JP2006189268A - Magnetic particle for diagnostic drug - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a magnetic particle for a diagnostic drug capable of realizing high sensitivity measurement, by avoiding optical absorption by a black or brown magnetic composite particle used hitherto for a diagnostic use using a photodetection system. <P>SOLUTION: This magnetic particle for the diagnostic drug includes a core including a magnetic body particle and a visible light scattering layer formed on the furthermore outside including silver than the core. The L value in a Lab color system is 60 or more, and residual magnetization is ≤30% of saturation magnetization, and the particle size is 0.5-15 μm. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention can be used for immunodiagnostic drug carriers, bacterial separation carriers, cell separation carriers, nucleic acid separation and purification carriers, protein separation and purification carriers, immobilized enzyme carriers, drug delivery carriers, etc. The present invention relates to magnetic particles for diagnostic agents that can be suitably used for separation, sorting and the like.

Magnetic composite particles are already used for the separation and collection or concentration of nucleic acids, viruses, proteins, cells, etc. by adsorption of specific substances on the surface of the particles and solid-liquid separation using chemical bonds and magnetic responsiveness. Widely used for molecular biology and medical diagnostics. As the assay method, a sandwich method, a competitive method and the like have been conventionally used.
Magnetic composite particles used for immunodiagnostic agents generally contain a magnetically responsive substance mainly composed of iron oxide, and are generally black or brown. Moreover, introduction of these magnetically responsive materials and pre-active functional groups that react with proteins is already known (see, for example, Patent Documents 1 to 5).
In the immunodiagnosis, various devices have been made in order to realize more sensitive detection by a simpler method. In particular, the detection system is greatly improved. Further, color development measurement (for example, fluorescence, phosphorescence, luminescence, chemiluminescence, etc.), which has conventionally been regarded as a high-sensitivity measurement system, has become the mainstream measurement system. However, despite the improvement of the detection system, provision of a solid phase carrier suitable for the characteristics of the detection system has been delayed, which has hindered high sensitivity of the entire diagnostic system.
For example, when magnetic composite particles are used for the solid phase carrier, as described above, the magnetic composite particles are generally black or brown. For this reason, the luminescence signal derived from the reaction between the labeled enzyme formed by capturing the target substance and the substrate may sometimes be reduced to half or less due to absorption by the brown magnetic composite particles themselves. It was.
In order to avoid such light absorption by the magnetic composite particles, in many cases, the magnetic composite particles and the substrate liquid layer are separated into solid and liquid using a magnet just before measuring the emission intensity. Measures are taken to pass light through only the layers. However, this countermeasure has two problems.

The first problem is that it is difficult to use the magnetic composite particles in a substrate system having a short color development or light emission time. When the antigen / antibody / enzyme complex formed on the surface of the magnetic composite particle is separated from the substrate liquid layer, the reaction rate is drastically reduced compared to the liquid layer reaction before separation, and the generated signal is also reduced. To do. Therefore, the method of performing solid-liquid separation and measuring light only through the liquid layer is not practical except for a substrate system having a long emission duration.
The second problem is time loss and separation accuracy caused by adding a solid-liquid separation operation. Specifically, in an assay system that requires rapid processing in a short time, it is desirable to reduce the number of solid-liquid separations as much as possible. In addition, noise is generated due to the presence of a minute amount of magnetic composite particles that remain without being separated, leading to a decrease in sensitivity. Therefore, it is strongly required to eliminate the solid-liquid separation operation.
The same problem arises when applying the above-described brown magnetic composite particles also in a fluorescence or phosphorescence detection system or a radioisotope detection system.

By the way, regarding the whitening of magnetic particles, proposals have been made to improve the appearance of magnetic toner, magnetic ink, etc. by combining silver on the surface of the ferromagnetic particles (see, for example, Patent Documents 6 and 7). . The magnetic composite particles used for these applications are required to have high saturation magnetization and residual magnetization, that is, are easily magnetized by an external magnetic field and have high coercivity. Such magnetic composite particles are required to be powder and to have a hydrophobic surface.
On the other hand, in the immunodiagnosis which is one of the uses of the present invention, the magnetic composite particles are generally used in the form of an aqueous suspension. For this reason, the magnetic composite particles are required to have particle colloidal stability. In particular, the magnetic composite particles used for immunodiagnosis are required to have at least a partially hydrophilic surface in order to avoid non-specific hydrophobic bonds such as proteins.

