglucamide:
Non-anoyl-N-methylglucami
de), MEGA-10 (decanoyl-N-methylglucamide: Decanoyl-N-methyl
-glucamide), etc. Examples of amphoteric surfactants include CI (APS (3-
((3-cholamidopropyl)dimethylammonio]-1
-Propanesulfonate: 3- ((3-Chola
midopropyl) dimethyl-ammon
io) -1-propanesulfonate)
, CIIAPSO(3-[(3-Cholamidopropyl)dimethylammonio]=2-hydroxy-1-propanesulfonate: 3((3-Cholamidopropyl)
pyl)dimethylaa+monio) -2-
hydroxyl-propane-5ulfonat
e) etc. As a cationic surfactant, dodecylamine
), hexadecyl trimethylammonium bromide
ium bromide).

Immobilization of a hydrophobic substance onto a carrier according to the method of the present invention is carried out by bringing a solution of a hydrophobic substance dissolved in a buffer, physiological saline, or purified water into contact with the immobilization carrier. As a result, the hydrophobic substance is first attracted to the cationic functional group on the surface of the carrier by ionic interaction, and then immobilized on the surface of the carrier by hydrophobic interaction with the carrier. The buffer used at this time.

Although any buffer known to those skilled in the art can be used.

It is desirable that the ionic strength is less than 0.1M. Since the method of the present invention utilizes ionic interaction as the first step in immobilizing a hydrophobic substance, it is not effective if the ionic strength is high. The pH of the buffer solution pt+ is adjusted to such a level that the cations on the carrier are dissociated, the substance to be immobilized can stably exist in the buffer solution, and is negatively charged. However, when a quaternary amino group is used as the cation on the carrier, a buffer solution having a relatively wide pH range can be used since the cation on the carrier is dissociated at any pH.

The carrier and method of the present invention are particularly suitable for RIA, EIA
.

It is suitably used in the preparation of immunodiagnostic reagents for latex methods and the like. For example, HBs antigen, HBc antigen.

It can be used to prepare various immunodiagnostic reagents in which antigens such as HBe antigen, syphilis antigen (treponemal antigen and lipid antigen), HIV antigen, and ATLV antigen are immobilized on appropriate carriers.

When preparing such an immunodiagnostic reagent according to the present invention, in some cases, choline chloride EDTA, sugars (polytars, dextran, etc.), polyethylene, etc. Hydrophilic polymers such as glycols can also be added to the reaction system. The immunodiagnostic reagent thus prepared can be widely used in fields such as clinical testing for disease diagnosis and treatment. Furthermore, it can be used in a method of preparing an immobilized enzyme and producing a specific substance using the same.

(Example) The invention is illustrated by the following examples.

[Immobilization of Trebonema pallidum antigen on a plastic plate in the presence of a surfactant] (A) Preparation of reagents and specimens The following reagents and specimens were prepared and used.

Phosphate buffer sodium diphosphate (dihydrate).

Disodium phosphate (dihydrate) and sodium chloride were prepared with the final 4 degrees of phosphoric acid and sodium chloride being 0, respectively.
．． 02M and 0.15M, and were mixed to have a pH of 7.4.

Phosphate-citrate buffer: 0.2M disodium phosphate and 0.1M citric acid were mixed and adjusted to pH 5.5.

1% polyamine sulfone aqueous solution: Polyamine sulfone (
Nittobo, PAS-A-5, average molecular weight 2,000-5
．． 000) was dissolved in water to make a 1% aqueous solution. The structural formula of this polyamine sulfone is represented by (U)2 in the specification.

1% polyethyleneimine aqueous solution = 30% polyethyleneimine P-70 (average molecular weight 70,000; manufactured by Wako Pure Chemical Industries, Ltd.) was diluted with purified water to obtain a 1% aqueous solution.

1mM hydrochloric acid: Hydrochloric acid was diluted with purified water to make a 1mM aqueous hydrochloric acid solution.

