JPS61167866A - Production of carrier for immune reaction - Google Patents

Abstract

PURPOSE:To make the quick and quantitative antigen-antibody reaction possible by depositing magnetosensitive powder on the surface or to the inside of a thermoplastic resin then immobilizing an antigen, antibody or physiologically active material thereto. CONSTITUTION:The magnetosensitive powder is deposited to the surface or the inside of the thermoplastic resin. The resin is then treated at least one kind of monomers having functional groups such as -OH group, -CHO group and -SH group or the polymers thereof or the mixture composed essentially thereof in order to immobilize the antigen, antibody or physiologically active material. The mixture is further treated by a crosslinking agent and/or surface treating agent. The antigen, antibody or physiologically active material is thus chemically and stably conjugated onto the solid phase and the surface concn. thereof is increased. The quick and quantitative antigen-antibody reaction is made possible.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing a carrier for immune reaction.

More specifically, the present invention relates to a method for producing an immunological diagnostic carrier for quantitatively and selectively measuring the amount of a trace substance in a human body fluid using an immune reaction.

(Prior Art) Immunoassay is generally widely used as a method for detecting trace amounts of substances contained in human body fluids, especially serum, and for measuring their concentrations. Immunoassay is an antigen and its response.
A method of measuring the concentration of an antigen or antibody by quantitatively detecting such an antigen-antibody reaction, taking advantage of the fact that the binding reaction between the antigen and antibody is extremely specific and occurs even at extremely low concentrations. be.

A number of measurement methods have been developed and used to quantitatively detect antigen-antibody reactions, but among them, the most widely used method is to label either the antigen or the antibody with something. is red blood cell.

Synthetic polymer particles, radioactive isotopes, enzymes, fluorescent substances, etc. are used.

For example, in radioimmunoassays labeled with radioactive isotopes, antigens are labeled and unlabeled antigens are reacted with antibodies against these antigens to form antigen-antibody conjugates (type B) and free antigens (type 1). Separate (B/? separation) and quantify the target antigen. In addition, in enzyme immunoassay using labeled enzymes, antibodies are labeled and antigen-antibody reactions are performed.
/7 After separation, type B is quantitatively detected and the target antigen is quantified.

However, the operation for B/1 separation in these measurement methods is extremely complicated, which greatly impedes rapid and accurate immunoassay.

For this reason, various efforts have been made to simplify the B/IF separation by immobilizing type B on a solid phase such as an antigen or antibody and performing an antigen-antibody reaction to insolubilize type B. However, in this case, since the antigen-antibody reaction is a solid-liquid reaction, a complete antigen-antibody reaction cannot be performed unless the density of the antigen or antibody on the solid phase is sufficiently high and sufficient stirring is performed. Furthermore, the reaction efficiency is better if the specific gravity of the solid phase is as close as possible to that of the reaction solution.

(Problems to be Solved by the Invention) The present invention has been made to solve the above problems.

(Action of the Invention) The present invention provides (1) supporting a magnetically sensitive powder on the surface or inside of a thermoplastic resin, and then supporting an antigen.

-OH for immobilizing antibodies or physiologically active substances.

A method for producing a carrier for immunoreaction, which comprises: e) one type of monomer or polymer, or a mixture containing these as the main component; and e) the mixture is further treated with a crosslinking agent and/or a surface treatment agent. .

The present invention will be explained in detail below.

Examples of thermoplastic resins include polyethylene, polypropylene, ethylene-vinyl acetate copolymer, nylon, polystyrene, styrene-isoprene copolymer, styrene-butadiene copolymer, polyvinyl chloride and At least one copolymer or mixture containing these as main components can be used.

Magnetically sensitive powders include iron, iron tetroxide, nickel,
Iron - Cobalge, silicon steel and MIF @, QB (M+ = chanting...

Zn, lii, O(L, Ou, Mg, Br,
At least one type of soft ferrite represented by Ba) having a particle size of 101 to 10 μm can be used. The thermoplastic resin is heated at ±50°C of its melting point, preferably ±50°C.
The thermoplastic resin is heated together with the magnetically sensitive powder at a temperature of 20° C., so that the magnetically sensitive powder is supported on the surface or inside of the thermoplastic resin. A thermoplastic resin supported with magnetically sensitive powder is hereinafter referred to as a magnetic resin.

The magnetic resin is processed in the following manner.

1) Magnetic resin is a monomer or polymer having a functional group such as a -sn group or an -OH group, or a mixture containing these as a main component (hereinafter referred to as "monomer or polymer having a functional group, or a mixture containing these as a main component") (referred to as "compounds with functional groups"). For example, methyl-L
- Polymerize cysteine or cellulose on the surface of a magnetic resin to support a thiol group or water group on the surface.

The polymerization conditions are determined in consideration of the melting point of the thermoplastic resin, the polymerization temperature, and the degree of polymerization progress.

