Magnetically responsive fluorescent polymer particles and application thereof

Description: TECHNICAL FIELD [Detailed description of the invention]

Field of invention The present invention relates to magnetically responsive fluorescent polymer particles. Background of the invention The present invention is a partial continuation of US Application No. 113294 filed October 26, 1987. In many biological techniques such as immunoassays, affinity purification, etc., it is necessary to separate the bound fraction from the free fraction. Magnetic particles have been used to promote the desired separation. Magnetic particles are made of various particulate magnetic materials using various steps and have various characteristics. For example, US Pat. No. 582622 of Ikeda et al. Discloses magnetic particles composed of gelatin, water-soluble polysaccharides, sodium phosphate and ferromagnetic substances, and US Pat. Nos. 4628037 and 4554088 are polymeric. Magnetic particles consisting of a magnetic metal oxide core surrounded by a silane coating are disclosed, and US Pat. No. 4,452,773 discloses a ferromagnetic polysaccharide (Fe) coated with a water-soluble polysaccharide or a derivative thereof having a functional group.₃O₄) Core, discrete colloid-sized particles are disclosed, and Mansfield US Pat. No. 4,297,337 discloses magnetic glass or crystal-containing materials as particulate carriers. Outline of the invention The present invention is a novel method for producing magnetically responsive fluorescent polymer particles (hereinafter referred to as magnetic fluorescent particles) from fluorescent polymer particles having an average size of about 1 to 100 μm in diameter regardless of shape and composition. Provide a method. The photomagnetic particles of the present invention first produce a magnetically responsive metal oxide (hereinafter referred to as metal oxide) having an average size of about 1 μm or less, and then the photopolymeric core particles are formed into a polymer layer containing the metal oxide. It can be prepared by coating with. The surface of these fluorescent particles can be further coated with another layer of polymer or polymer having functional groups to give the desired surface properties. The spectral properties of these fluorescent magnetic particles can be altered by using core particles incorporating various fluorescent dyes, which are a single fluorescent dye or a combination of several fluorescent dyes. Separately, the fluorescent magnetic particles of the present invention are a single fluorescent dye or number that is soluble in the monomer and capable of withstanding polymerization conditions in the presence of non-fluorescent polymer core particles, metal oxides and monomers. It can be prepared by incorporating various fluorescent dyes that are a combination of different dyes. The fluorescent particles produced according to the present invention are monodisperse in size, have a rough surface, and have a magnetic metal oxide content of 5% to 50%, preferably 10% to 25%. The fluorescence intensity of these fluorescent magnetic particles can be determined by varying the magnetic metal oxide content to alter the shading of the metal oxide and / or by varying the amount of fluorescence dye incorporated into the fluorescence polymer core particles. Can be adjusted by Particles with these characteristics have been found to be useful in immunoassays and a wide variety of biomedical applications. These photomagnetic particles can be used for passive or covalent binding of biological materials such as antigens, antibodies, enzymes or DNA / RNA, as well as various types of immunoassays, DNA / RNA hybrids. It can be used as a solid phase for damping assay, affinity purification, cell isolation, feeding and other biomedical applications. These fluorescent particles with or without binding of biological material are used to ensure that the correct number of particles are released into the wells and to check for particle loss during the assay. It can be incorporated in different proportions to non-magnetic particles for different assays to serve as a marker for. Purpose and benefits An object of the present invention is as follows. To develop a method for easily producing magnetically responsive fluorescent polymer particles from readily available polymer particles. To develop a method for producing magnetically responsive fluorescent polymer particles with moderate sedimentation and fast magnetic separation. To develop a method for producing magnetically responsive fluorescent polymer particles having various surface charges and functional groups for binding biological materials by passive adsorption or covalent bonding. To develop medical, biological, diagnostic and industrial applications using these magnetically responsive fluorescent polymer particles. Advantages of the present invention include: A wide range of species with a size of about 1-100 μm can be varied such fluorescent polymeric