JPS637786A - Carrier for immobilizing protein - Google Patents

Abstract

PURPOSE:To obtain a carrier for immobilizing a protein suitable as a carrier used for immunological diagnostic agent, affinity chromatography and other biological applications, by modifying the surface of a polyamide with N- alkoxymethyl groups. CONSTITUTION:Formalin and an alcohol, e.g. methyl alcohol, ethyl alcohol, etc., are reacted with a polyamide which is molded into a desired shape, e.g. nylon 6, nylon 66, nylon 8, etc., as a carrier to introduce the N-alkoxymethyl groups into the surface of the polyamide.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to carriers for immobilizing proteins, and in particular to immunodiagnostic agents using antigen-antibody reactions, affinity chromatography for separating and purifying antigens and antibody hormones, etc., and cells. The present invention relates to a protein immobilization carrier suitable for use in biological applications such as culture, artificial organs, biosensors, and bioreactors.

[Conventional technology]

Conventionally, methods for immobilizing proteins on carriers for these biological uses include (a) -Coo)T, -NH! ,
-Chemical bonding method, in which a substance having a functional group such as OR and a protein are immobilized using chemical bonds; (b) Immobilization using physical adsorption between the protein and the surface of a substance such as polystyrene or glass; A physical adsorption method, and (c) an electrostatic adsorption method in which a charged polymer substance and a protein are immobilized using electrostatic attractive force are known.

However, the conventional immobilization method using the chemical bonding method (a) has the problem that the immobilization conditions are complicated, many processes are required to immobilize the protein, and the operations are complicated. Furthermore, in the immobilization methods using (b) physical adsorption method or (c) electrostatic adsorption method, protein immobilization is insufficient and incomplete, and as a result, there is a problem that the adsorbed protein is easily desorbed. be. Furthermore, in cases where the protein is easily desorbed, it is usually necessary to insolubilize the adsorbed protein in water in order to completely fix the protein, and the problem is that the insolubilization process causes denaturation of the protein. There is.

[Problem that the invention seeks to solve]

The purpose of the present invention is to solve the above-mentioned problems, and to provide a protein immobilization method that has excellent protein immobilization ability, does not cause desorption of the protein after immobilization, and has a simple protein immobilization method that does not require complicated operations. The objective is to provide carriers for

[Means for solving problems]

The above problem is caused by the N-alkoxymethyl substituent, e.g.
The problem is solved by a protein immobilization carrier whose surface is formed of a modified polyamide having -methoxytyl groups, N-ethoxymethyl groups, etc.

The present invention will be explained in detail below.

Modified polyamide having an N-alkoxymethyl group used in the present invention (hereinafter simply referred to as "modified polyamide")
is an alcohol-soluble polyamide constructed by introducing an alkoxymethyl group into the amide bond -Co-NH- constituting the polyamide.

This modified polyamide is a polyamide, such as 6-nylon, 6.6-nylon, 4,6-nylon, 6.1o-
Introducing an alkoxymethyl group into the amide group of nylon by the conventional method of reacting formalin with an alcohol such as methyl alcohol, ethyl alcohol, or butyl alcohol into nylons such as nylon, 8-nylon, 11-nylon, and 12-nylon. ("Plastic Materials Course [16] Polyamide Resins" edited by Osamu Fukumoto, Nikkan Kogyo Shimbun, etc.). Among the above nylons, 6-nylon, 6.6-nylon,
8-nylon and the like, and among alcohols, lower alcohols such as methyl alcohol and ethyl alcohol are preferred.

The number average molecular weight of the modified polyamide used in the present invention is not particularly limited, but is usually 10,000 to 100,000 in terms of polystyrene. This modified polyamide is, for example, rFRl0IJ (f
It is commercially available from llll lead company.

The carrier for protein immobilization of the present invention has the following characteristics: (1) When immobilizing, for example, an immunologically reactive substance to modified polyamide, the particle shape has a particle size of about 0.05 to 50 μm, and a thickness of 0.1 to 50 μm. A flat plate shape of about 200 mm, a microtiter plate shape, etc. Also, when fixing keratin derivatives, collagen, gelatin, fibroin, etc. for cell culture, a petri dish shape, a flat plate shape, or a particle shape with a particle size of about 50 to 250 groups. , a method of forming into fibrous, hollow fibrous, etc. shapes.

(2) Method of molding polyamide into the shape as exemplified in (1) and then introducing an alkoxymethyl group (3) Synthesis of polystyrene, polypropylene, polyethylene, etc. molded into the shape as exemplified in (1) It can be obtained by a method of forming a modified polyamide film on the surface of a polymer material, glass, ceramics, inorganic material such as metal, etc.

Specific methods for forming the modified polyamide film in method (3) above include, for example, fluidized bed drying method, spray drying method, etc. in case of particle shape, dipping method, etc. in case of fiber shape. In the case of petri dish shapes, casting methods can be used, and an alcohol solution of modified polyamide may be applied to the surface of a synthetic polymer material, inorganic material, etc. using a coating method depending on the shape, and then dried. Examples of the alcohol at this time include lower alcohols such as methanol and ethanol, and the concentration of the modified polyamide is usually 0.1 to 30% by weight.

In the protein immobilization carrier of the present invention, the N-alkoxymethyl group constituting the modified polyamide is chemically bonded to a protein functional group, such as -〇〇OH, -NH, -〇H1-3H1, especially -N Hz. It is thought that the protein is immobilized by binding.

