JPH0593727A - Magnetic solid phase carrier - Google Patents

Abstract

PURPOSE:To enable highly sensitive measurement with a handy operation in shorten specified time by forming a magnetic layer on the surface of resin particles of a solid phase carrier using a composite film comprising a mixture of a high polymer material and a magnetic metal. CONSTITUTION:A magnetic layer about 0.05-1mum thick is formed on the surface of resin particles with a particle size of about 0.1mum-1mm using a composite film comprising a high polymer material made by a plasma polymerization and a magnetic metal by sputtering. The high polymer material is formed by the plasma polymerization using an organic monomer as start material. The organic monomer herein used is not limited specifically and, for example, is ethylene preferably. Any magnetic metal will do only if it is sensitive to an external magnetic field and the content thereof in the magnetic layer is preferably in a range of about 10-15% in terms of the number of atoms. The formation of the magnetic layer on the surface of the resin particles facilitates the coupling of an antigen and an antibody by hydrophobic interaction and the size and surface area thereof can be controlled evenly thereby enabling the separation of an antigen-antibody reaction medium utilizing magnetic property easily with no delay in an antigen-antibody reaction.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a solid-phase carrier used for various detection methods, measurement methods and the like utilizing an antigen-antibody reaction.

[0002]

2. Description of the Related Art Conventionally, various detection methods and measurement methods utilizing the antigen-antibody reaction have been used in the fields of research and diagnosis. In order to simplify these detection methods and measurement methods, the antigen Alternatively, an antibody previously bound to a solid phase carrier (hereinafter referred to as a solid phased antigen or a solid phased antibody) is used, and the solid phased antigen or the solid phased antibody and the antibody or the antigen in the antigen-antibody reaction medium are used. Is reacting.

The binding of the antigen or antibody to the solid phase carrier is generally carried out by physical adsorption utilizing hydrophobic interaction. Examples of the solid phase carrier utilizing the above hydrophobic interaction include synthetic polymer moldings having a hydrophobic surface (for example, moldings such as beads, plates, and membranes). Beads made of synthetic polymer having a particle size of 2 to 10 mm (for example, polystyrene beads)
Is preferably used. However, since the above bead-shaped solid phase carrier has a large particle size, it is difficult to suspend it in the antigen-antibody reaction medium, and even if it is suspended by adjusting the specific gravity of the medium, unlike the fine particles, Brownian motion is caused. Since it does not move, it is difficult to move in the medium unless external vibration is applied. As a result, there is little chance of collision between the solid-phased antigen or the solid-phased antibody and the antibody or the antigen in the medium, and the antigen-antibody reaction takes time, as early as 1 hour, if slow, 1-2 days, usually There has been a problem that measurement cannot be performed unless the reaction is performed for about 3 hours.

On the other hand, a solid-phase carrier composed of synthetic polymer microparticles can shorten the time for the antigen-antibody reaction, but when separating the medium and the solid-phase carrier, it is necessary to add an operation such as centrifugation, which is practical. Not.

In order to solve the above-mentioned drawbacks, various proposals have been made, for example, those in which magnetic fine particles are bound to the surface of synthetic polymer microparticles are proposed. The reaction time is short, and it can be easily separated from the medium by fixing with a magnet, but there is a drawback that the magnetic fine particles are separated from the surface of the fine particles of the synthetic polymer during the measurement operation. In addition, JP-A-60-79266
The publication discloses magnetic particles in which a magnetic core is covered with a resin such as polystyrene. However, since it is difficult to make the size and surface area of the magnetic particles uniform, it is difficult to carry out the antigen-antibody reaction. However, there is a drawback that it is difficult to constantly and uniformly perform the treatment under a certain condition.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above drawbacks, and an object thereof is to easily bind an antigen or an antibody by hydrophobic interaction,
To provide a magnetic solid-phase carrier capable of uniformly controlling the size and surface area, having no delay in antigen-antibody reaction, and easily separating from an antigen-antibody reaction medium by utilizing magnetism. It is in.

[0007]

The magnetic solid phase carrier of the present invention is a solid phase carrier having a magnetic layer formed on the surface of resin particles, and the magnetic layer is a polymer substance formed by a plasma polymerization method. The composite film is composed of a mixture of magnetic metals formed by a sputtering method, and the above object is achieved thereby.

The present invention will be described in detail below. The resin particles used in the present invention are not particularly limited as long as they bind to an antigen or an antibody, and examples thereof include polystyrene, polyethylene, polypropylene, cellulose acetate, nitrocellulose, and a single resin such as a fluororesin. Coalescing,
Examples thereof include particles made of a copolymer of styrene and another monomer.