core particles easily magnetically responsive particles. The content of the metal oxide can be changed according to the application. The surface can be derivatized for covalent bonds to introduce various functional groups. Various monomers can be used in the final coating to impart different surface properties to the resulting polymer. Any of the crosslinked or uncrosslinked magnetically responsive fluorescent polymer particles can be produced. Monodisperse magnetically responsive fluorescent polymer particles can be produced. Detailed description of the invention The fluorescent magnetic particles of the present invention can be prepared by first producing a metal oxide having an average size of about 1 μm or less. Metal oxides are produced by heating and precipitating a mixture of divalent or trivalent metal salts, preferably ferrous and ferric sulfates or chlorides and sodium hydroxide solutions. In order to obtain the desired size and magnetic properties of the metal oxide, the molar ratio of the divalent metal salt to the trivalent metal salt can be varied in the range of 0.5 to 2.0, preferably 0.5 to 1.0. It has been observed that the molar ratio of divalent metal salts to trivalent metal salts affects the size of metal oxides. That is, the smaller the molar ratio of the divalent metal salt to the trivalent metal salt, the smaller the size of the metal oxide. The molar ratio of the divalent metal salt to the trivalent metal salt also affects the color of the resulting magnetic particles. That is, the smaller the molar ratio, the brighter the brownish color of the obtained magnetic particles. Preferably, the metal oxide is superparamagnetic or paramagnetic, but ferromagnetic metal oxides can also be used. However, in this case, centrifugation is used instead of magnetic separation during cleaning. Other divalent transition metal salts, such as manganese, magnesium, cobalt, nickel, zinc and copper salts, may replace the ferrous salt. After the metal oxide has settled, wash it several times with a 250 xg centrifuge until the pH of the supernatant is neutral. The metal oxide is resuspended in deionized water and mechanically stirred at high speed to crush the aggregates of metal oxide crystals. Furthermore, even if the metal oxide is further settled at 250 × g, all the metal oxides do not become pellets. The supernatant containing small size metal oxide crystals is collected and the pellet is resuspended in deionized water. This operation is repeated at least 3 times or until most of the metal oxides are no longer pelletized at 250 xg. The size of the metal oxide obtained by this method is usually less than 2.0 μm. A slow centrifuge of 100 xg to remove large crystals results in a size of less than 0.8 μm. A metal oxide with an average size of 1.0 μm or less is mixed with the monomer and coated on photopolymer core particles, preferably polystyrene particles, with a size of 1-100 μm in the presence of an initiator. The addition of a small amount of emulsifier will help prevent the particles from sticking. The fluorescent particles are then coated with a protective layer of polymer, preferably polystyrene, to prevent the metal oxides from falling off. If a photomagnetic particle having a functional group is desired, it is better than a polymer having a functional group in order to impart a functional group such as a carboxyl group, an amino group or a hydroxyl group for covalently bonding a biological material. The magnetic particles can be further coated with another layer. Fluorescent magnetic particles prepared according to the present invention can be shown in FIG. Where 1 represents a fluorescent core particle, 2 represents a metal oxide / polymer coating, 3 represents a protective polymer coating and 4 represents a polymer coating having a functional group. FIG. 2 shows a transmission electron micrograph of 0.08 to 0.1 μm sections of magnetic particles prepared according to the present invention. FIG. 3 shows a scanning electron micrograph of 6.8 μm magnetic particles prepared according to the present invention. Figure 3a is 1000x magnification and Figure 3b is 5000x magnification. The photopolymeric core particles useful in the present invention can be obtained as a dispersion system of small particles and absorb a monomer, thereby coating a mixture of a metal oxide and the monomer on the surface of the core particles. It may be made of any polymer as long as it can produce. The core particles may be of any size and shape, but are preferably 1 to 100 μm in size and have a spherical shape. When using monodisperse core particles, the resulting magnetic particles will also be monodisperse in size. Core particles can be obtained by emulsion polymerization, suspension polymerization or other polymerization means with or without a cross-linking agent such as divinylbenzene. Monomers that can be used to prepare core particles include, for example, styrene, methyl methacrylate, vinyltoluene and the like. A