Seven volumes of bovine serum albumin (1%) were dissolved in 1% BSA diphosphate buffer.

Triton X- in 1% Triton X-100 diphosphate buffer
100 was dissolved to give a concentration of 1%.

Syphilis antigen solution: Trebonema palitum cultured for 10 to 14 days in a domestic rabbit laboratory;
Center for Disease Control
l, Public Health Service,
11. s, DeparLmer+L of IIea
LLh, Education and Welfare
, ALanta, Georgia) was used to inoculate domestic rabbits, and 8! 1 using the subculture was suspended in physiological saline at a density of 109 cells/ml, and centrifuged in phosphate buffer (6,000 rpm for 5 minutes). 23 times). Next, 1 ml of 1% Triton X-100 was added to the obtained precipitate, and the mixture was incubated at 37°C for 30 minutes. Thereafter, this was applied to an ultracentrifuge (50,000 rpm + x 1 hour) and the supernatant was collected.

It was diluted 1,000 times with 1% Triton X400 and used.

Syphilis-positive rabbit serum: Serum was collected from rabbits raised for 45 days after being inoculated with Trebonema pallidum. Commercially available TP) IA kit (Celodia TP (Xushishi Bio),
When the titer was measured using Serocrit TP (Kaketsuken), the titer was 10,000 for all kits.

This serum was diluted 100 to 400 times with 1% BSA and used.

Normal rabbit serum: Trebonema pallidum t, f fi
Serum collected from untreated domestic rabbits was used. When the titer was measured using a commercially available TP II A kit in the same manner as above, the result showed negative. 1 of this serum
% BSA and diluted 100 to 400 times.

Peroxidase-labeled anti-rabbit rgc: Peroxidase-labeled anti-rabbit IgG (Miles Laboratories) was diluted 1,00 times with 1% BSA and used.

Microquiter plate: 96 holes made of plastic (
A microtiter plate (Nunc) was used.

Peroxidase substrate: 0-phenylenediamine (dihydrochloride) and aqueous hydrogen peroxide were dissolved in a phosphoric acid-citric acid buffer solution to a concentration of 2/all and 0.03%, respectively. Substrate preparation was performed immediately before use.

IN sulfuric acid: Concentrated sulfuric acid was diluted with purified water to obtain an IN sulfuric acid aqueous solution.

(B) Treatment of microtiter plate [Polyamine treatment] Add 50 μl of 1% polyamine sulfone aqueous solution or 1% polyethyleneimine aqueous solution (hereinafter, polyamine sulfone and polyethyleneimine are collectively referred to as polyamine) to each well of the microtiter plate. Dispense,
It was left at room temperature for 1 hour. Thereafter, remove the polyamine aqueous solution using an aspirator and fill each well with a 200 μm solution.
twice with purified water, then once with 200 μl of 1sM hydrochloric acid,
Finally, suction washing was performed once with 200 μl of phosphate buffer.

[Immobilization of antigen]

Syphilis antigen solution 5 is added to the well of this microquiter plate.
0 μl was added and incubated for 1 hour at room temperature.

As a control, 50 μl of 1% BSA was added instead of the syphilis antigen solution.
1 well was prepared. After incubation, the syphilis antigen solution and 1% BSA were removed by suction, and 200 μm
The tube was washed with 1% BSA three times by suction. Then 200μm
1% BS^ was added thereto and allowed to stand at room temperature for 1 hour for blocking. Thereafter, the 1% BSA was removed by aspiration and used immediately for ELISA analysis.

(C) εLISA analysis As the first antibody, 100 times, 200 times and 400 times the above
100 μm of syphilis-positive rabbit serum diluted twice was used.

This was dispensed into each well of the syphilis antigen-immobilized microtiter plate prepared as described above. Control wells (
Syphilis-positive rabbit serum was similarly dispensed into the cells (treated with 1% BSA instead of syphilis antigen).