Japanese Patent No. 2672151 Japanese Patent No. 2844263 JP 7-316466 A JP-A-6-265550 International Publication No. 84/02031 Pamphlet JP 2004-043950 A JP 2004-346272 A

  An object of the present invention is to provide magnetic particles for diagnostic agents that can avoid light absorption by the black or brown magnetic composite particles that have been conventionally used in diagnostic applications using a light detection system and can realize high-sensitivity measurement.

  As a result of intensive studies by the present inventors, it has been concluded that the above-mentioned problems can be solved by eliminating the color of conventionally used brown magnetic composite particles and switching to light or white magnetic composite particles. Thus, whitening and lightening of the magnetic composite particles has been found to be the most effective solution for improving the sensitivity of immunoassays using magnetic composite particles.

The magnetic particle for diagnostic agent of the present invention comprises a core containing magnetic particles, and a visible light scattering layer containing silver formed outside the core,
The L value in the Lab color system is 60 or more, the residual magnetization is 30% or less of the saturation magnetization, and the particle diameter is 0.5 to 15 μm.
Here, the Lab color system means a color system defined in JIS Z8729.
In the diagnostic magnetic particles, the visible light scattering layer may contain at least one of titanium oxide and zinc oxide.

The magnetic particle for diagnostic agent of the present invention includes a core containing magnetic particles and a visible light scattering layer containing silver formed outside the core, and has an L value of 60 or more in the Lab color system, and residual magnetization. Is not more than 30% of the saturation magnetization and the particle diameter is 0.5 to 15 μm, which suppresses visible light absorption, and is particularly suitable for applications such as light measurement, light detection, and light sorting. The magnetic particles for diagnostic agents of the present invention are particularly preferable materials for biochemical carriers such as carriers for diagnostic agents. Other uses include cells, genes, carriers for gene therapy, electronic materials, electrophotography, cosmetics, pharmaceuticals, agricultural chemicals, It can be used in a wide range of fields such as food and catalysts. As the use of the magnetic particle for diagnostic agent of the present invention, a medical diagnostic agent is preferable, and it is more preferable to apply as a particle corresponding to an automatic measuring instrument.
The magnetic particles for diagnostic agents of the present invention are particularly suitable as solid phase carriers for nucleic acids such as DNA and RNA, nucleotides, or physiologically active substances such as antigens / antibodies, haptens and enzymes. By using the magnetic particle for diagnostic agent of the present invention as a solid phase carrier, optical measurement and optical operation can be efficiently performed in a system containing the particle.

Hereinafter, each component of the present invention will be described in detail.
The magnetic particle for diagnostic agent of the present invention includes a core containing magnetic particles and a visible light scattering layer containing silver formed outside the core, and has an L value of 60 or more in the Lab color system and a residual magnetization. Is 30% or less of the saturation magnetization, and the particle diameter is 0.5 to 15 μm. Since the visible light scattering layer containing silver is formed outside the core, light absorption can be efficiently suppressed in the detection system.

[color]
The magnetic particles for diagnostic agents of the present invention (hereinafter also simply referred to as “magnetic particles”) differ in appearance color or colorimetry measured with a colorimeter depending on the content of the particles in the suspension. The appearance of a 10% by weight aqueous dispersion of diagnostic magnetic particles is usually white, milky white or light (light gray). Generally, when the particle suspension is air-dried, the color changes.
The color of the magnetic particles for diagnostic agents of the present invention is measured using a 10% by weight dispersion thereof. Specifically, 100 μL of a 10 wt% dispersion of magnetic particles for diagnostic agents of the present invention is gently dropped on a microscope slide glass, and then allowed to stand for 10 minutes or more and air dried. The air-dried particles are then measured with a colorimeter. The measurement result is expressed in the Lab color system defined by JIS Z8729.
In the diagnostic magnetic particles of the present invention, the L value in the Lab color system is preferably 60 or more, and more preferably 70 or more. If the L value is less than 60, light absorption by the detection system may not be efficiently avoided.

  The hue (a, b value) of the magnetic particle for diagnostic agent of the present invention is not particularly limited, and can be selected according to the emission wavelength of the substrate and the wavelength of the phosphor selected during the actual assay. . That is, in order to suppress light absorption efficiently, the color of the surface of the magnetic particle can be selected according to the wavelength of light to be avoided, in addition to making the surface of the magnetic particle for diagnostic agent of the present invention white. For example, in an assay system using a substrate that emits light having a wavelength of 450 nm, the same effect can be obtained even when the surface of the magnetic particle for diagnostic agent of the present invention is white or light blue-purple.