mixture of monomers can also be used. Fluorescent core particles can be obtained by incorporating a fluorescent dye into the core particles using various techniques known to those skilled in the art. The monomer used for coating or protective coating with magnetic metal oxide may or may not be of the same type as the fluorescent core particles. The weight ratio of the monomer used for coating with the metal oxide to the fluorescent core particles can be 0.1 to 12, preferably 0.2 to 6, depending on the desired thickness of the metal oxide / polymer layer. When a metal oxide prepared from a mixture of ferrous salt and ferric salt is used for coating, it is preferable to use a monomer at a weight ratio of about 0.1 to 0.5 with respect to the brilliant core particles. However, when a metal oxide prepared from a mixture of a manganese (divalent) salt and a ferric salt is used for coating, the monomer weight ratio to the core particles can be 0.1-12. As a result, when seeking crosslinked photomagnetic particles that are inactive in ordinary organic solvents, 2% or more of the metal oxide prepared from a mixture of manganese (divalent) salt and ferric salt is added. It contains a cross-linking agent in a weight ratio of 10%, preferably 8% to 10%, and a monomer weight ratio to core particles of 3 to 12, It is preferably 4 to 6, and is preferably used together with a monomer having a monomer weight ratio of 0.1 to 10 with respect to the fluorescent dye. If a lower monomer weight ratio to the core particles (ie 0.1-0.5) is used during the metal oxide / polymer coating, the polymer coating is used to adhere the metal oxide more tightly to the surface of the photomagnetic particles. It is preferable to protectively coat the obtained photomagnetic particles with a protective layer made of. However, when using a high monomer ratio to core particles (ie 3-12), coating with a protective polymer is not necessary. The polymerization temperature may be 55 ° C to 90 ° C, preferably 55 ° C to 65 ° C. The polymerization initiator may be a water-soluble agent such as potassium persulfate or a water-insoluble agent such as benzoyl peroxide. Other polymerization initiation means such as irradiation and ionization can also be used. Surprisingly, it was found that when a metal oxide prepared from a mixture of a manganese (divalent) salt and a ferric salt was used for the coating, fluorescent magnetic particles could be produced without using any emulsifier. It was issued. However, when a metal oxide prepared from a mixture of ferrous salt and ferric salt is used for coating, such as sodium dodecyl sulfate, Aerosol 22, Tween 20 or Nonidet P-40 (NP 40). A small amount of the emulsifier has been found to be useful in preventing excessive agglomeration of particles between metal oxide / polymer coatings. Other emulsifiers with the same ability can also be used. By using different amounts of metal oxide during the metal oxide / polymer coating, the content of magnetic metal oxide is 5% to 50%, It can be preferably varied in the range of 10% to 25%. Metal oxide / polymer multicoating can also be performed to increase the content of the metal oxide. Other components usually used for polymerization may be added as long as the magnetic particles having the desired properties are obtained. The components for the metal oxide / polymer coating may be added all at once at the beginning of the metal oxide / polymer coating step, or may be added in stages. When using a metal oxide made from a mixture of ferrous salt and ferric salt, it is preferable to add the components step by step. The components can be mixed under vacuum or under an inert gas such as argon by mechanical stirring, shaking or other stirring means. The functional group is a photomagnetic particle by using a mixture of a monomer and a monomer having a functional group during the metal oxide / polymer coating, or by finally recoating the magnetic particle with a thin layer of the monomer having a functional group. Can be incorporated on the surface of. The functional group-bearing monomer used can be selected from one or a mixture of the following: 2-hydroxyethyl methacrylate, 2-aminoethyl methacrylate, trimethylammonium methylmethosulfate methacrylate, dimethylaminoethyl methacrylate, methacrylic. Acid, undecylene acid, methylpropensulfonic acid, undecylene alcohol, oleylamine, glycidyl methacrylate, achlorein, glutaaldehyde, etc. The magnetic fluorescent particles can also be recoated with a layer of polymer different from that used for the metal oxide / polymer coating or protective coating to give the surface properties of that polymer. Glutaraldehyde, etc. The magnetic fluorescent particles can also be recoated with a layer of polymer different from that used for the metal oxide / polymer coating or protective coating to give the surface properties of that polymer. Glutaraldehyde, etc. The magnetic fluorescent particles can also be recoated with a layer of polymer different from that