JPH06109735A - Measuring method for in vivo material by antigen-antibody reaction - Google Patents

Abstract

PURPOSE:To qualitatively or quantitatively measure an in vivo material simply at high sensitivity by reacting an antigen in the in vivo material with an antibody of mixed particles carried with the antibody or antigen on two kinds of magnetic particles having the ratios between magnetization quantities and grain sizes within the specific ranges and particular specific weights. CONSTITUTION:The antibody of mixed particles and an antigen in an in vivo material, or the antigen of the mixed particles and an antibody in the in vivo material are reacted with the mixed particles carried with the antibody or antigen on the magnetic particles having the ratio between the magnetization quantity and grain size within the ranges of A, B, C, D, E and the specific weight of 1-2 and the magnetic particles having the ratio between the magnetization quantity and grain size within the ranges of C', D', E', F, G and the specific weight of 1-2. The magnetic field is applied to the reaction mixture. The absorbance or transmittance is measured with the wavelength of 350nm or above on unmagnetized magnetic particles, and the antigen or antibody in the in vivo material is qualitatively or quantitatively measured. Cross-linked polymerization particles, organic polymer material, or inorganic fine powder is used for the magnetic particles.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to an in-vivo substance by combining particles having a magnetization amount that is not magnetized and particles having a magnetization amount that is magnetized when the magnetic particles are in a monodispersed state with a particle size in a specific range. In the antigen / antibody assay method described above, the present invention relates to an immunoassay method by the agglutination method, which is highly sensitive and reliable in an extremely short time.

[0002]

2. Description of the Related Art For the measurement of various antibodies, antigens, bacteria, viruses, cancer markers and the like of substances in a living body, a large number of specimens must be sensitive in a short time and highly reliable such as reproducibility. Moreover, from the viewpoint of analysis cost, a small amount of analysis using a small amount of sample or reagent is desired. Various methods using specific magnetic particles to which an antigen or an antibody is immobilized have been adopted.

In JP-A-1-193647, a method for measuring an antigen / antibody using insoluble carrier particles containing a magnetic substance and insoluble carrier particles containing no magnetic substance, the insoluble carrier particles are particles made of an organic polymer substance. The use of ceramic particles, silica particles, or the like is described.
In JP-A-3-59459, in an antigen / antibody measuring method using insoluble carrier particles containing a magnetic substance and insoluble carrier particles not containing a magnetic substance, the average particle size of the particles, the use ratio and the measurement wavelength are specified. It is described to do. JP-A-3-193647 and JP-A-3-19367
Japanese Patent No. 59459 uses magnetic particles and insoluble carrier particles containing no magnetic material, and is a combination of magnetic particles and non-magnetic particles, and utilizes the difference in specific gravity between two types of particles. There is no mention of improving the dispersibility by the method.

US Pat. No. 4,177,253 describes an immunoassay reagent and a measurement method using magnetic particles. This description relates to EIA (enzyme immunoassay), RIA (radioisotope immunoassay) and the like that require B / F separation, and does not describe an aggregation method that does not require B / F separation. Further, there is no mention of using two types of magnetic particles in combination.

Further, in Japanese Patent Laid-Open No. 122222/1990,
A reaction solution containing magnetic particles on which a substance to be measured and a substance that specifically reacts with or competes with the substance is immobilized is placed in a reaction vessel, and a fluid such as an air stream or a water stream is supplied to the reaction solution from a nozzle. The reaction liquid is swirled, and a magnetic field is applied so that the magnetic particles converge along the wall surface of at least a part of the reaction container, thereby forming a distribution pattern of the magnetic particles and measuring a binding state between substances. Immunological assay methods have been described. Further, JP-A-2-2811
In Japanese Patent Laid-Open No. 42-42, an antigen or antibody to be measured is brought into contact with an antigen or an antibody capable of specifically binding to the antigen or magnetic microparticles immobilized with the antigen in a liquid to cause an antigen-antibody reaction. In the method for measuring an antigen or an antibody, which comprises a step of producing an antigen-antibody-magnetic fine particle conjugate, the antigen-antibody reaction described above,
It describes a method for measuring an antigen or an antibody, which is carried out with an alternating magnetic field applied to the reaction system. However, the method of JP-A-2-122265 has a problem that the material of the microplate is affected because the distribution pattern formed by applying a magnetic field to the aggregated particles is measured,
The method of JP-A-2-281142 has a problem that a special device for applying an alternating magnetic field is required.