Separately, in order to examine the presence or absence of substances exhibiting nonspecific adsorption in serum,
Normal rabbit serum diluted twice was dispensed into wells in the same manner as the syphilis-positive rabbit serum described above. After incubating these wells for 1 hour at room temperature, 1. Suction the liquid and remove the 200μm
The tube was washed with 1% BSA three times by suction. Next, add 100 μl of peroxidase-labeled anti-rabbit rgG as a second antibody.
The solution was dispensed into each well and incubated at room temperature for 1 hour. after that.

The liquid in the well was removed by suction, and the well was prepared for 20 minutes in the same manner as above.
After aspiration washing with 0 μl of 1% BSA three times, 100 μm of peroxidase substrate was added to each well.

Incubate for exactly 15 minutes at room temperature. As a substrate blank, a well to which neither the first antibody nor the second antibody had been added was prepared, and the substrate solution was similarly added and incubated. Thereafter, the enzyme reaction was stopped by adding 100 μl of IN sulfuric acid. After the reaction has stopped,
Microtiter plate reader (MTP-100,
492 using the substrate blank as a control.
The absorbance at nm was measured. The results are shown in Table 1.

Example 1 was repeated using microtiter plates that were not treated with the base polyamine. The results are shown in Table 1 together with the results of Example 1.

Table 1 Average value when measured with sleeves = 4.

As is clear from the results in Table 1, it is difficult to immobilize the syphilis antigen on a plastic plate in the presence of a surfactant using the conventional physical adsorption method. However, according to the present invention, a syphilis antigen can be effectively immobilized on a plastic plate even in the presence of a surfactant.

1 Benefit ± or [Immobilization of Treponema pallidum antigen to latex in the presence of surfactant] (A) Preparation of reagents and specimens Unless otherwise specified, reagents and specimens with the same names as in Example 1 are the same as in Example 1. It was prepared as follows.

Latex; 0.23 μm polystyrene latex (
Solid content 10%, Block Chemical Industry ■) was used.

(B) Latex treatment [Polyamine sulfone treatment] 1 ml of latex and 1% polyamine sulfone aqueous solution 5a
+l was mixed and left at room temperature for 1 hour. Thereafter, the aqueous polyamine sulfone solution was removed by centrifugation AI (I5,000 rpm x 1 hour), and centrifugal washing was performed three times with 5 ml of 1 mM hydrochloric acid (I5,000 rpm x 1 hour). Furthermore, after centrifugally washing three times with 5 ml of purified water, the latex was suspended in purified water to a solid content of 10% and stored in this state until use.

[Immobilization of antigen] Latex 2 treated with polyamine sulfone as described above
00 μm and 800 μl of syphilis antigen solution were mixed and stirred at room temperature for 1 hour. Thereafter, 5 ml of 1% BSA was added and centrifuged at 15,000 rpm+ for 1 hour.

Further, 5 ml of 1% BSA was added to the obtained precipitate, and the precipitate was washed by centrifugation in the same manner. 4 ml of 1% BSA was added to this precipitate and dispersed well to obtain a latex reagent. The latex reagent thus prepared was stored at 4°C.

(C) Analysis by immunoagglutination method 50 μl of each of the syphilis-positive rabbit serum and the latex reagent prepared as described above were placed on a glass plate, stirred and mixed, and reacted for 3 minutes. As a control, normal rabbit serum was also reacted in the same manner.

After the reaction, the presence or absence of agglutination of the latex reagent was determined by visual observation, and if agglutination was observed, it was evaluated as positive (+).
The case where no agglutination was observed was considered negative (-). The results are shown in Table 2.

Comparative Plate I Example 2 was repeated using the latex reagent without polyamine sulfone treatment. The results are shown in Table 2 together with the results of Example 2.

Table 2 As is clear from the results in Table 2, it is difficult to immobilize the syphilis antigen on latex in the presence of a surfactant using the conventional physical adsorption method. In contrast, according to the present invention, a syphilis antigen can be effectively immobilized on latex even in the presence of a surfactant, and it can be used as an immunodiagnostic reagent.