used for the metal oxide / polymer coating or protective coating to give the surface properties of that polymer. Utilization of fluorescent magnetic particles The use of various photomagnetic particles as a solid phase for various applications such as 1982 immunoassay, radioimmunoassay, enzyme immunoassay, cell isolation, enzyme immobilization and affinity purification is illustrated in the following papers. Considered in the literature: Hirschbein et al, Chemical Technology, March 1982, 172-179 (1982); Pourfarzaneh, The Ligand Quarterly, 5 (1): 41-47 (1982); Halling and Dunnill, Enzyme Microbe Technology , 2: 2-10 (1980); Mosbach and Anderson, Nature, 270: 259-261 (1977); Guesdon et al, J, Allergy Clinical immunology, 61 (1), 23-27 (1978). Also for use, for enzyme immobilization, 1982, 198210 and 4343901 for enzyme immobilization, 1982, 3970518, 4230685, and 42672343 for cell isolation, and 4554088, 4628037, and 4628037 for immunoassay. Each is disclosed in No. 3933997. Some magnetic particles are useful in some applications but not in others. For example, the magnetic particles disclosed in US Pat. Nos. 4554088 and 4638037 usually consist of a superparamagnetic metal oxide core surrounded by a coating of polymeric silane, due to its large surface area and slow precipitation rate. It is useful for immunoassays and affinity purification, but not for cell separation such as bone marrow lavage. Due to the small size of the magnetic particles disclosed in these two patents, it is very difficult to effectively remove all the magnetic particles from the cell suspension. Moreover, smaller magnetic particles have much higher non-specific binding to normal cells. In the use of magnetic particles for purification of bone marrow cells, the magnetic particles are coated with an antibody such as sheep anti-mouse IgG, and the bone marrow is treated with a mixture of several monoclonal antibodies against the surface antigens of cancer cells. The magnetic particles bind only to the cancer cells and can separate the cancer cells from the normal cells by passing a strong magnetic field through them. The washed cells are then returned to the patient. By using the methods of the present invention, the size, surface area, metal oxide content and surface properties of magnetic particles can be optimized for a wide variety of biomedical applications. The magnetic particles produced by the present invention can be used as a solid phase for enzyme immunoassay, fluorescence immunoassay, radioimmunoassay, DNA / RNA hybridization assay and other diagnostic applications. Immunoassays can be performed in a variety of forms apparent to those of skill in the art, such as sandwich assays and competitive binding assays. DNA / RNA hybridization can also be performed in various forms such as solid phase hybridization or liquid phase hybridization. In the form of solid-phase hybridization, the DNA or RNA probe (catcher probe) is first immobilized on the magnetic particles. The immobilized catcher probe is used to hybridize with complementary DNA strands from the sample (sample DNA). Finally, another probe (signal probe) labeled with a fluorescent, radioactive or enzyme tracer and capable of hybridizing with another portion of the DNA sample is used for signal generation. In the liquid phase hybridization form, the catcher probe and signal probe are first hybridized with the sample DNA in the liquid phase and then immobilized on magnetic particles. Alternatively, the signal probe can be labeled with one or several biotin groups to increase the sensitivity of the assay, in which case the biotin group is bound to avidin labeled with a fluorescent, radioactive or enzyme tracer. The signal is detected by. Immunoassays and DNA / RNA hybridization assays can be used to measure a wide variety of substances such as drugs, hormones, antibodies, peptides, DNA, RNA, nucleic acids, viral antigens and carbohydrates in biological samples. it can. The magnetic particles produced by the present invention can also be used for affinity purification, cell separation, enzyme fixation and other biomedical applications. In cell separation, magnetic particles are used for the removal of unwanted cells (negative selection) by an immune or non-immune reaction or for the enrichment of desired cells (positive selection). This principle can be used to remove cancer cells from bone marrow (bone marrow lavage), purify cell populations by positive or negative selection for tissue culture, and perform various cell immunoassays. In affinity purification, magnetic particles are used to purify a wide variety of biological materials such as antibodies, antigens, enzymes, inhibitors, cofactors, single-stranded DNA, binding proteins, haptens and carbohydrates, polyacrylamide gels, It is used in place of the conventional solid phase such as Sepharose gel or other cellulose beads. In other applications similar to affinity purification, magnetic particles can be used to cross-adsorb