2) Treating the magnetic resin with a compound having a functional group such as an -oHO group and a crosslinking agent. For example, acrolein and a crosslinking agent are mixed, adhered to the surface of a magnetic resin, and polymerized by applying heat or light to support aldehyde groups on the surface. Polymerization conditions are determined taking into account the degree of progress of the reaction.

S) The magnetic resin is treated with a compound having a functional group such as a monoOH group, and then treated with a surface treatment agent. For example, polyvinyl alcohol is polymerized on the surface of a magnetic resin, and then treated with, for example, a silane coupling agent having an amino group to support the amino group on the surface. The polymerization conditions and the treatment conditions with the coupling agent are determined in consideration of the reaction progress conditions and the like.

It is further treated with another surface treatment agent. For example, a magnetic resin is coated with glycidyl methacrylate and polymerized by heating. Next, hydroxyl groups are supported on the surface by, for example, poppying with an alkali, and then treated with another surface treatment agent, such as a silicic acid bubbling agent having amino groups, to support amino groups on the surface. The polymerization conditions are determined by considering the melting point and polymerization temperature of the thermoportable resin. Furthermore, the conditions for treatment with the surface treatment agent are determined by taking into account the conditions for the reaction to proceed.

Treat with a chemical. For example, the resin surface is coated with a mixture of glycidyl methacrylate and a crosslinking agent and polymerized by heating. Next, it is reacted with a diamine compound such as ethylenediamine, and the amide 7 group is supported on the surface. The polymerization conditions are determined by taking into consideration the polymerization temperature, etc. The reaction conditions for the diamine compound are determined in consideration of the reaction progress conditions.

By treating the magnetic resin as described above, it is possible to stably support antigens, antibodies, or physiologically active substances on the carrier by chemical bonding using a conventional method, and it is also possible to quickly and thoroughly stir the antigen, antibody, or physiologically active substance using a simple magnetic stirrer or the like. can be done.

In each case, at least one monomer such as polyvinyl alcohol, acrolein, methyl-L-cysteine, glycidyl methacrylate, a polymer obtained by polymerizing these by a known method, or a mixture containing these as a main component is used. can.

As the crosslinking agent, those used in ordinary polymerization reactions can be used.

Examples of surface treatment agents include sodium hydroxide.

Diamines such as ethylenediamine, silane coupling agents, etc. can be used as necessary. The processing conditions may be determined in consideration of the melting point of the thermoplastic resin and the degree of progress of the processing.

It is desirable that the radius is in the range of 11 to 20 degrees. If the radius is smaller than 11-, the operability of B/7 separation will be poor. Moreover, if it is larger than 20 cm, it will not be possible to stir it sufficiently using the usual stirring method.

Specific gravity is α95 to 150. Preferably 1.5 from tOO
It is clear that it is in the 0 range. If it is smaller than α95, the carrier will float in the reaction solution, resulting in poor reactivity. 55 again
If it is heavier than 0, it will not be possible to stir it sufficiently using the normal method.

(Effects of the Invention) As described above, by using the immunoreaction carrier of the present invention, antigens, antibodies, or physiologically active substances can be stably bound to a solid phase in a chemically balanced manner, and the surface density can be reduced. It can be made large enough.

Furthermore, sufficient stirring can be performed using a simple magnetic stirrer, and the B/A separation procedure, which was extremely complicated in the past, can be extremely simplified, making it possible to perform antigen-antibody reactions extremely quickly and quantitatively. Now I can.

(Examples) The present invention will be described below with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Nyen sphere 1009 with an average particle size of 7.8 and an average particle size α1
The mixture was mixed with iron tetroxide 209 μm and heated at 220° C. for 10 minutes. After the reaction product was brought back to room temperature, thoroughly washed and dried, aquilein 109 and tetraethylene glycol dimethacrylate α19 were added and heated at 100° C. for 20 minutes.

A 2% (V/V) glutaraldehyde solution was added to the carrier thus obtained, and the mixture was shaken at 4°C for 16 hours.

After washing three times with carrier 11M phosphate buffer, α5q/
-200% of the 125-labeled chemical gG solution per carrier.
μ! The mixture was then shaken at 4°C for 16 hours.

The carrier was washed five times with α1M Linzen buffer, and the radiation dose was measured using an r-counter.

&1 μ9 of G was bound per carrier.

Example 2 Ethylene vinyl acetate copolymer 100 with average particle size 2.2
9 and 15 g of soft 7 elite expressed as (Mn-zn)7e and Qs with an average particle size of α5 pm were mixed and heated at 110°C.
and heated for 20 minutes. After the reaction product was returned to room temperature and thoroughly washed and dried, 11 g of glycidyl methacrylate was added and heated at 90° C. for 15 minutes. Next, the carrier was heated in a sodium hydroxide solution of an appropriate concentration at 40 to 50°C for 30 minutes, and then thoroughly washed with water and dried. 10% (
V/V) A 5-aminopropyltriethoxysilane solution was added and the mixture was shaken at room temperature for 4 hours, then thoroughly washed with water and dried. Next, a 2% (V/V) glutaraldehyde solution was added and the mixture was shaken at room temperature for 2 hours, followed by washing three times with 11M 9-phosphate buffer.