The shape of the resin particles is not particularly limited, but it can be easily produced by emulsion polymerization, suspension polymerization, etc., and in consideration of a classification technique for making the particle diameter uniform, spherical or spherical shape The elliptical shape is preferable.

When the particle size of the resin particles is small, the surface area of the hydrophobic portion of the obtained magnetic solid phase carrier is small and the binding amount with the antigen or antibody is small, and when it is large, the magnetic layer formed on the surface is small. Is relatively smaller than that of resin particles, and it becomes difficult to separate from the antigen-antibody reaction medium by magnetic force during measurement. Therefore, it is limited to the range of 0.1 μm to 1 mm, preferably 0.5 to It is 100 μm.

The magnetic solid phase carrier of the present invention has a composite film formed of a mixture of a polymer substance and a magnetic metal as a magnetic layer on the surface of the resin particles, the composite film comprising: It can be obtained by performing formation of a polymer substance by plasma polymerization and formation of a magnetic metal by sputtering in the same container.

The above polymer substance is formed by plasma polymerization using an organic monomer as a starting material, and the organic monomer is not particularly limited, and examples thereof include ethylene, styrene, formaldehyde, naphthalene, pyrene, perylene, Biphenyl, terphenyl, quarterphenyl and the like are preferably used.

The elements contained in the organic monomer can be changed depending on the type of antigen or antibody used. For example, when a nitro group is required, a nitrogen-containing monomer such as nitromethane or nitrobenzene is used as a starting material, and when an amino group is required, a nitrogen-containing monomer such as aniline or a derivative thereof is used as a starting material to perform plasma polymerization.

In the plasma polymerization of the above-mentioned organic monomer, if the organic monomer is a gas, it is vaporized as it is, and if it is a liquid, it is vaporized in a container adjusted to a constant pressure with a carrier gas.
In the case of a solid, it is sublimated by heating and electric power is supplied between the electrodes.

As the carrier gas, for example,
An inert gas such as N ₂ or Ar is preferably used, and the pressure at that time is preferably 10 ⁻¹ to 10 ⁻⁴ Torr, more preferably 10 ⁻² to 10 ⁻³ Torr.

The type of the electric power supplied is not particularly limited, and for example, the frequency is 10 to 35.
KHz or 10 to 50 MHz, or direct current can be preferably used, and the output is preferably 50 W to 1 KW.

As the magnetic metal to be sputtered, any one can be used as long as it is sensitive to an external magnetic field.
Examples thereof include ferromagnetic metals such as iron, cobalt and nickel; or alloys of ferromagnetic metals; metal oxides such as garnet and ferrite; rare earth metals such as Gd.

The sputtering of the metal is performed by using a metal as an anode electrode material and supplying electric power between the electrodes in a container in which the carrier gas is adjusted to a constant pressure to ionize and accelerate the carrier gas. It is performed by sputtering the above metal.

Since the sputtering is carried out simultaneously with the plasma polymerization of the organic monomer in the same container, the conditions are the same as the plasma polymerization conditions. The magnetic layer in the present invention preferably covers the surface of the resin particles uniformly, but it does not necessarily have to be a continuous film, and the thickness thereof does not show sufficient magnetism when it becomes thin, and it becomes magnetic when it becomes thick. Since the layer peeling and the particle size become non-uniform,
0.05 to 1 μm is preferable, and more preferably 0.1 to
It is 0.5 μm.

The content of the magnetic metal in the magnetic layer can be adjusted by controlling the type of carrier gas, the pressure, the high frequency output, and the type of the monomer, and can be arbitrarily changed. When the content is low, sufficient magnetism is not exhibited, and when the content is high, the magnetic metal particles are condensed with each other.

The magnetic layer used in the present invention is formed by using the above-mentioned materials and methods. In order to form the magnetic layer with a uniform film thickness on the surface of resin particles, the magnetic layer is vibrated in a container holding the resin. It is desirable to provide a device or a stirrer to form the magnetic layer while stirring the resin particles.

In the magnetic solid-phase carrier of the present invention, when the surface of the resin particles is completely covered with the magnetic layer, the polymer substance formed by plasma polymerization becomes a hydrophobic surface and the surface of the resin particles becomes magnetic. When not completely covered by a layer, the surface of the resin particles becomes a hydrophobic surface, and in any case, hydrophobic interaction can be used to bind the antigen or antibody to the solid phase carrier. ..