and remove unwanted protein components from antisera or clinical samples. In enzyme immobilization, the enzyme is immobilized on the magnetic particles by various binding means so as to retain the enzyme activity and allow the immobilized enzyme to be reused. Magnetic particles carrying immobilized enzymes are glass beads commonly used in immobilized enzyme systems for producing a wide variety of materials such as carbohydrates, amino acids and proteins, controlled porous glass, silica gel and cellulose beads. It can be used in place of other solid phase such as. These fluorescent particles with or without biological material serve as markers to ensure that the correct number of particles are released into the wells and to check for particle loss during the assay. Therefore, it can be incorporated in different proportions to the non-magnetic particles for the various assays described in Examples 42 and 43. Both of these uses are streamlined by the ease of separation, fast reaction rate and large surface area common to most magnetic particles. The following examples are provided to further illustrate the versatility and advantages of the present invention, the details of which should not be construed in a restrictive manner. It is clear that the implementation of various equivalents, modifications and modifications can be made without departing from the spirit and scope of the invention, and specific examples of such equalities are intended to be included in the invention. Because there is. General method of preparing metal oxides Example 1 0.361 mol ferrous sulfate and 0.369 mol ferric sulfate (Fe) in 400 ml deionized water in a three-necked round-bottom flask equipped with a mechanical stirrer, condenser, thermometer, dropping funnel and heating mantle.⁺⁺/ Fe⁺⁺⁺A mixture of ratio = 1) was added. The mixture was heated to 85-90 ° C with stirring and 850 ml of 6N sodium hydroxide was added dropwise over 90 minutes. The mixture was stirred at 85-90 ° C for an additional hour and cooled to room temperature. The metal oxide precipitate was centrifuged at 250 xg for 10 minutes. The clear supernatant was tilted and discarded and the pellet was resuspended in 90 ml of deionized water using a mechanical stirrer. This washing step was repeated 6 times or until the pH of the supernatant was almost neutral. The supernatant was tilted and discarded and resuspended in 200 ml of deinsulated water. Further centrifugation at 250 xg does not result in all of the metal oxide precipitates becoming pellets. The supernatant containing the small size metal oxide crystals was collected and the pellet was resuspended in 200 ml of deionized water. This step was repeated at least 3 times or until most of the metal oxides no longer produced pellets at 250 xg. The metal oxides obtained by this method usually have a size of less than 2.0 μm. The metal oxide suspensions were combined and centrifuged at 100 xg for 10 minutes. The supernatant was recovered to give 800 ml of an 8.6% w / v suspension of magnetic metal oxide with a size less than 0.8 μm. Example 2 0.235 mol of ferrous sulfate in 400 ml of deionized water, 0.297 mol of ferric sulfate ((Fe)⁺⁺/ Fe⁺⁺⁺The same procedure as described in Example 1 was followed, except that a ratio of 0.79) and 2000 ml of a 2.8% w / v suspension of magnetic metal oxide were obtained using 480 ml of 6N sodium hydroxide. Example 3 0.178 mol of ferrous sulfate in 400 ml of deionized water, 0.298 mol of ferric sulfate ((Fe)⁺⁺/ Fe⁺⁺⁺The same procedure as described in Example 1 was followed, except that a ratio = 0.59) and 1500 ml of a 2.98% w / v suspension of magnetic metal oxide was obtained using 520 ml of 6N sodium hydroxide. Example 4 0.15 mol ferrous sulfate in 400 ml deionized water, 0.276 mol ferric sulfate ((Fe)⁺⁺/ Fe⁺⁺⁺The same procedure as described in Example 1 was followed, except that a ratio = 0.54) and 700 ml of a 6.88% w / v suspension of magnetic metal oxide was obtained using 520 ml of 6N sodium hydroxide. Example 5 0.116 mol of manganese sulfate in 255 ml of deionized water, 0.146 mol of ferric sulfate ((Mn)⁺⁺/ Fe⁺⁺⁺The same procedure as described in Example 1 was followed, except that 1700 ml of a 1.8% w / v suspension of magnetic metal oxide was obtained using a ratio = 0.79) and 240 ml of 6N sodium hydroxide. Preparation of magnetic particles Example 6 A mixture of 600 ml of deionized water, 6 ml of styrene and 80 ml of 8.6% w / v magnetic metal oxide prepared as described in Example 1 was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 1 hour. To the mixture was added 12 g potassium persulfate and 850 ml of 5% w / v 4.0 μm polystyrene particles. The bottle was resealed, aspirated and rotated for 1 hour, and 50 ml of 2% sodium dodecyl sulfate was added. After an additional 5 hours, 6 ml of styrene and 10 g of potassium persulfate were added to the mixture. The mixture was spun for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 1.6 liters, and a 2.5% w / v suspension having