The carrier thus obtained was treated in the following three ways.

1) α5■/aj's 12! s! 20μ per 1+ labeled 1gG solution carrier! The mixture was then shaken at 4°C for 16 hours.

The carrier was washed three times with 11M 9-phosphate buffer, and the radiation dose of the carrier was measured using an r-counter.

1.2 μ2 of GG was bound to each carrier.

2) Add 100 μl of GA solution at the required concentration per 1+ carrier.
After shaking at 4° C. for 16 hours, the mixture was washed three times with CLIM phosphate buffer. Next, add 200 μl of alkaline 7-mole-7 atase camphor antibody gG antibody solution at an appropriate concentration, and stir at 37°C for 30 minutes using a magnetic stirrer.
Stir for a minute. After washing three times with 5 (laM2-methyl-2-amino-1-propatur buffer, 1!II
200 μl of M p-nitrophenylphosphorus II solution was added and shaken at 37° C. for 30 minutes. The reaction was stopped by adding 1N sodium hydroxide solution, and the absorbance at 40 liters was measured.

The results are shown in Figure 1.

3) 500 μl of 250C) 0 engineering V1 anti-human 1gl solution per carrier! After washing the carrier 3 times with aIMIJ acid buffer, 500 μl of 1% bovine serum albumin solution was added to each carrier and shaken at 4°C for 16 hours.

The carrier (after washing 5 times with 11M 9-phosphate buffer, the appropriate concentration) was added to 100μ! of 1gll solution. In addition, the mixture was stirred at 57° C. for 1 hour using a magnetic stirrer.

After washing three times with 11M 9-phosphate buffer, the appropriate concentration of β
-D-galactosidase-labeled anti-human Egl antibody solution (300 μl per carrier) was added and stirred at 37° C. for 1 hour using a magnetic stirrer. After washing three times with 11M phosphorus buffer, 500 μl of 0-nitro722-β-D-galactoside solution was added and shaken at 37° C. for 1 hour. α2M sodium charcoal solution 1JIL7! ! was added to stop the reaction, and the absorbance at 42 onm was measured.

The results are shown in Figure 2.

Example 3 Ethylene vinyl acetate copolymer 1 with average particle size of 1.6 sIl
(MneZn)IFe2 with 009 and average particle size α5 pm
110
Heated at ℃ for 20 minutes. After the reaction product was returned to room temperature, thoroughly washed with water and dried, glycidyl methacrylate 109 and tetraethylene glycol dimethacrylate (Ll 9) were added and heated at 90° C. for 15 minutes.

Next, a 65% (V/V) ethylenediamine solution was added to the carrier, which was shaken at room temperature for 16 hours, and then thoroughly washed with water and dried. The carrier thus obtained was treated in the following two ways.

1) Add 20 μl of 15 m9/- of 125-labeled chemical gG solution per carrier, shake at 4°C for 16 hours,
The carrier was washed three times with 11 M IJ acid buffer, and the radiation dose of the carrier was measured using an r-counter.

1.5 μ90 IgG was bound per carrier.

2) 500μ of a 15m9/d anti-7 eritin antibody solution was added to each carrier, shaken at 4°C for 16 hours, and then washed five times with Tris-HCl buffer. 10 μl of a 7-erithin solution of the required concentration and 75 μl of an alkaline phosphatase-labeled anti-7-erythin antibody solution of an appropriate concentration were added, and the mixture was stirred at 37° C. for 20 minutes using a magnetic stirrer.

After washing three times with Tris-HCl buffer, 100 μl of 1 mM 4-methylumbelliferyl phosphate solution was added, and 37
Shake for 5 minutes at °C.

Next, the reaction was stopped by adding α14M phosphate buffer containing 1M EDTM for 2.9 d. Excitation wavelength 360n
The fluorescence intensity was measured at a fluorescence wavelength of 450 mm.

The results are shown in Figure 3.

[Brief explanation of drawings]

FIGS. 1 to 5 are diagrams showing the results of immune reactions performed using the immune reaction carrier obtained in the present invention. Patent applicant: Toyo Ichida Kogyo Co., Ltd. Drawings) 11° 2 (i' (no changes) Fig. 1
G) Figure 2 090180360 730 1450 rU/
m1 (19E) Figure 3 (Ferritin) Procedural Amendment May 30, 1985

Claims (1)

[Claims] 1) A magnetically sensitive powder is supported on the surface or inside of a thermoplastic resin, and then -OH group, -CHO group, -SH group is used to immobilize an antigen, antibody, or physiologically active substance. A method for producing a carrier for immunoreaction, which is characterized by treating with at least one monomer or polymer having a functional group such as ▲ mathematical formula, chemical formula, table, etc., or a mixture containing these as main components. 2) The method according to claim 1, characterized in that the mixture is further treated with a crosslinking agent and/or a surface treatment agent.