[0006]

SUMMARY OF THE INVENTION Therefore, the present invention solves the above-mentioned problems in the measurement of the prior art and provides a highly sensitive, convenient and highly reliable qualitative or quantitative measurement method for an in-vivo substance. With the goal.

According to the present invention, (1) A (1,10) and B (1,10) in the magnetization amount-particle diameter diagram.
00), C (X1,10), D (X2,300) and E
Magnetic particles (I) having a specific gravity of 1-2 in the range of (X3,1000), and C '(X1,10), D' (X
2,300), E '(X3,1000), F (70,1)
0) and G (70,1000) within the range of magnetic particles (II) having a specific gravity of 1 to 2 and carrying an antibody or an antigen, mixed particles, the mixed particles of the antibody and an in-vivo substance. Reacting the antigen in the mixture or the antigen in the mixed particles with the antibody in the in-vivo substance (where X1, X2 and X3 are X1
>X2> X3, 18 <X2 <35 Magnetic particles (I)
Lower limit amount of magnetism collected when the particles are monodispersed, X-axis of points C, D and E of magnetic particles (II)> X1, X2, X3, specific gravity of magnetic particles (I) and magnetic particles (II) The difference is 0 to 0.
It is 5. (Magnitude of magnetization: emu / g, particles: nm) (2) Step of applying a magnetic field to the reaction mixture (3) Absorbance or transmittance of the amount of magnetic particles that are not magnetically collected is measured at a wavelength of 350 nm or more to measure in-vivo substance There is provided a method for measuring an in-vivo substance by an antigen-antibody reaction, which sequentially undergoes a step of qualifying or quantifying an antigen or antibody therein.

According to the method of the present invention, magnetic particles (I) which are not magnetized when monodispersed and magnetic particles (II) having a specific particle size and a specific amount of magnetization having a magnetization amount which is magnetized even in a monodispersed state are prepared.
By reacting the mixed particles supporting the antibody or antigen with the in-vivo antigen or antibody to be measured, the magnetism in the magnetism collecting step can be performed more quickly and reliably, and the absorbance, transparency or turbidity can be measured. The present invention has been completed by finding that the antigen or antibody in the substance in the living body can be qualitatively or quantitatively determined by performing the above. In the present invention, the state in which magnetic particles are monodispersed means that there is no in-vivo substance in the reaction system.
The antibody reaction does not occur and the magnetic particles are dispersed.

As the magnetic particles (I) and magnetic particles (II) used in the present invention, cross-linked polymer particles, organic polymer substances, inorganic fine powders and the like are used.

Various methods have been proposed for producing crosslinked polymer particles, one of which is an ethylenically unsaturated monomer and a crosslinkable copolymerizable monomer in a suspension medium in an aqueous medium. Emulsion polymerization is used to make a dispersion of fine resin particles, and water is removed by solvent replacement, azeotropic distillation, centrifugal separation drying, etc. to obtain crosslinked polymer particles. SP organic solvent or ester,
In a non-aqueous organic solvent that dissolves monomers but not polymers, such as high-SP organic solvents such as ketones and alcohols,
Copolymerize an ethylenically unsaturated monomer and a cross-linked polymer,
There is a method called a NAD method or a precipitation method in which the obtained fine resin particle copolymerization is dispersed. Further, fine resin particles synthesized from a monomer having an amphoteric group described in JP-A-58-129066 of the present applicant may be used. Cross-linked polymer particles produced in an aqueous medium or a non-aqueous organic medium, the particle size of the fine resin particles are selected by filtration, spray drying, freeze drying, etc. It can also be used after crushing.