[Residual magnetization]
The residual magnetization of the diagnostic magnetic particles of the present invention is 30% or less of the saturation magnetization, more preferably 20% or less, and even more preferably 10% or less. If the remanent magnetization is higher than 30% of the saturation magnetization, the magnetic particles are attracted to each other and aggregate even after the magnetic field is removed from the outside, which may be inconvenient for the subsequent redispersion of the magnetic particles.
The immunodiagnostic agent is one of the uses of the magnetic particles for diagnostic agents of the present invention (other uses will be described later). When the magnetic particle for diagnostic agent of the present invention is used as an immunodiagnostic agent, the property of quickly responding to the application of an external magnetic field and the property of aggregation, the property of rapidly disappearing the magnetization due to the removal of the external magnetic field, and the redispersion of the particles It is desirable to have both.
The property of aggregating by application of an external magnetic field is necessary for recovering only the diagnostic magnetic particles and the components specifically bound to the diagnostic magnetic particles and removing unnecessary components.
In addition, the characteristic that the magnetization disappears rapidly by removing the external magnetic field is such that, for example, magnetic particles are uniformly dispersed in a dispersion medium (for example, water) to measure luminescence, or magnetic properties are used to cause multiple specimens to act. Necessary to disperse the particles. If strong residual magnetization exists in the magnetic particles after removing the external magnetic field, it may be inconvenient because it may take a long time to redisperse the magnetic particles or may require external energy such as strong stirring. .
Magnetic properties such as saturation magnetization, residual magnetization, and coercive force of the magnetic particles for diagnostic agents of the present invention can be generally measured with a vibrating sample magnetometer. Here, it is assumed that the maximum external applied magnetic field is saturation magnetization at 15 K Oe, and the residual magnetization is obtained from the magnetization hysteresis curve obtained when the external magnetization is returned to zero.

[Particle size]
The particle diameter of the magnetic particles for diagnostic agents of the present invention is 0.5 μm to 15 μm, more preferably 1 μm to 10 μm. If the particle size is smaller than 0.5 μm, disturbance due to the Brownian motion of the magnetic particle itself cannot be ignored other than the response to the external magnetic field, which is not desirable because it takes a long time for magnetic separation. On the other hand, if the particle diameter is larger than 15 μm, the shielding factor of the magnetic particle itself against the excitation light, signal color development, light emission, etc. used in the detection system becomes large, and the protein that can be introduced because the surface area relative to the volume of the magnetic particle becomes too small. As a result of limiting the amount, the detection sensitivity may be lowered, or the natural sedimentation rate of the magnetic particles is increased, which makes it difficult to maintain a uniformly dispersed state of the magnetic particles.

[Production method]
The method for producing diagnostic magnetic particles of the present invention comprises a step of producing magnetic core particles, a step of subsequently coating the magnetic core particles with a visible light scattering layer containing silver, and various functions as required. A step of covering the visible light scattering layer with a layer. That is, by the step of producing the magnetic core particle, the “core containing magnetic particles” in the magnetic particle for diagnostic agent of the present invention is formed, and the step of coating the magnetic core particle with a visible light scattering layer containing silver, A visible light confusion layer containing silver formed outside the core is formed.
The magnetic core particles may be formed of only an inorganic substance, but it is preferable that an organic substance is included because the sedimentation in water is delayed by making the specific gravity low, and the dispersion in water becomes easy. Alternatively, in order to reduce the specific gravity, porous particles made of an organic material or an inorganic material may be used.
Examples of magnetic core particles include those in which magnetic particles are dispersed in polymer particles, those in which magnetic particles are physically attached to the surface of polymer particles, and magnetic particles deposited on the surfaces of polymer particles and porous particles. Can be mentioned. Monomers for obtaining such an organic polymer substance include styrene, vinyl naphthalene, vinyl anthracene, methyl acrylate, ethyl acrylate, acrylic acid-n-butyl, acrylic acid-2-hydroxyethyl, and acrylic acid. Acrylics such as polyoxyethylene, glycidyl acrylate, ethylene glycol diacrylate, tribromophenyl acrylate, lauryl acrylate, tridecyl acrylate, stearyl acrylate, 2-ethylhexyl acrylate, cyclohexyl acrylate, benzyl acrylate, etc. Acid ester, methyl methacrylate, ethyl methacrylate, methacrylate-n-butyl, 2-hydroxyethyl methacrylate, polyoxyethylene methacrylate, glycidyl methacrylate, ethylene glycol dimethacrylate Acid esters, tribromophenyl methacrylate, lauryl methacrylate, tridecyl methacrylate, stearyl methacrylate, methacrylate-2-ethylhexyl, cyclohexyl methacrylate, benzyl methacrylate, and the like, chlorostyrene, chloromethylstyrene, α- Examples include methylstyrene, divinylbenzene, sodium styrenesulfonate, and the like. These can be used alone or in combination of two or more. Of these, copolymers of styrene, vinyl naphthalene and (meth) acrylic acid are particularly suitable for the present invention.