Actually, the operation of binding an antigen or antibody to a magnetic solid-phase carrier to form a solid-phased antigen or a solid-phased antibody is carried out by combining the antigen or antibody and the solid-phase carrier in an aqueous medium such as a buffer solution. It can be easily performed by bringing them into contact with each other. At this time, excess antigens and antibodies can be easily removed by decanting while holding the solid phase carrier in the container by bringing a magnet close to the container from the outside.

Next, as an example of the measurement using the magnetic solid phase carrier of the present invention, a method for measuring an antigen by the enzyme immunoassay method by the one-step sandwich method using a magnetic solid phase immobilized antibody will be described.

First, a magnetic solid-phased antibody to which an antibody against a substance to be measured (antigen) is bound, a sample containing the substance to be measured, and an enzyme-labeled antibody obtained by labeling the antibody to the substance to be measured with an enzyme are contained in the same container. After mixing inside to carry out the antigen-antibody reaction, bring the magnet close to the outside of the container to hold the magnetically immobilized antibody in the container and remove unreacted antigen and enzyme-labeled antibody by decantation method. To do.

Next, the immobilized antibody after the above reaction is washed with an appropriate buffer to remove the remaining unreacted enzyme-labeled antibody, and then an enzyme substrate is added to carry out the enzyme reaction.
After the enzyme reaction is completed, the amount of the substance to be measured is measured by measuring the enzyme activity bound to the immobilized antibody.

By using the magnetic solid phase carrier of the present invention in the above measurement process, it is possible to react the solid phased antibody in a suspended state in the antigen-antibody reaction and the enzyme reaction. Since the reaction rate is faster than that of the solid phase carrier, the measurement time can be shortened. Further, since the solid-phased antibody can be held in the container by bringing the magnet closer from the outside, the reaction medium and the solid-phased antibody can be easily separated.

[0028]

EXAMPLES Examples of the present invention will be described below. (Example) 1) Production of solid phase carrier.

A Petri dish having a diameter of 40 mm was placed on the lower electrode (cathode) in the reaction vessel in which a parallel plate electrode made of a stainless steel plate (SUS-304) was placed, and polystyrene particles (Sekisui Fine Chemical) as resin particles were placed therein. "Micropearl SP", average particle size 10 μm) was supplied.

Next, 4,4'-dihydroxy-p-quaterphenyl was supplied as an organic monomer to a tungsten board separately installed in the reaction vessel, and the pressure inside the vessel was reduced to 1 × 10 ^-5 Torr. After that, Ar gas was supplied so that the total pressure in the container was 5 × 10 ⁻³ Torr. Then, pass an electric current through the tungsten board to make 4,4'-
Didoxy-p-qua-terphenyl was heated to 300 ° C, and a low frequency of 13 KHz was applied between the electrodes at 100 W.
Was applied and the resin particles were deposited for 150 minutes while stirring to obtain a solid phase carrier having a magnetic layer on the surface of the resin particles.

Elemental analysis of the surface composition of this solid phase carrier by X-ray photoelectron spectroscopy revealed that the atomic ratios of the main elements were C: 41%, O: 35%, Fe: 11%, C
r: 4%, which was shown to be a composite film of a polymer substance and stainless steel. 2) Preparation of reagents The following reagents were prepared.

Phosphate buffer solution (PBS): monosodium phosphate, disodium phosphate, and sodium chloride, the final concentrations of phosphoric acid and sodium chloride were 0.
It was prepared by mixing 02M, 0.15M, and pH to be 6.90.

Bovine serum albumin (BSA) / PBS: B
SA was dissolved in PBS to a concentration of 1% (w / v) for preparation. Anti-insulin antibody: Semi-synthetic human insulin [Actrapid Human MC, 1.4 mg / m 2 in guinea pig
1, (400 U / ml), manufactured by Novo Co., Ltd.] and Freund's complete adjuvant were mixed at a ratio of 1: 1, and 0.5 ml of the emulsified mixture was immunized 6 times every 2 weeks to obtain an antiserum. The antiserum was subjected to ammonium sulfate fractionation with 38% saturated ammonium sulfate to obtain a γ-globulin fraction partially containing ammonium sulfate. Γ − obtained
The globulin fraction is dissolved in PBS, and this PBS solution is
Gel filtration was performed using a Sephadex G25 column equilibrated with PBS to obtain an anti-insulin antibody-free PBS solution of ammonium sulfate. This was diluted with PBS, and the γ-globulin concentration was adjusted to 20 μg / ml before use.