a magnetic metal oxide content of about 11% and an average size of 4.3 μm was obtained. Example 7 1.6 liters of 2.5% w / v magnetic particles prepared according to Example 6 were mixed with 1 g of sodium dodecyl sulfate, 10 g of potassium persulfate, and 0.98 ml of undecylenic acid and 0.02 ml of divinylbenzene in 4 ml of methanol. It was carboxylated by adding a solution containing. The mixture was added to a sealed bottle, aspirated and rotated at about 60 rpm in an oven at 55 ° C for 5 hours. The obtained carboxyl magnetic particles were magnetically separated and washed with deionized water several times until the supernatant became transparent. The carboxyl magnetic particles were resuspended in deionized water to make 680 ml, and a 5.8% w / v suspension having a magnetic metal oxide content of about 11% and an average size of 4.3 μm was obtained. Example 8 600 ml of deionized water, 6 ml of styrene and 80 ml of 8.6% w / v magnetic metal oxide prepared according to the description of Example 1 were added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 1 hour. To the mixture was added 12 g potassium persulfate and 850 ml of 4.78% w / v 6.1 μm polystyrene particles. The bottle was resealed, aspirated and rotated for 5 hours, and 6 ml styrene and 10 g potassium persulfate were added. The mixture was rotated for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in 1.5 l of deionized water and contained 1 g of sodium dodecyl sulfate, 10 g of potassium persulfate, and 0.98 ml of undecylenic acid and 0.02 ml of divinylbenzene in 4 ml of methanol. It was carboxylated by adding the solution. The mixture was added to a sealed bottle, aspirated and rotated at about 60 rpm in an oven at 55 ° C for 5 hours. The obtained carboxyl magnetic particles were magnetically separated and washed with deionized water several times until the supernatant became transparent. The carboxyl magnetic particles were resuspended in deionized water to make 800 ml, and a 4.3% suspension having a magnetic metal oxide content of about 11.6% and an average size of 6.8 μm was obtained. Example 9 A mixture containing 600 ml of deionized water, 6 ml of styrene and 60 ml of 8.6% w / v magnetic metal oxide prepared according to Example 1 was added to a three-necked round bottom flask at 67 ° C. under an argon atmosphere. Stirred for 1 hour. To the mixture was added 12 g potassium persulfate and 470 ml of 5% w / v 2.7 μm polystyrene particles. The mixture was stirred at 67 ° C. for 1 hour and 30 ml of 2% sodium dodecyl sulfate was added. After stirring at 67 ° C. for another 5 hours under an argon atmosphere, 6 ml of styrene and 6 g of potassium persulfate were added to the mixture. The mixture was stirred at 67 ° C. under an argon atmosphere for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 90 ml, and in 0.6 g of sodium dodecyl sulfate, 10 g of potassium persulfate, and 2.4 ml of methanol, 0.598 ml of undecylenic acid and 0.012 ml of divinylbenzene were added. It was carboxylated by adding a solution containing. The mixture was added to a sealed bottle, aspirated and rotated at about 60 rpm in an oven at 55 ° C for 5 hours. The resulting carboxyl magnetic particles were magnetically separated and washed several times with deionized water until the supernatant became clear. The carboxyl magnetic particles were resuspended to 500 ml to give a 6.5% w / v suspension with a magnetic metal oxide content of about 14% and an average size of 4.0 μm. Example 10 A mixture containing 600 ml of deionized water, 6 ml of styrene and 60 ml of 8.6% w / v magnetic metal oxide prepared as described in Example 1 was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 1 hour. To the mixture was added 12 g potassium persulfate and 470 ml of 5% w / v 2.7 μm polystyrene particles. The bottle was resealed, aspirated and rotated for 1 hour and 30 ml of 2% sodium dodecyl sulfate was added. After an additional 5 hours, 6 ml of styrene and 10 g of potassium persulfate were added to the mixture. The mixture was spun for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were suspended in deionized water to make 500 ml, and a suspension of 6.8% w / v having a magnetic metal oxide content of about 14% and an average size of 4.0 μm was obtained. Example 11 A mixture containing 180 ml of deionized water, 2 ml of styrene and 20 ml of 8.6% w / v magnetic metal oxide prepared as described in Example 1 was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 1 hour. To the mixture was added 4 g of potassium persulfate and 160 ml of 6.8% w / v magnetic particles (3.0 μm, metal oxide content 14%) prepared as described in Example 10. The bottle was resealed, aspirated and rotated for 1 hour and 10 ml of 2% sodium dodecyl sulfate was added. After an additional 5 hours, 2 ml of styrene and 2 g of potassium persulfate were added to the mixture. The mixture was rotated for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were suspended in deionized water to make 160 ml, and