In the present invention, the material of the magnetic particles used for forming the magnetic core particles is not particularly limited, but iron oxide-based substances are typical, and MnFe ₂ O ₄ (Mn = Co, Ni, Mg, Cu, Li _0.5 Fe _0.5 or the like), magnetite represented by Fe ₃ O ₄ , or γFe ₂ O ₃ . Moreover, you may use combining the said magnetic body. In particular, magnetic particles composed of γFe ₂ O ₃ and Fe ₃ O ₄ are preferable as magnetic materials having strong saturation magnetization and low residual magnetization. Moreover, the diameter of 50 nm or less is preferable as a single magnetic domain composed of these magnetic materials. If the single magnetic domain exceeds 50 nm, the residual magnetization of the obtained diagnostic magnetic particles may exceed 30% of the saturation magnetization, which is not preferable.
Next, the visible light scattering layer formed outside the core containing the magnetic particles (magnetic core particles) is a layer that reflects or scatters visible light. The visible light confusion layer preferably has a high refractive index for visible light.
In the present invention, as a production method for forming a visible light scattering layer containing silver outside the magnetic core particle, for example, the magnetic core particle is dispersed in a solvent, and a solution of silver salt or a complex solution is used. A method of depositing metallic silver on the surface can be exemplified. In particular, a method of precipitating titanium oxide on the surface of the magnetic core particle before precipitating metallic silver is preferable.
By forming a visible light scattering layer containing silver outside the magnetic core particles, the appearance of the magnetic particles for diagnostic agents of the present invention can be white or light. That is, by covering the magnetic core particles with a visible light scattering layer containing silver, the magnetic core particles, which are usually black or brown, are concealed by the visible light scattering layer, and the function to suppress the attenuation of the emission signal derived from the substrate is given. can do.

In the present invention, if necessary, the polymer coating layer is reintroduced on the surface of the visible light scattering layer, whereby the magnetic particles can be prevented from being exposed on the surface of the magnetic particle for diagnostic agent of the present invention.
As a method for measuring the degree of exposure of the magnetic particles to the surface of the magnetic particles for diagnostics of the present invention, it is one of the substrates used in normal enzyme immunoreaction, and it reacts sensitively with iron. Phenylenediamine (OPD) can be used. Since OPD is a substrate for enzyme immunoassay, the influence of magnetic particles exposed on the surface of magnetic particles for diagnostics of the present invention can be confirmed in a form closer to an actual assay system. In order to facilitate dispersion in aqueous systems or to introduce functional groups for binding proteins such as antibodies and antigens to the surface of the magnetic particles, polymers and oligomers are formed on the outermost layer of the diagnostic magnetic particles of the present invention. It is particularly preferable to form a coating layer.

The magnetic particle for diagnostic agents of the present invention is a physiologically active substance mainly composed of a protein or a physiologically active substance that can be easily bound to DNA, oligonucleotides, lectins, polysaccharides and the like on the surface. And can be combined without damage.
Examples of the method of binding the magnetic particle for diagnostic agent of the present invention and a physiologically active substance include a physical adsorption method using a hydrophobic bond between the magnetic particle and the protein, and a chemical bond between the functional group present on the surface of the magnetic particle and the protein. Law. From the viewpoint of simplicity, the physical adsorption method is promising, but in view of the long-term stability of the immobilized substance, the chemical bonding method is preferable for stable use in an environment containing a surfactant. The chemical bonding method generally utilizes a carboxyl group, amino group, SH group, amino acid amino group on the protein side, or a functional group at the tip of the spacer to be introduced. Therefore, it is preferable to introduce at least one carboxyl group, amino group, aldehyde group, epoxy group, and tosyl group on the surface of the magnetic particle. As a specific immobilization method, for example, a method of bonding amino groups (diisocyanate method, glutaraldehyde method, difluorobenzene method or benzoquinone method), a method of bonding carboxyl groups (succinimide method), or the like can be used. (Edited by Yuji Ishikawa et al., “Enzyme Immunoassay 3rd Edition”, 1987, published by Medical School).
Needless to say, these functional groups can be introduced on the surface of the magnetic particle for diagnostic agents of the present invention. Specific methods include, for example, a method in which a reactive monomer having an unsaturated double bond is polymerized on the surface of the magnetic particle, or a compound containing another type of functional group is bonded to the introduced functional group to form the surface of the magnetic particle. The functional group can also be introduced on the surface of the magnetic particle for diagnostic agent of the present invention by a method of modifying the above.