As the organic polymer substance, polystyrene,
Styrene-butadiene copolymer particles, polyvinyl chloride, polymethyl methacrylate, polyamide, polyester, polycarbonate and the like can be used. Besides this,
Polysaccharides, protein anti-synthetic polymers, natural polymers and mixtures thereof can also be used.

As the inorganic fine powder, ceramic fine powder,
Silica, alumina, silica-alumina and the like can be used. Regarding the magnetized portion of the particle, the particle may be magnetized in the surface layer or in the inside thereof, or in the entire particle.

To magnetize, for example, metallic iron, Fe ₃
O ₄ , γ-Fe ₂ O ₃ , Co-γ-Fe ₂ O ₃ , (Ni
CuZn) O. (CuZn) O.Fe ₂ O ₃ , (MnZ
_{n) O · Fe 2 O 3} , (NiZn) O · Fe 2 O 3, S
rO · 6Fe ₂ O ₃ , BaO · 6Fe ₂ O ₃ and Si
Various ferrites such as Fe ₃ O ₄ coated with O ₂ are added in an amount of 3-1.
The thing prepared so that it may be contained at 00%, preferably 30 to 50% is used. As the particles having the above-mentioned magnetism, JP-A-63-65085 and JP-A-2-2 by the present applicant.
What was obtained by the method of 37019 gazette etc. can be used. The magnetic particles may be magnetized colored particles, colored magnetic particles, or may not be further colored.

The magnetic particles (I) have a magnetization amount (emu) on the X axis.
/ G) A (1,10), B (1,100) in the magnetization amount-particle size diagram (FIG. 1) having coordinates of particle size (nm) on the Y axis
0), C (X1,10), D (X2,300) and E
The magnetic particles (II) are contained in the range of (X3,1000), and the magnetic particles (II) include C ′ (X1,10) and D ′ (X2,
300), E '(X3,1000), F (70,100)
0) and G (70,1000), except that the X axis at the C′D′E ′ point of the magnetic particle (II)> X1, X
2, X3,).

Regarding the amount of magnetization, the magnetic particles (I) are
1 emu / g ≦ magnetic particles (I) <X1, X2, X3em
u / g, and the magnetic particles (II) have X1, X
2, X3 ≦ magnetic particles (II) ≦ 70 emu / g are used. X1, X2, X3 represent the X-axis of points C, D and E in the magnetization amount-particle size diagram, and represent the conditions of the magnetism collecting step in the present invention (for example, the magnetic force of the magnet, the arrangement condition of the magnet and the device, The maximum value of the amount of magnetization at which magnetic particles having a particle size of 10, 100 and 1000 nm are not collected when the magnetic particles are monodispersed in (magnetic time, etc.). It is the amount of magnetization that the dispersed magnetic particles are magnetized. Therefore, the magnetization amounts of X1, X2, and X3 are values determined by the magnetic flux collection conditions, and take different values when the conditions are changed. For example, the magnetizing conditions are set at the center of a bar magnet (2700 gauss) having a diameter of 3 mm, in which wells (analyte solution storage part) having an inner diameter of 7 mm are arranged at equal intervals (center interval 9 mm) in four directions, and magnetism is collected for 5 minutes. As a result, X1, = 35, X2
= 26 and X3 = 20.

Various methods can be used to control the amount of magnetization and there is no restriction. For example, the above-mentioned JP-A-63-65085 and JP-A-3-23701 are available.
According to the method described in Japanese Patent Publication No. 9, the concentration of ferrous ions,
It is possible by changing the concentration of the oxidant, the reaction temperature, the ferrous ion, the supply ratio of the oxidant into the solution, etc. Among the above factors, in order to reduce the magnetization amount, the ferrous ion In order to increase the amount of magnetization by decreasing the concentration, increasing the concentration of the oxidizing agent, lowering the reaction temperature, and increasing the amount of the oxidizing agent supplied, the above conditions can be obtained in the opposite direction. The above conditions may be used alone or in combination.

The amount of magnetization was measured using a vibrating sample magnetometer. Such a magnetometer is commercially available as MODEL BHV-3.5 from Riken Denshi Co., Ltd., for example.