The method of immobilizing proteins on modified polyamide in the present invention is carried out by bringing the surface of the modified polyamide into contact with a protein solution.

The contact temperature at this time is 20 to 100°C, preferably 30 to 100°C.
The temperature is 60°C, and the contact time is 10 minutes or more, preferably 30 to 180 minutes.

When applying the protein immobilization agent of the present invention, the protein to be immobilized is not limited in any way,
Proteins such as gelatin, collagen, fibroin, keratin derivatives and their denatured products, hepatitis B surface antigen CH
BS antigen), anti-HBS antibody, human chorionic gonadotropin (HCG antigen), anti-HCG antibody, immunoglobulin G1
mycoplasma antigen, nucleic acid, nuclear protein, estrogen,
Examples include immunoreactive substances such as anti-estrogen antibodies, enzymes, etc., and can be appropriately selected depending on the purpose.

〔Example〕

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1 Modified polyamide rFR10 in which N-methoxymethyl groups were introduced into each formula of a polystyrene microtiter plate having 24 holes (recesses) with a diameter of 17III11.
10% methanol solution of 100-
were injected and dried to form a film with a thickness of about 4 decoys, and a film-like protein immobilization carrier (hereinafter simply referred to as "
A film carrier was obtained.

06 of gelatin in each formula in which this film-like carrier was formed.
5% water by weight! 200 d of solution was added to each, and the gelatin was fixed on the film carrier while shaking at 60° C. for 2 hours.

The amount of gelatin immobilized on the film carrier was measured using the fluorescamine method and was found to be 7.0 W/cm''.Next, the plate with gelatin fixed on the film carrier was immersed in warm water at 60°C. After shaking for 2 hours, the amount of gelatin immobilized on the film carrier was measured again using the fluorescamine method in the same manner as above.6.
5 R/cm".

From the above, the gelatin fixed on the film-like carrier according to this example is almost never desorbed by hot water, and therefore gelatin after fixation does not need to be insolubilized, and the film-like carrier according to this example was confirmed to be a good carrier for protein immobilization.

Example 2 Modified polyamide rFRt was prepared in the same manner as in Example 1.
A film-like carrier of oll (manufactured by Nippon City) was obtained. And 0.5% water by weight of carboquine methylated keratin? Carboxymethylated keratin was fixed on the film-like carrier in the same manner as in Example 1 using 8 liquids. Thereafter, the amount of carboxymethylated keratin immobilized on the film carrier was measured by the same method as in Example 1, and it was found that 34 k
/cm”.

Next, the plate on which this film-like carrier was formed was heated to 6
After immersing in warm water at 0°C and shaking for 2 hours, the amount of carboxymethylated keratin immobilized was measured again in the same manner as described above, and it was found to be 3111 g/cm''.

From the above, the carboxymethylated keratin immobilized on the film-like carrier according to this example is almost never desorbed by hot water, and therefore the carboxymethylated keratin after immobilization does not need to be insolubilized. It was confirmed that the film-like carrier of the example is a good carrier for fixing carboxymethylated keratin.

Example 3 Polystyrene particles rDR502J (with an average particle diameter of 5 wa)
Hg manufactured by Japan Synthetic Rubber Co., Ltd. and 0.1 wt% methanol of rFRtoll (manufactured by @Tenichi)? ? ! A particulate protein immobilization carrier (hereinafter simply referred to as "particulate carrier") having a coating layer of methoxymethylated polyimide with a thickness of about 0.2 a was obtained by dispersing the solution in a liquid and spray drying.

An aqueous dispersion of this particulate carrier with a solid content of 7% and 1% was prepared, and an equal amount of heat-denatured H)-r-globulin 20011 g/-phosphate buffer (pH 7°2) solution was prepared. The heat-denatured human-r-globulin was immobilized on the particulate carrier while stirring at 37° C. for 2 hours.

This particulate carrier was mixed with phosphoric acid at 1 (pl+ 7.2) for 2
After washing twice, heat-denatured human T-
When the amount of globulin immobilized on the particulate carrier was measured,
3 Jlg/c@”.

Furthermore, after storing the particulate carrier on which heat-denatured human-r-globulin was immobilized at room temperature for one month, the amount of heat-denatured human-r-globulin immobilized was measured again in the same manner as described above. C11', and it was confirmed that heat-denatured human-γ-globulin was not detached even after storage for one month.

〔Effect of the invention〕

Since the surface of the protein immobilization carrier of the present invention is formed of a specific modified polyamide, it has the following effects, as is clear from the above examples.

■Excellent ability to fix various proteins.

■Protein fixation is easy; for example, protein can be fixed by simple operations such as immersion, and no complicated operations are required.

(2) Once fixed, the protein is in a stable state and is difficult to desorb; therefore, there is no need for insolubilization of the protein after fixation, and this insolubilization does not cause denaturation of the protein.

By having the above effects, the protein immobilization carrier of the present invention can be used for various biological purposes, such as immunodiagnostic agents, affinity chromatography, cell culture, artificial organs, etc.
It is suitable as a carrier for biological applications such as biosensors and bioreactors.

Claims (1)

[Claims] 1) A carrier for protein immobilization, the surface of which is formed of a modified polyamide having an N-alkoxymethyl group.