In addition to the chemical reaction, a method of introducing the functional group onto the surface of the magnetic particle for diagnostic agent of the present invention by precipitation method, precipitation method, or impact is also included in the scope of the present invention. From the viewpoint of compactness, a chemical reaction or a method combining the introduction method and the chemical reaction is preferable. Moreover, you may introduce | transduce the molecular spacer which has a suitable length as needed.
The detection target that can use the magnetic particle for diagnostic agent of the present invention is not particularly limited. The measurement object covers a wide range of nucleic acids, antigens, antibodies, viruses and the like. Specific immunodiagnostics include, for example, CEA, immunoglobulin (IgG, IgA, IgM, IgD, IgE), complement (C3, C4, C5, C1q), C-reactive protein, α ₁ -antitrypsin, α ₁ Microglobulin, β ₂ -microglobulin, haptoglobulin, transferrin, ceruloplasmin, ferritin, albumin, hemoglobin A ₁ , hemoglobin A _1C , myoglobin, myosin, dupan-2, α-fetoprotein (AFP), tissue polypeptide antigen ( TPA), apolipoprotein _{A 1,} apolipoprotein E, rheumatoid factor, anti-streptolysin O (ASO), fibrin degradation products (FDP), fibrin degradation product D fraction (FDP-D), fibrin degradation product D-D fraction (FDP-D Dimer), fibrin degradation product E fraction (FDP-E), protein such as antithrombin-III (AT-III), amylase, prostate-derived acid phosphatase (PAP), nerve specific enolase (NSE), fibrinogen, elastase, plasminogen, creatine kinase myocardium Type (CK-MB) and other enzymes, insulin, thyroid stimulating hormone (TSH), 3,5,3′-triiodothyronine (T ₃ ), thyroxine (T ₄ ), corticotropin (ACTH), growth Hormones (GH), hormones such as luteinizing hormone (LH), hepatitis B virus related antibodies, hepatitis B virus related antigens, hepatitis C virus antibodies, HTLV (adult T cell leukemia virus) antibodies, HIV (AIDS virus) Various infectious diseases such as antibodies, chlamydia antibodies, syphilis antibodies, toxoplasma antibodies Cause the virus, and the like.

There are no particular limitations on the type of specimen that can be used with the magnetic particles for diagnostic agents of the present invention. Specific examples include blood, serum, plasma, urine, body fluid, saliva, bacilli and cell culture fluid. The treatment method of these specimens is in accordance with the usual immunodiagnostic method. In gene detection, it is preferable to extract and purify genes from these specimens in advance.
Depending on the object to be detected, an antibody, an antigen, a hapten, or the like can be immobilized on the surface of the magnetic particle for diagnostic agent of the present invention. Examples of antibodies corresponding to the antigen to be tested include antibodies that can react with normal antigens such as polyclonal antibodies and monoclonal antibodies ["Single Coulomb Antibody Experimental Manual" edited by Tomiyama Shinji et al., Kodansha Scientific, 1987. : New Chemistry Laboratory, Vol. 12, “Molecular Immunology III Antigen, Antibody, Complement”, edited by the Japanese Biochemical Society, published by Tokyo Chemical Dojinsha, 1992]. The antibody may be composed of a plurality of antibodies, and may be a product obtained by limited degradation or protein modification of the antibody.
These antibodies, which are covalently immobilized on the surface of the magnetic particles for diagnostic agents of the present invention, dispersed and washed with a bovine serum albumin or milk skin-containing buffer, can be used as they are for immunodiagnosis. Examples of the buffer solution used for antibody sensitization, particle washing, and dispersion include phosphate buffer solution, Tris-HCl buffer solution, glycine buffer solution, borate buffer solution, acetate buffer solution, Good's buffer solution and the like. If necessary, a salt such as sodium chloride, a stabilizer such as bovine serum albumin, a preservative such as sodium azide, or a surfactant such as polyoxyethylene sorbitan monolaurate (Tween 20) may be added.
Examples of the labeling substance include horseradish peroxidase (POD), enzymes such as alkaline phosphatase, β-galactosidase and glucose oxidase, luminescent substances such as acridium-I, and fluorescent substances such as fluorescein. In many cases, these labeling substances are generally bound to a secondary antibody and used as an antibody / label conjugate.