Enzyme-labeled antibody: Anti-insulin / monoclonal antibody (manufactured by Cosmo Bio Inc.) was added to N- (γ-Male).
imidobutyroxy) succimide was used in the conventional method ["Protein and nucleic acid enzyme", separate volume No. 31.3
35-343 (1987)], labeling with β-D-galactosidase was performed. It was diluted with BSA / PBS containing 1 mM magnesium chloride and used for the measurement.

Standard insulin: The above-mentioned semi-synthetic human insulin was diluted with BSA / PBS, and the insulin concentrations were 10, 20, 40, 80, 160 and 320 μU, respectively.
/ Ml standard insulin was prepared. BSA / PBS was used as it was as a standard of 0 concentration.

Enzyme substrate: o-nitrophenyl-β-D-
Galactopyranoside was dissolved in BSA / PBS containing 1 mM magnesium chloride to a concentration of 0.1% (w / v) and used.

Saline: 0.9% aqueous sodium chloride solution was used. Enzyme reaction stop solution: A 1% sodium carbonate aqueous solution was used. 3) Preparation of immobilized antibody 1 g of the solid phase carrier prepared in 1) was added with anti-insulin antibody 50
ml was added and incubated at 37 ° C. for 1 hour. After removing the unreacted anti-insulin antibody by decantation while approaching the magnet from the outside of the container and holding the solid phase carrier inside the container, 50 ml of BSA / PBS was added, and the mixture was again incubated at 37 ° C. for 1 hour.

The same decantation as above is performed and B
After removing SA / PBS, an additional 20 ml of BSA
/ PBS was added to wash the solid phase carrier, and the supernatant was removed by decantation. This operation was repeated 3 times to prepare a solid-phased antibody.

Then, the above immobilized antibody was added to 10 ml of BS.
It was suspended in A / PBS, transferred to a new container, and used for measurement. 4) Measurement of insulin by enzyme immunoassay 50 μl of the above-mentioned solid-phased antibody suspension was put into each of 7 test tubes, and the concentration prepared in 2) was 0, 10,
100 μl of standard insulin of 20, 40, 80, 160, 320 μU / ml was added, and then 300 μl of enzyme-labeled antibody was further added and incubated at 37 ° C. for 1 hour.

Then, bring the magnet close to the outside of the test tube,
The reaction solution was removed by decantation, and saline solution 2
After adding and suspending ml, the operation of bringing the magnet close to and decanting was repeated twice.

Next, 0.5 ml of enzyme substrate was added to each test tube and incubated at 37 ° C. for 1 hour to carry out an enzyme reaction, and then 2 ml of an enzyme reaction stop solution was added to stop the enzyme reaction.

In addition, 0.5 m of enzyme substrate was placed in an empty test tube.
After adding 1 liter and incubating at 37 ° C. for 1 hour, 2 ml of the enzyme reaction stopping solution was added, and this was used as a substrate blank. From the outside of the test tube, bring the magnet close to the supernatant to collect the immobilized antibody so that the solid-phased antibody does not float, and measure the absorbance at 420 nm for standard insulin at each concentration, and the results are shown in Fig. 1. Indicated. (Comparative Example) Except that 100 plastic beads made of polystyrene whose surface was polished (polystyrene beads # 80 manufactured by Sekisui Chemical Co., Ltd., diameter 6.4 mm) were prepared and no magnetic layer was formed on the surface. Similarly, a solid-phased antibody was prepared.

However, anti-insulin antibody, BSA at the time of washing
The removal of / PBS and the like was performed by suction removal instead of decantation. Using one of the above-mentioned solid-phased antibodies, the enzyme immunoassay was carried out by the same operation as in Example, and then the absorbance for standard insulin at each concentration was measured,
The results are shown in Fig. 1.

Compared to the comparative example, the example gives a larger absorbance, and a smaller amount of insulin can be measured.

[0045]

INDUSTRIAL APPLICABILITY The magnetic solid phase carrier of the present invention can easily bind antigens and antibodies by hydrophobic interaction, and its size and surface area are uniformly controlled, so that there is no delay in antigen-antibody reaction. The measurement time can be shortened.

Moreover, highly sensitive measurement can be performed by a simple operation,
The magnetism can be used to easily separate from the antigen-antibody reaction medium.

[Brief description of drawings]

FIG. 1 is a graph showing the absorbance for standard insulin used in the enzyme immunoassay.

Claims (1)

[Claims] 1. A magnetic layer is formed on the surface of resin particles having a particle diameter of 0.1 μm to 1 mm, and the magnetic layer is formed from a mixture of a polymer substance formed by a plasma polymerization method and a magnetic metal formed by a sputtering method. A magnetic solid-phase carrier, which is a composite film comprising