N. Amaniwa [Immobilization of 1Bs antigen on plastin plate in the absence of surfactant 1 (A) Preparation of reagents and specimens HBs antigen solution: HBs-positive human plasma, rabbit-produced anti-IBs antibody, and Sepharose. Affinity purification was performed using an affinity column coupled to CL4B.

This purified HBs antigen was dissolved in phosphate buffer and 11Bs
This was used as an antigen solution, and the a degree was measured by the Lowry method.

) The IBs antigen solution was diluted with phosphate buffer immediately before use for antigen immobilization, and the concentration was adjusted to 1 to 10 μg/ml before use.

Anti-HBs antiserum: Anti-llBs obtained by immunizing a rabbit with purified HBs antigen together with Freund's complete adjuvant.
The serum was absorbed by a CNBr-activated Sepharose CL4B column (Pharmacia) bound with normal human serum. Absorption treatment with this column was performed according to the manufacturer's instructions. The anti-HBs serum subjected to absorption treatment was diluted 100 times to 40 times with 1% BSA as in Example 1.
It was used after being diluted 0 times.

(B) Treatment of microtiter plate [Polyamine sulfone treatment] Each well of the microtiter plate was treated with polyamine sulfone according to Example 1. Immediately add H to the polyamine sulfone-treated microtiter plate.
The as antigen was immobilized.

[Immobilization of antigen]

The fixation process was the same as in Example 1 except that an HBs antigen solution was used instead of the syphilis antigen solution. This HB
s Antigen-immobilized microtiter plate is immediately subjected to EL.
Used for IS^ analysis.

(C) ELISA analysis Measurement was performed according to Example 1, except that anti-HBs antiserum was used instead of syphilis-positive rabbit serum as the first antibody. Table 3 shows the results of measuring the absorbance at 492 nm.

Example 3 was repeated using a microtiter plate without polyamine sulfone treatment. The results are shown in Table 3 together with the results of Example 3.

(Margin below) Table 3 Average value when measured at village = 4.

As is clear from the results in Table 3, according to the present invention, a larger amount of HBs can be effectively absorbed than the conventional simple physical adsorption method.
It is possible to immobilize antigens on plastic plates.

Next, a case where cardiolipin was immobilized on a carrier using polyamine sulfone (Example 4).

A comparison was made with a case where cardiolipin was immobilized on a carrier using polylysine as described in Publication No. 63-501902 (Comparative Example 4).

Preparation of Jose Charcoal Earth (A) Reagents and Samples Unless otherwise specified, reagents and samples with the same names as in Examples 1-3 were prepared in the same manner as in Examples 1-3.

Cardiolipin (calciolipin) solution: Cardiolipin, lecithin and cholesterol were each added to 0.
Ethanol solutions containing 0.03%, 0.21%, and 0.9% were prepared.

(B) Latex Treatment The same procedure as in Example 2 (B) was carried out except that cardiolipin solution was used instead of the syphilis antigen solution.

(C) Analysis by immunoagglutination reaction Using the latex reagent obtained in section (B), Example 2 (
The operation was performed according to section C). The results are shown in Table 4.

Comparison ↓ Same as Example 4 except that a 1% aqueous solution of polyOL-lysine was used instead of a 1% aqueous polyamine sulfone solution. The results are shown in Table 4.

Table 4 This twelve positive m: Negative As is clear from Table 4, according to the present invention, cardiolipin can be immobilized with an efficiency equal to or higher than that of polylysine. Polyamine sulfone is advantageous in that, unlike polylysine, it is non-toxic.

Next, the pH conditions when cardiolibin was immobilized using polyamine sulfone (Example 5) and polylysine (Comparative Example 5) were compared.

Unless otherwise specified, reagents and specimens with the same names as in Examples 1 to 4 were prepared in the same manner as in Examples 1 to 3.