Regarding the magnetic particles (I) and (II), it is difficult to use particles having a specific gravity of 1 to 2 and particles having a specific gravity of less than 1 are difficult to make. If the specific gravity exceeds 2, Dispersibility becomes poor in the reaction solution. Further, regarding the specific gravity, the difference in specific gravity between the two is 0 to 0.5 regardless of the relative magnitude of the specific gravity.
Is. When the difference in specific gravity exceeds 0.5, the cohesiveness of the magnetic particles (I) and the magnetic particles (II) due to an immunoreaction decreases.
It is preferably 0 to 0.2.

Regarding the particle size, both the magnetic particles (I) and the magnetic particles (II) have a particle size of 10 to 1000 nm. If the particle size is less than 10 nm, the agglomerates are hard to be magnetized even if the magnetization amount is large, and if it exceeds 1000 nm, the dispersibility deteriorates and the immune reaction becomes slow. The particle size is determined by the particle size of the above-mentioned crosslinked polymer particles, which are the core of the magnetic particles, the organic polymer substance, and the inorganic fine powder. The particle diameter in the present invention is obtained by measuring the primary particles with an electron microscope.

In the method of the present invention, the magnetic particles (I) and C'in the range of A, B, C, D and E in the magnetization amount-particle diameter diagram,
The magnetic particles (II) in the range of D ', E', F, G are used to generate the agglomerates. For the magnetic particles (I), preferably m (1,200), n (1,400). ),
The inner range surrounded by the line connecting the points of o (18,400) and p (18,200), preferably for magnetic particles (II) q (35,200), r (35,40)
0), s (45,400) and t (45,200) are inside ranges surrounded by a line connecting the points. The unit is such that the amount of magnetization is emu / g and the particle size is nm.

In the method of the present invention, when either one of the magnetic particles is out of the measurement range, aggregates due to immune reaction are hard to grow or aggregates are not attracted by magnetic force. As a result, it causes a decrease in sensitivity.

The preferred use ratio of the magnetic particles (I) and the magnetic particles (II) is: magnetic particles (I) / magnetic particles (II) =
It is preferably 1/20 to 2/1 and more preferably 1/8 to 1/1. The ratio is smaller than 1/20 (magnetic particles (II)
Too much), the measurement range that can be quantified becomes narrower and 2 /
If it exceeds 1 (there are too many magnetic particles (I)), the magnetic particles (II) due to the immune reaction cannot attract the magnetic particles (I), and the sensitivity of the absorbance due to the immune reaction becomes small.

As the step (1) of the present invention, the magnetic particles (I) are used.
As a method for supporting the antibody or antigen on the magnetic particles (II), there are a physical adsorption method, a covalent bond method, and an entrapment method. The physical adsorption method is a method in which magnetic particles are immobilized by a hydrophobic interaction that works between an antibody or an antigen, and the magnetic particles can be obtained by simply immersing the particles in an antigen solution or an antibody solution. Adsorb. The covalent bond method is a magnetic particle in which functional groups such as amino groups and carboxyl groups are attached to the surface, and an antigen or antibody is shared by using a cross-linking agent such as glutaraldehyde or an activator such as water-soluble carbodiimide. It is a method of combining. The encapsulation method is an intermediate method between the physical adsorption method and the covalent binding method, in which the antigen or antibody is transferred to HEMA (Hydr
oxymethylmethacrylate) and HPM
A (Hydroxypropy lmethacryla
It is a method of mixing with a monomer such as te), polymerizing with γ rays or the like, and binding.