As the method for measuring the labeling substance, when the labeling substance is an enzyme, the enzyme activity is measured using a substrate. When the labeling substance is a luminescent substance, the luminescence photometry is performed using a luminescence photometer. Fluorophotometric measurement method using, and in the case of a radioisotope, measurement method using a liquid scintillation counter. Among the enzyme activity measurement methods, o-phenylenediamine, hydroxyphenylpropionic acid and the like are used as substrates for measuring POD activity, and 3- (2′-spiroadamantane) -4-methoxy-4 as a substrate for measuring alkaline phosphatase activity. -(3 ″ -phosphopyroxy) phenyl-1,2-dioxetane [AMPPD], p-nitrophenyl phosphate, p-nitrophenyl-β-galactoside, 4-methylum as a substrate for the measurement of β-galactosidase activity Veriferyl-β-galactoside and the like are used. For glucose oxidase activity, POD is used to detect hydrogen peroxide produced when glucose is oxidized.
The measurement principle of the immunoassay using the magnetic particle for diagnostic agent of the present invention can be carried out by a known method. For example, a sandwich method, a competitive method, etc. are mentioned. The difference from the conventional method is that optical measurement can be performed while containing the magnetic particles for diagnostic agents of the present invention without performing solid-liquid separation between the solid phase carrier and the substrate reaction solution.

Examples Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.
In this example, the particle size measurement is performed by an optical diffractometer SOLD2000V (manufactured by Shimadzu Corporation), and the amount of functional groups on the particle surface is expressed in moles of functional groups with respect to the weight of the particles. It was determined from (manufactured by Metrohm). Moreover, the color of the particle dispersion was determined by the following method. 10 μl of a 10% by weight dispersion of particles is placed on a glass slide to form a particle dispersion drop having a diameter of 1 to 2 mm, air-dried at room temperature for 10 minutes, and then the color difference at three locations in the circle including the center of the drop is displayed. After measuring twice with a meter (Minolta color difference meter, CR241) and confirming that the CV value of these measured values was within 10%, the average value was taken as the color value of the particles. Saturation magnetization and residual magnetization were measured with a vibrating sample magnetometer (VSM). The magnetic separation property is shown by the time when the magnetic particles are separated 100% (100% particle separation time). Specifically, 3 ml of 0.1% particle dispersion was placed in a 1 cm square glass absorbance measurement cell, a 3500 gauss neodymium magnet was placed on the side of the cell, and the 100% particle separation time was determined by the change in absorbance over time. . The protein binding amount (antibody binding amount) immobilized by covalent bonding was quantified using a BCA reagent (Pierce) according to the reagent protocol.

Example 1 Production of Magnetic Core Particles 1 and 2 Crosslinked polymethylmethacrylate (PMMA) particles having the particle sizes shown in Table 1 were synthesized to obtain dry particles. 10 g each of the dried particles and ferrite superparamagnetic fine particles (average particle size: 0.01 μm) obtained by the solvent precipitation method of the oil-based magnetic fluid “FV55” (manufactured by Matsumoto Yushi Co., Ltd.) This mixture was treated for 4 minutes at a peripheral speed of 100 m / sec (16200 rpm) of a blade (stirring blade) using a hybridization system NHS-0 type (manufactured by Nara Machinery Co., Ltd.). By this treatment, magnetic core particles 1 and 2 in which the surfaces of the dry particles (PMMA particles) were coated with a magnetic material were obtained. The obtained magnetic core particles 1 and 2 were each dispersed with a 0.1% SDS soap solution, and the particle diameter was measured. Table 1 shows the relationship between the particle diameter of the PMMA particles used in this example and the obtained magnetic core particles.

[Example 2] Production of light-colored magnetic particles 1 and 2 20 g of the magnetic core particle 1 or 2 obtained in Example 1 above was 0.1% by weight of the nonionic emulsifier "Emulgen 150" (manufactured by Kao Corporation). 1 g of a 5 wt% titanium sulfate (IV) aqueous solution was added to 400 g of the aqueous solution using an ultrasonic disperser and stirred for 4 hours, and then the supernatant was replaced with 400 g of ion-exchanged water. In a separate container, 20 g of silver nitrate is dissolved in 500 g of ion-exchanged water, 500 g of 2% aqueous sodium hydroxide and 20 g of 25% aqueous ammonia are added thereto, and the above-described dispersion of magnetic core particles is added. Stir. Subsequently, 500 g of a 10% glucose aqueous solution was added and stirring was continued for 1 hour to precipitate silver on the magnetic core particles. The particles were washed three times with ion exchange water to obtain light-colored magnetic particles 1 and 2. Table 2 shows the results of measuring the particle diameters of the obtained light-colored magnetic particles 1 and 2 respectively.