a suspension of 7.78% w / v having a metal oxide content of about 19% and an average size of 4.2 μm was obtained. Example 12 A mixture containing 90 ml of deionized water, 1 ml of styrene and 10 ml of 8.6% w / v magnetic metal oxide prepared as described in Example 1 was added into a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 1 hour. To the mixture was added 1 g of potassium persulfate and 80 ml of 7.78% w / v magnetic particles (3.2 μm, metal oxide content 19%) prepared as described in Example 11. The bottle was resealed, aspirated and rotated for 4 hours, and 5 ml of 2% sodium dodecyl sulfate was added. After an additional 5 hours, 1 ml of styrene and 1 g of potassium persulfate were added to the mixture. The mixture was rotated for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 160 ml, and a 4.5% suspension having a metal oxide content of about 23% and an average size of 4.5 μm was obtained. Example 13 400 ml deionized water, 1.92 ml styrene, 0.08 ml divinylbenzene, 4 g potassium persulfate, 20 g 200-400 mesh 4% divinylbenzene crosslinked polystyrene beads and 8.6% w / v prepared according to Example 1. A mixture containing 10 ml of the magnetic metal oxide of benzene was added into a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was precipitated and the supernatant was tilted and discarded. The obtained magnetic beads were resuspended in 200 ml of deionized water and precipitated again. This process was repeated several times until the supernatant became transparent. The obtained magnetic beads were resuspended in 200 ml of deionized water, and 0.1 g of sodium dodecyl sulfate, 2.0 g of potassium persulfate, 0.48 ml of styrene and 0.02 ml of divinylbenzene were added. The bottle was resealed, aspirated and rotated in an oven at 55 ° C. at about 60 rpm for 1 hour, and a solution containing 0.098 ml undecylene acid and 0.002 ml divinylbenzene was added to 0.4 ml methanol. The mixture was washed by spinning for an additional 4 hours and sedimenting by weight as described above. Water was removed by filtration and the carboxyl magnetic beads were dried to obtain 20 g of carboxyl magnetic beads of 200 to 400 mesh. Example 14 100 ml of deionized water, 0.5 ml of styrene, 2 g of potassium persulfate, 75 ml of 5% w / v 4.0 μm polystyrene particles, and 10 ml of 6.88% w / v magnetic metal oxide prepared as described in Example 4. The containing mixture was added in a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 150 ml, and a 2.5% w / v suspension having a metal oxide content of about 14% and an average size of 4.3 μm was obtained. Example 15 A suspension of 2.5% w / v with a metal oxide content of about 18% and an average size of 4.3 μm using 20 ml of 6.88% w / v magnetic metal oxide prepared as described in Example 4. The same procedure as described in Example 14 was followed except that 160 ml was obtained. Example 16 A mixture containing 2000 ml of deionized water, 13 ml of styrene and 550 ml of 2.98% w / v magnetic metal oxide prepared as described in Example 3 was added into a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 1 hour. To the mixture was added 20 g potassium persulfate and 950 ml of 10% w / v 3.0 μm polystyrene particles. The bottle was resealed, aspirated and rotated at 60 rpm for 1 hour and 60 ml of 2% sodium dodecyl sulfate was added. After an additional 5 hours, 8 ml of styrene and 10 g of potassium persulfate were added to the mixture. The mixture was rotated for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 3000 ml, and a suspension of 3.38% w / v having a magnetic metal oxide content of about 12% and an average size of 3.2 μm was obtained. Example 17 Magnetic particles prepared according to the description of Example 16 (3.2 μm, 3.38% w / v, metal oxide content 12%) 150 ml, 2 ml 1% NP40, 0.5 ml methyl methacrylate or styrene, 1 g potassium persulfate and , A mixture containing trimethylammonium ethyl methacrylate (40% aqueous solution) of methylsulfate, which is a monomer having a functional group, was added to a sealed bottle. The bottle was rotated at about 60 rpm in an oven at 55 ° C for 4 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 200 ml, and a suspension of 2.5% w / v of magnetic particles having a trimethylammonium functional group on the surface was obtained. Example 18 It is described in Example 17, except that 1 ml of 2-aminoethyl methacrylate, which is a monomer having a functional group, was used to obtain 200 ml of a suspension of 2.5% w / v of magnetic particles having an amino group on the surface. I followed the same procedure as before. Example 19 It is described in Example 17, except that 1 ml of 2-hydroxyethyl methacrylate, which is a monomer having a functional group, was used to obtain 200 ml of a 2.5% w / v suspension of magnetic particles having a hydroxyl group on the surface. I followed the same procedure. Example 20 The same procedure as described in Example 17 was followed, except that 1 ml of 1-vinyl-2-pyrrolidinone