The pH of the boric acid buffer diboric acid aqueous solution was adjusted with sodium hydroxide, and the pH was adjusted to 0. OIM borate buffer (pH 9,2) was manufactured by IAI.

Bovine serum albumin was added to 1% BSA/boric acid buffer solution (to a concentration of 1%).

(B) Treatment of Latex Latex was treated with polyamine sulfone in the same manner as in Example 2 (B) and stored in a suspended state in a borate buffer so that the solid content was 10%. No latex aggregation was observed under this storage condition.

200 μ2 of latex treated with polyamine sulfone as described above and suspended in borate buffer and 800 μ of cardiolipin solution! and stirred at room temperature for 1 hour. Add 5 d of 1% BSA/borate buffer to this, 15
Centrifugation was performed at 000 rpm for 1 hour. 1 to the obtained precipitate
Add %BSA 5rn1, 1 at 15000 rpm
The precipitate was washed by centrifugation for hours. 4 ml of 1% BSA was added to this precipitate, and the mixture was well dispersed to obtain a latex reagent. Latex reagents were stored at 4°C.

(C) Analysis by immunoagglutination method Using the latex reagent obtained in section (B), Example 2 (
The operation was performed according to section C). The results are shown in Table 5.

Flame Stop] The same as Example 5 except that a 1% aqueous solution of polyOL-lysine was used instead of the 1% aqueous polyamine sulfone solution.

In this comparative example, aggregation was observed when the latex treated with polylysine was suspended in a boric acid buffer, but the same operation as in Example 5 was performed.

The results are shown in Table 5.

Table 5 Mainland: Positive: Negative In Example 5, latex treatment with polyamine sulfone did not cause latex aggregation.

In Comparative Example 5, latex aggregation was observed. Furthermore, in Example 5, the antigen-antibody reaction after cardiolibin immobilization was strong, and it was confirmed that cardiolipin was immobilized more efficiently than with conventional primary amines even at bright pl+.

Next, using a polymer material into which amino groups have been introduced, 1
An example of implementing the present invention will be described using nylon as the polymer material.

(A) Preparation of reagents and specimens Unless otherwise specified, reagents and specimens with the same names as in Examples 1-5 were prepared in the same manner as in Examples 1-3.

Ethylenediamine: A special grade reagent was used.

Hydrochloric acid; IN to hydrolyze the surface of nylon beads
of hydrochloric acid was used.

EDC (I-ethyl-3(3-dimethylaminopropyl)carbodiimide): Used as a condensing agent (carboxyl group activator) for bonding the amino group of ethylenediamine and the carboxyl group of nylon.

Nylon beads: 6-nylon molded into a spherical shape with a diameter of 6.40 (manufactured by Block Chemical Industry ■, nylon beads, #
0) was used.

(B) Immobilization of syphilis antigen Soak 100 nylon peas in 100 m of hydrochloric acid.

Hydrolysis was carried out at 37° C. for 1 hour, thoroughly washed with purified water, and air-dried. This was immersed in 50 ml of O.1M ethylenediamine, and 1 g of EDC was added as a powder while stirring.

After reacting at room temperature for 1 hour, the mixture was thoroughly washed with purified water and air-dried. 20 nylon beads surface-treated in this manner were placed in a 50 tnft beaker, 10 m of syphilis antigen solution was added, and the mixture was stirred at room temperature for 1 hour. Next, the syphilis antigen solution was removed by suction, and 1% Torin After adding 25 ml of 100, the operation of suctioning and removing was repeated three times for washing. B
25 d of SA was added to form syphilis antigen-immobilized nylon beads. The syphilis antigen-immobilized nylon beads were stored at 4°C. As a control, a sample was prepared in which the same procedure was performed using 1% BS8 instead of the antigen solution.

(C) Detection of syphilis antibodies using El^ As the first antibody, the above-mentioned 100x, 200x and 40x
500 μβ of syphilis-positive rabbit serum diluted 0 times was dispensed. Separately, 100 times as described above to check for the presence of substances exhibiting nonspecific adsorption of serum.