The in-vivo substance to be measured in the present invention is blood,
Antibodies or antigens in body fluids such as lymph, urine, saliva,
Alternatively, it is a microorganism, virus or its component that has invaded into the body fluid, the type of which has an antibody or antigen relationship with a specific antigen or antibody of the in-vivo substance to be measured, Is selected according to the antigen or antibody.
Examples of such antigens or antibodies include the followings. Antigens: IgG, IgA, IgM, IgE, albumin, hCG, AFP, cardiolivin antigen, blood group substance, concanavalin A, DNA, prostaglandin,
CRP, HBs, human growth hormone, steroid hormone, CEA, IgD, etc. Antibodies: anti-albumin antibody, anti-hCG antibody, anti-IgG antibody, anti-IgA antibody, anti-IgM antibody, anti-IgE antibody, anti-I
gD antibody, anti-AFP antibody, anti-DNA antibody, anti-prostaglandin antibody, anti-human coagulation factor antibody, anti-CRP antibody, anti-HBs antibody, anti-human growth hormone antibody, anti-steroid hormone antibody, etc., and serum containing these, and Monoclonal antibody etc.

In the present invention, when the absorbance and the transmittance are measured in the step (2), the agglomerates are magnetized to the side of the analysis solution storage portion of the analysis container, and the magnetic force is applied in the step (3). By analyzing the non-magnetized magnetic particles, the interference from agglomerates can be eliminated very simply and efficiently from the light source-detector optical path of the spectrophotometer.

In the present invention, in order to further improve the efficiency, it is preferable that the analysis liquid storage bottom portion of the analysis container is not V-shaped or U-shaped, but flat-shaped. The arrangement of the magnets is preferably such that the magnets are arranged on the support plate so that the magnetic force is applied at equal intervals in four directions around the analysis liquid storage portion of the analysis container, but the magnets are not limited thereto, and the magnets may be arranged only on the support. However, the agglomerate is not limited as long as it is arranged to collect magnetism on the side wall of the analysis liquid storage portion of the analysis container, and it can be achieved by the number of at least one magnet. In addition, magnets may be arranged around the entire circumference of the analysis liquid container. Further, the magnet may have any shape such as a round bar shape, a square shape, a ring shape, and a sheet shape.

The magnet for collecting the magnet may be a permanent magnet or an electromagnet. In the case of the permanent magnet, the magnet can be collected by bringing the support plate on which the magnet is arranged close to the analysis container when the magnetism is required. In the case of an electromagnet, magnetism can be collected by electrically generating a magnetic force when necessary. A more efficient and preferred method of using a permanent magnet is, for example, 9
Plastic plate with 6 micro holes (well) (commercially available "Griner Microplate")
Can be used) and use this as an analysis container,
The magnet support plate is used by covering the microplate so that the magnet support plate fits inside the lower peripheral edge projection of the microplate. A rubber magnet sheet can also be used.
The method of arranging the magnets on the support plate is such that the magnets are fixed so that the magnetic force is applied to the side portion of the analysis liquid storage portion in the 3-4 directions of the portion corresponding to the storage portion, preferably at equal intervals. Good.

In the present invention, the absorbance and the transmittance are measured by a spectrophotometer, and the measurement wavelength is selected to be 350 nm or more.

The method for measuring a substance in a living body according to the present invention has been described mainly for quantitative measurement, but it can be applied to qualitative measurement. For example, if the cutoff value is AFP,
The amount of magnetic particles, the buffer concentration, the salt concentration in the buffer, and the surfactant concentration are adjusted so that the absorbance at that time is set to about 1.0. Using the wavelength of 350 nm or more with the cutoff value as the boundary, the absorbance is 1.
Qualitative measurement is carried out by judging that those exceeding 0 are positive, and those not exceeding 0 are negative.

In the present invention, magnetic particles (I) having a specific amount of magnetization, a specific particle size and a specific gravity generate an aggregate of an antigen-antibody-magnetic particle or an antibody-antigen-magnetic particle complex. It is presumed that the magnetic particles (I) are not magnetized in the monodisperse state, but are magnetized when the agglomerates are generated, based on the principle of Stokes' law. The force acting according to Stokes 'law is gravity, but in the present invention, a magnetic force acts instead of gravity according to Stokes' law to collect magnetism when an aggregate is formed.

The present invention will be described in more detail based on the following examples, but it goes without saying that the technical scope of the present invention should not be limited to these examples.