[Example 3] Preparation of diagnostic magnetic particles 1 and 2 30 g (solid content) of the light-colored magnetic particles 1 and 2 obtained in Example 2 above, and a nonionic emulsifier “Emulgen 150” (Kao (as a dispersing agent) 900 g of 0.5% by weight aqueous solution manufactured by Kogyo Co., Ltd. was charged into a 1 L separable flask and sufficiently dispersed. To this was added 3 g of styrene as a monomer, 0.9 g of methacrylic acid, and 0.6 g of t-butylperoxy-2-ethylhexanate (manufactured by NOF Corporation; Perbutyl O ™) as a polymerization initiator, and N ₂ The reaction was carried out by stirring for 8 hours at 80 ° C. at a rotation speed of 200 rpm using a squid type stirring blade under a gas stream. Through the above steps, magnetic particles 1 and 2 for diagnostic agents of this example having a carboxyl group on the surface were obtained.

[Example 4] Preparation of magnetic particles 3 for diagnostic agents 30 g (solid content) of the light-colored magnetic particles 1 obtained in Example 1 above, and a nonionic emulsifier "Emulgen 150" (manufactured by Kao Corporation) as a dispersant. Was added to a 1 L separable flask and sufficiently dispersed. To this was added 3 g of styrene as a monomer, 2.0 g of glycidyl methacrylate, 0.08 g of divinylbenzene, and 0.12 g of 2,2′-azobis (2-aminopropane) dihydrochloride as a polymerization initiator, and glycidyl methacrylate after 2 hours. 0.3 g was added, and the reaction was carried out by stirring at 80 ° C. for 8 hours at a rotation speed of 200 rpm using a squid stirring blade under a N ₂ gas stream. Through the above steps, magnetic particles 3 for diagnostic agents of this Example having a particle diameter of 4.8 μm and having an epoxy functional group on the surface were obtained.

[Example 5] Preparation of magnetic particles 4 for diagnostic agents 1 g of the light-colored magnetic particles 1 obtained in Example 1 above was washed 3 times with 5 mM MES buffer, and then 5 mM MES buffer in which 0.5 g of lysine was dissolved. 1 ml of liquid, 10 mg / ml EDC (1-ethyl 3-dimethylaminopropylcarbodiimide hydrochloride, manufactured by Doujin Chemical Co., Ltd.) 0.5 ml was added, and the mixture was rotated and stirred at room temperature for 12 hours. Subsequently, the plate was washed three times with PBS, dispersed in 10 nM phosphate buffer so that the particle solid content was 10% by weight, and magnetic particles 4 for diagnostic agents of this example having amino groups on the surface were obtained. Similarly to the antibody binding measurement, the amount of lysine introduced was measured by an assay using a BCA reagent.

[Comparative example]
A magnetic composite particle 1 of a comparative example was prepared by the method of Example 1 and then Example 3 using PMMA particles having a particle diameter of 3.7 μm. Since this magnetic composite particle 1 is obtained by omitting the lightening step corresponding to Example 2, it is brown. The particle diameter of the magnetic composite particle 1 was 4.4 μm.

Example 6 Characteristic Evaluation of Magnetic Particles for Diagnostic Agents 0 so that the solid content of the magnetic particles for diagnostic agents prepared in Examples 1-4 and the magnetic composite particles 1 obtained in the above Comparative Examples is 5%. The above particles are dispersed in a 0.01% SDS solution, transferred to a glass container having a length of 20 cm, a width of 15 cm, and a height of 5 cm. The wide surface of the container is brought into contact with a 3500 gauss magnet, and magnetic separation is performed twice. From this, 100% particle separation time was measured. The measurement results are shown in Table 4. Further, the L value, the amount of surface functional groups (μmol / g), and the ratio (%) of the residual magnetization to the saturation magnetization of the magnetic particles for diagnostic agents obtained in Example 1-5 were measured by the methods described above. The measurement results are also shown in Table 4.