Normal rabbit serum diluted 200-fold and 400-fold was similarly dispensed into test tubes.

Two of the syphilis antigen-immobilized nylon beads described above were added to each test tube. As a control, surface-treated nylon beads on which 1% BSA was immobilized instead of the antigen were also added in the same manner.

After incubating the test tube for 1 hour at room temperature.

The reaction solution was removed by suction and washed three times with 2 ml of 1% BSA. 500 μβ of peroxidase-labeled anti-rabbit IgG was dispensed into each test tube and incubated at room temperature for 1 hour. Aspirate and remove the reaction solution.

Suction washing was performed three times with 2 d of 1% BSA.

Next, 500 μl of enzyme substrate was added to each test tube and incubated at room temperature for 15 minutes.

As a substrate blank, the substrate was similarly dispensed into an empty test tube and incubated. 2 d of IN sulfuric acid was added to this to stop the enzyme reaction. Care was taken to keep the enzyme reaction time constant for each test tube. After stopping the reaction, the absorbance at 492 nm was measured using a spectrophotometer (Hitachi, υV3200) using the substrate blank as a control (n.
2). The results are shown in Table 6.

Nylon beads that were only hydrolyzed with hydrochloric acid (i.e., ethylenediamine and ED in Example 6)
The same operation as in Example 6 was performed using nylon beads (not treated with C). The results are shown in Table 6.

Comparison 1 Using polystyrene beads (polystyrene beads #0 manufactured by Block Chemical Industry Co., Ltd.) molded in the same mold as the nylon beads, one mulberry crop was produced in the same manner as in Example 6. The results are shown in Table 6.

(Leaving space below) Table 6 Average values when measured at n・2 As is clear from Table 6, syphilis antibodies were detected with high sensitivity even when a cationic functional group was introduced by covalent bond, and It was confirmed that non-specific reactions were low.

In Example 7, as compared to Comparative Example 6, a certain degree of reaction with syphilis-positive rabbit serum was observed. this is.

This is thought to be due to the presence of amino groups generated by hydrolysis of nylon beads. Nylon beads also generate carboxyl groups through hydrolysis.

No particular interference was observed during measurement due to this carboxyl group (which is an anionic functional group).

(Effects of the Invention) According to the present invention, a hydrophobic substance is effectively immobilized on a carrier in this way. For example, there are physiologically active substances that cannot be immobilized by conventional physical adsorption, or the amount of immobilization per unit area is small.

They are effectively immobilized on inert carriers such as latex, plastic plates, and inorganic carrier red blood cells.

Therefore, the carrier and immobilization method of the present invention can be used, for example, in measuring various substances using immune reactions. In particular, it can be widely used in fields such as bed tests for diagnosis and treatment of diseases.

that's all

Claims (1)

[Scope of Claims] 1. A carrier for immobilizing a hydrophobic substance, which is an inert carrier into which a cationic functional group is introduced or a compound having a cationic functional group is adsorbed. 2. The carrier according to claim 1, wherein the cationic functional group includes a quaternary amino group. 3. The carrier according to claim 1, wherein the compound having a cationic functional group is a compound having the following structural formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼... (I) Here, R_1 to R_1_0 are hydrogen or alkyl groups,
Y_1 and Y_2 are an alkyl group or an alkylene group, and Y_1 and Y_2 may contain S, N or O. 4. A method for immobilizing a hydrophobic substance, which comprises bringing the hydrophobic substance into contact with the carrier according to claim 1, and physically adsorbing the hydrophobic substance onto the surface of the carrier. 5. The immobilization method according to claim 4, wherein the cationic functional group includes a quaternary amino group. 6. The immobilization method according to claim 4, wherein the compound having a cationic functional group is a compound having the following structural formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼・...(I) Here, R_1 to R_1_0 are hydrogen or alkyl groups,
Y_1 and Y_2 are an alkyl group or an alkylene group, and Y_1 and Y_2 may contain S, N or O.