(Production Example 1) Production of polymer particles Distilled water: 1100 g Sodium styrenesulfonate: 0.28 g 4, 4 -Azobis (4 cyan valeric acid) ... 3.75 g Dimethyl ethanolamine ... 2.40 g was charged into a reaction vessel, the temperature was set to 83 ° C, and nitrogen gas (N ₂ ) was introduced. After stirring, 100 g of styrene was added dropwise from the dropping funnel over 60 minutes to carry out polymerization for a total of 6 hours. When the particle size was measured with a scanning electron microscope (S-510: manufactured by Hitachi, Ltd.), the average particle size was 110 nm.
This solution is called latex solution (1).

(Production Example 2) Production of polymer particles Distilled water: 1100 g Sodium styrenesulfonate: 0.28 g 4, 4 -Azobis (4 cyan valeric acid) ... 3.75 g Dimethyl ethanolamine ... 2.40 g was charged into a reaction vessel, the temperature was set to 83 ° C, and nitrogen gas (N ₂ ) was introduced. After stirring, 300 g of styrene was added dropwise from the dropping funnel over 60 minutes to carry out polymerization for a total of 6 hours. When the particle size was measured with a scanning electron microscope, the average particle size was 290 nm. This solution is referred to as a latex solution (2).

(Production Example 3) Production of polymer particles Distilled water: 1100 g Sodium styrenesulfonate: 0.28 g 4, 4 -Azobis (4 cyan valeric acid) ... 3.75 g Dimethyl ethanolamine ... 2.40 g was charged into a reaction vessel, the temperature was set to 83 ° C, and nitrogen gas (N ₂ ) was introduced. After stirring, 500 g of styrene was added dropwise from the dropping funnel over 60 minutes to carry out polymerization for a total of 6 hours. When the particle size was measured with a scanning electron microscope, the average particle size was 620 nm. This solution is called latex solution (3).

(Production Example 4) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex solution (1) of (Production Example 1) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl ₂ was added and the pH was adjusted to 6.9 with 0.1N-NaOH. After that, the temperature in the container was adjusted to 70
Warmed to ° C. Deoxidized ion-exchanged water 1
2 g of a solution of 20 g of sodium nitrite dissolved in 1 liter
It was supplied at a rate of 5 ml / min. During this time, the pH was kept constant, and after about 20 minutes, magnetic particles of fematite were produced.
When the particle size was measured with a scanning electron microscope, the average particle size was 1
It was 20 nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. Magnetization amount is 10e
It was mu / g.

(Production Example 5) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex solution (1) of (Production Example 1) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added to this
It was supplied at a rate of 1 / min. During this period, keep the pH constant,
After about 20 minutes, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 120.
was nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. The amount of magnetization is 20 em
It was u / g.

(Production Example 6) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex solution (1) of (Production Example 1) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added to this product to 10 m.
It was supplied at a rate of 1 / min. During this period, keep the pH constant,
After about 20 minutes, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 120.
was nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. The amount of magnetization is 31 em
It was u / g.

Production Example 7 Production of Magnetic Particles 0.9 L of ion-exchanged water was charged into a reaction vessel. To this, 100 g (solid content 10 g) of the latex solution (1) of (Production Example 1) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. 5 ml of a solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added.
/ Min. During this time, the pH was kept constant, and after about 20 minutes, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 120n.
It was m. This was filtered and washed with water to obtain magnetic particles.
The particles obtained were brown. The amount of magnetization is 42 emu /
It was g.

(Production Example 8) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex solution (2) of (Production Example 2) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water in advance was added to this product for 30 m.
It was supplied at a rate of 1 / min. During this period, keep the pH constant,
After about 20 minutes, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 31.
It was 0 nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. The amount of magnetization is 1 emu
/ G.

(Production Example 9) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex (2) solution of (Production Example 2) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added to this product for 25 m.
It was supplied at a rate of 1 / min. During this period, keep the pH constant,
After about 20 minutes, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 30.
It was 0 nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. Magnetization amount is 9 emu
/ G.