[Example 7] Preparation of AFP antibody-immobilized particles 100 μl of 10% aqueous dispersion of diagnostic magnetic particles 1 of the present invention prepared in Examples 1 to 3 above was dispensed into a 1 ml microtube, and 1 ml of 5 mM MES (pH 6). And washed three times by magnetic separation. 50 μl of 10 mg / ml EDC (1-ethyl 3-dimethylaminopropylcarbodiimide hydrochloride, manufactured by Doujin Chemical Co., Ltd.) solution was added, and the mixture was rotationally stirred at room temperature for 2 hours, and then the supernatant was removed by magnetic separation. Subsequently, 20 μl of monoclonal antibody IgG1 (Biotest Corp., clone NB-013) collected from 100 μg / ml AFP BALB / C mouse transplanted ascites was added, and the volume was made up to 1 ml with 5 mM MES (pH 6). After re-dispersing the particle dispersion, it was reacted at room temperature for 5 hours with rotary stirring. Then 0.1 ml of 1M ethanolamine was added and stirred overnight at room temperature. This reaction solution was washed twice with PBS buffer, and finally the magnetic particles were dispersed in 1 ml of 0.1% casein / PBS solution. Subsequently, it adjusted with PBS so that particle solid content might be 1%. As a result, a suspension of diagnostic drug magnetic particles (immobilized diagnostic drug magnetic particles) 1 immobilized with an anti-AFP antibody was obtained. This suspension was stored in a refrigerator until use. Further, the antibody binding amount of the immobilized diagnostic magnetic drug magnetic particle 1 prepared by the above method was measured. The obtained antibody binding amounts are shown in Table 5.
Moreover, using the magnetic particles 2 for diagnostic agents prepared in Examples 1 to 3 and the magnetic composite particles 1 prepared in Comparative Examples, antibody sensitization was performed in the same manner as described above, and immobilized with an anti-AFP antibody. The magnetic particles for diagnostic agents (immobilized magnetic particles for diagnostic agents) 2 and the magnetic composite particles (immobilized magnetic composite particles) 1 immobilized with an anti-AFP antibody as a comparative example were obtained.
Furthermore, 100 μl of an aqueous dispersion of 10% by weight particles of magnetic particles 3 for diagnostic agents prepared in Example 4 above was washed twice with 0.1 M PBS (pH 7), so that the antibody solution and 0.1M phosphate buffer (pH 7) was added and allowed to react overnight at room temperature. For other diagnostic parts immobilized with anti-AFP antibody, the operation part other than the above, the antibody compounding ratio, etc. are handled in the same manner as in the case of particles (magnetic particles 1 and 2 for diagnostic drugs) containing carboxyl groups on the surface. Magnetic particles (immobilized diagnostic drug magnetic particles) 3 were obtained.

Example 8 AFP Immunoassay Characteristic evaluation of the immobilized diagnostic magnetic particle 1 obtained in Example 7 was performed by a two-step immunoassay chemiluminescence method. 10 μl of a 1% by weight dispersion of magnetic particles for immobilized diagnostic drug 1 obtained by binding to an AFP monoclonal antibody in Example 7 above, and 50 μl of a serum sample having an AFP Ag (antigen) concentration of 0 to 1000 ng / ml In addition to a plate well having a volume of 300 μl, 40 μl of PBS solution was further added, and the mixture was gently shaken and allowed to stand at room temperature for 5 minutes. Subsequently, the magnetic particles were brought to the well wall with a magnet to remove the solution. After washing once with 100 μl PBS buffer, 100 μl of PBS solution containing mouse monoclonal antibody (Biotest, clone NB-017) conjugated with 10 ng / ml bovine small intestine alkaline phosphatase was added, and after shaking dispersion, 5 μm at room temperature was added. Let stand for a minute. This reaction solution was solid-liquid separated with a magnet, washed twice with a PBS / 0.1% casein-containing solution, 100 μl CDPstar substrate solution (manufactured by Tropix) was added to the particle pellet, and the mixture was stirred and allowed to stand at room temperature for 3 minutes. Then, the chemiluminescence value was measured. The measurement results are shown in Table 6. Further, the immobilized diagnostic magnetic drug particles 2 and 3 obtained in Example 7 and the immobilized magnetic composite particle 1 of the comparative example were also sensitized with an antibody in the same manner as described above, and an AFP immunoassay was performed. . The results are also shown in Table 6.
As a control example, in the above step, the immobilized diagnostic diagnostic magnetic particles and the serum sample are added to the plate well, and further PBS solution is added and stirred and shaken at room temperature for 3 minutes. Table 7 shows an example in which the chemiluminescence value was measured by immediately separating with a magnet and dispensing the supernatant.

Thus, in the enzyme immunoassay system using the immobilized diagnostic magnetic drug particles of Example 7, the result of measuring the magnetic particles as they were without B / F separation (see Table 6) It was found that there was almost no difference from the measurement result after removal (see Table 7).
On the other hand, in the case of the immobilized magnetic composite particle 1 of the comparative example, the measurement result (see Table 6) in the state containing the immobilized magnetic composite particle 1 in the whole antigen concentration range shows that the immobilized magnetic composite particle 1 It was revealed that the signal was reduced by about ½ to 比較 as compared with the result measured in the state not containing (see Table 7).

Claims (1)

A core containing magnetic particles, and a visible light scattering layer containing silver formed outside the core,
Magnetic particles for diagnostic agents having an L value in the Lab color system of 60 or more, a residual magnetization of 30% or less of saturation magnetization, and a particle size of 0.5 to 15 μm.