Production Example 10 Production of Magnetic Particles 0.9 L of ion-exchanged water was charged into a reaction vessel. In this, 100 g (solid content 1) of the latex solution of (Production Example 2)
0 g) was charged and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added and 0.1N was added.
-Adjusted to pH 6.9 with NaOH. Then, the temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was supplied to this in advance at a rate of 20 ml / min. During this period, keep the pH constant and keep
After a minute, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 310 nm.
Met. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. Magnetization amount is 19 emu / g
Met.

(Production Example 11) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex (2) solution of (Production Example 2) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. 5 ml of a solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added.
/ Min. During this time, the pH was kept constant, and after about 20 minutes, magnetic particles of fematite were produced. When the particle size was measured with a scanning electron microscope, the average particle size was 300.
was nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were black. Magnetization amount is 40 em
It was u / g.

Production Example 12 Production of Magnetic Particles 0.9 L of ion-exchanged water was charged into a reaction vessel. To this, 100 g (solid content 10 g) of the latex (3) solution of (Production Example 3) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added to this product for 25 m.
l / min. It was supplied at a rate of. During this time, the pH was kept constant, and after about 20 minutes, magnetic particles of fematite were produced.
When the particle size was measured with a scanning electron microscope, the average particle size was 630 nm. This was filtered and washed with water to obtain insoluble carrier particles containing a magnetic material. The particles obtained were brown. The amount of magnetization was 9 emu / g.

(Production Example 13) Production of magnetic particles A reactor was charged with 0.9 liter of ion-exchanged water. To this, 100 g (solid content 10 g) of the latex (3) solution of (Production Example 3) was added, and deoxidation was performed with nitrogen gas. After sufficiently deoxidizing, 10 g of FeCl _{2 was} added,
The pH was adjusted to 6.9 with 0.1 N NaOH. After this,
The temperature in the container was heated to 70 ° C. while stirring under the flow of nitrogen gas. A solution prepared by dissolving 20 g of sodium nitrite in 1 liter of deoxygenated ion-exchanged water was previously added to this product to 10 m.
l / min. It was supplied at a rate of. During this time, the pH was kept constant, and after about 20 minutes, magnetic particles of fematite were produced.
When the particle size was measured with a scanning electron microscope, the average particle size was 630 nm. This was filtered and washed with water to obtain magnetic particles. The particles obtained were brown. Magnetization amount is 30e
It was mu / g.

(Sensitization method) The magnetic particles prepared in (Production Examples 1 to 13) are dispersed in 1 ml of a phosphate buffer (pH 5.0) so that the particle concentration becomes 4%. 1% EDC for this
(1-Ethyl-3- (3-Dimethylylami
nopropyl) Calbody, hyd
200 ml of aqueous solution was added and stirred. Then, the supernatant is removed by centrifugation (12000 rpm × 20 minutes),
The separated particles were redispersed in 1 ml of distilled water, and this operation was repeated twice, and 20 mM phosphate buffer solution (pH 7.5) was added to the particles of the precipitate from which the supernatant had been removed at the third time, and the particle concentration was adjusted to 4 It was dispersed so as to be%. Anti-human AFP goat-derived antibody (ATA) partially purified by salting out
After adding 200 μl (manufactured by Company B) and stirring, sensitize in a refrigerator for 3 days. Then, centrifuge (12000 rpm x 2
(0 min), the supernatant was removed, and the separated particles were mixed with 1 ml of “0.1% Tween 20, 1.5% polyethylene glycol (PEG6000), 1% bovine serum albumin /
20 mM phosphate buffer (pH 7.5) ”(abbreviated as PB solution) is re-dispersed, and this operation is repeated 3 times to adjust the particle concentration to 4%. Table 1 shows the particle size, the amount of magnetization and the specific gravity of each sensitized particle.

Example 1 0.125 ml of the sensitized particle 9 solution as the magnetic particles (I) in Table 1 and 0.5 ml of the sensitized particle 11 solution as the magnetic particles (II) were added to the PB solution 1.375 m.
A solution prepared by mixing 1 and stirring well is used as a reagent. 0.1 ml of this reagent is dispensed into the wells of a flat-bottom 96-well microplate. An AFP product (manufactured by Biotest) is diluted with physiological saline to have a concentration of 500 ng / ml, which is used as a standard solution. A series prepared by diluting the standard solution by 2 times with physiological saline was prepared and used as a sample. 0.1 ml of these samples
Each was dispensed into the wells of the above flat-bottom 96-well microplate and reacted at room temperature for 50 minutes. Next, attach the magnetic separation device manufactured by our company to the outside 5
The particles agglomerated by the antigen-antibody reaction are attracted for a minute to collect the magnetism. The remaining unreacted magnetic particles were measured with a microplate reader (MPRA4i manufactured by Tosoh Corporation) at a wavelength of 57
The absorbance was measured at 0 nm.

Example 2 The solution of the sensitized particles 11 as the magnetic particles (II) in Example 1 was added to 0.125 ml,
The operation method is the same as in Example 1 except that the PB solution was changed to 1.75 ml.

Example 3 The solution of the sensitized particles 11 as the magnetic particles (II) in Example 1 was added to 0.125 ml.
The operation method is the same as in Example 1 except that the PB solution was changed to 0.175 ml.

(Examples 4 to 11, Comparative Example 1) Examples 4 to
In Example 11 and Comparative Example 1, experiments were carried out by combining magnetic particles (I) and magnetic particles (II) as shown in Table 3, and the operation method was the same as in Example 1.

[0050]

[0051]

[0052]

Results The results of absorbance and AFP solution concentration are shown in Table 3. In Comparative Example 1, since the magnetic amounts of both magnetic particles were high, the unreacted magnetic particles were attracted by the magnetic force. It was impossible to measure.

[0054]

INDUSTRIAL APPLICABILITY According to the present invention, by using two types of magnetic particles having different amounts of magnetization, an aggregate of antigen-antibody-magnetic particle or antibody-antigen-magnetic particle is generated and the aggregate is collected. Since it is magnetized, B / F separation is not required, the target substance can be quantitatively or qualitatively simpler than in the past, the magnetism can be collected more reliably, and the analysis with higher accuracy becomes possible.
A useful clinical diagnostic method is obtained.

[Brief description of drawings]

FIG. 1 is a graph showing the amount of magnetization of a magnetic particle used in the present invention.

[Explanation of symbols]

A, B, E, D, C ......... The conditions for magnetic particles (I) in this range C ', D', E ', G, F ... ... Magnetic particles (II) in this range Condition m, n, o, p ………………………… This range is preferable condition for magnetic particles (I) q, r, p, t …………………… Preferred conditions for (n)

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Makoto Anan 4-1-1-15 Minami-Shinagawa, Shinagawa-ku, Tokyo Inside Nippon Paint Co., Ltd.

Claims (1)

[Claims] 1. A method for measuring an in-vivo substance by an antigen-antibody reaction, which comprises sequentially performing the following steps (1) to (3). (1) Amount of magnetization vs. grain size A (1,10), B (1,10)
00), C (X1,10), D (X2,300) and E
Magnetic particles (I) having a specific gravity of 1-2 in the range of (X3,1000), and C '(X1,10), D' (X
2,300), E '(X3,1000), F (70,1)
0) and G (70,1000) within the range of magnetic particles (II) having a specific gravity of 1-2 and carrying an antibody or an antigen; mixed particles; Step of reacting the antigen of the above, or the antigen of the mixed particles with the antibody in the in-vivo substance (where X1, X2, X3 are X
1>X2> X3, 18 <X2 <35 The lower limit amount of magnetization collected when the magnetic particles (I) are monodispersed, the X axis of point C′D′E ′ of the magnetic particles (II)> X1 , X2, X
3, the specific gravity difference between the magnetic particles (I) and the magnetic particles (II) is 0
~ 0.5. (Magnetization amount: emu / g, particle size: nm) (2) Step of applying magnetic field to the reaction mixture (3) Absorbance or transmittance of the amount of magnetic particles that are not magnetically collected is measured at a wavelength of 350 nm or more to measure in vivo Process of qualitatively or quantitatively determining antigen or antibody in substance