was used as the monomer to obtain 200 ml of a 2.5% w / v suspension of magnetic particles with polyvinylpyrrolidinone on the surface. .. Example 21 The same procedure as described in Example 17 except that 1 g of methylpropene sulfonic acid, which is a monomer having a functional group, was used to obtain 200 ml of a 2.5% w / v suspension of magnetic particles having a sulfonic acid group on the surface. Followed. Example 22 Same as in Example 17 except that 1 ml of dimethylaminoethyl methacrylate, which is a monomer having a functional group, was used to obtain 200 ml of a 2.5% w / v suspension of magnetic particles having a dimethylamino group on the surface. I followed the procedure. Example 23 7.0% w / v 2.11 μm polystyrene particles 20 ml, 1.8% w / v metal oxide 100 ml prepared according to Example 5, 50 ml deionized water and 0.15 g benzoyl peroxide in 7.5 ml styrene The mixture containing the solution containing the above was added to a sealed bottle. The bottle was aspirated and rotated at 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained magnetic particles were resuspended in deionized water to make 200 ml, and a suspension of 5.0% w / v having a metal oxide content of about 16.8% and an average size of 3.6 μm was obtained. Example 24 7.0% w / v 2.11 μm polystyrene particles 20 ml, 1.8% w / v metal oxide 100 ml prepared according to Example 5, 50 ml deionized water and 0.15 g benzoyl peroxide in 6.75 ml styrene. A mixture containing a solution containing 0.75 ml of divinylbenzene and 0.75 ml of divinylbenzene was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained crosslinked magnetic particles were resuspended in deionized water to make 200 ml, and a suspension of 5.0% w / v having a metal oxide content of about 16.8% and an average size of 3.6 μm was obtained. It was found that the crosslinked magnetic particles thus prepared had a uniform size and were inactive to ordinary organic solvents such as acetone, acetonitrile and dimethylformamide. Example 25 20 ml of 7.0% w / v 2.11 μm polystyrene particles, 150 ml of 1.8% w / v metal oxide prepared as described in Example 5, 0.15 g of benzoyl peroxide and 0.75 ml of divinylbenzene in 6.75 ml of styrene. A mixture containing a solution containing and was added into a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained crosslinked magnetic particles were resuspended in deionized water to make 200 ml, and a suspension of 5.4% w / v having a metal oxide content of about 23% and an average size of 4.0 μm was obtained. The crosslinked magnetic particles thus prepared were found to be uniform in size and inactive to ordinary organic solvents such as acetone, acetonitrile and dimethylformamide. Example 26 9.16% w / v 3.2 μm polystyrene particles 15 ml, 1.8% w / v metal oxide 100 ml prepared according to Example 5, 55 ml deionized water and 0.15 g benzoyl peroxide in 6.75 ml styrene. A mixture containing a solution containing 0.75 ml of divinylbenzene and 0.75 ml of divinylbenzene was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained crosslinked magnetic particles were resuspended in deionized water to make 200 ml, and a suspension of 4.7% w / v having a metal oxide content of about 16.8% and an average size of 5.5 μm was obtained. It was found that the crosslinked magnetic particles thus prepared had a uniform size and were inactive to ordinary organic solvents such as acetone, acetonitrile and dimethylformamide. Example 27 30 ml of 4.5% w / v 4.1 μm polystyrene particles, 100 ml of 1.8% w / v metal oxide prepared according to Example 5, 40 ml of deionized water, and 0.15 g of benzoyl peroxide in 6.75 ml of styrene. A mixture containing a solution containing 0.75 ml of divinylbenzene and 0.75 ml of divinylbenzene was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained crosslinked magnetic particles were resuspended in deionized water to make 200 ml, and a suspension of 4.5% w / v having a metal oxide content of about 16.9% and an average size of 6.7 μm was obtained. The crosslinked magnetic particles thus prepared were found to be uniform in size and inactive to common solvents such as acetone, acetonitrile and dimethylformamide. Example 28 20 ml of 7.0% w / v 2.11 μm polystyrene particles, 100 ml of 1.8% w / v metal oxide prepared according to Example 5, 50 ml of deionized water, and 0.15 g of benzoyl peroxide in 6 ml of styrene. A mixture containing a solution containing 0.75 ml undecylenyl alcohol and 0.75 ml divinylbenzene was added into a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 55 ° C for 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained crosslinked hydroxyl magnetic particles were filtered and dried to obtain 9 g of powder having a metal oxide content of about 16.8% and an average size of 3.9 μm. The crosslinked hydroxyl magnetic particles thus prepared were found to be uniform in size and inactive to common solvents such as acetone, acetonitrile and dimethylformamide. Bonding of biological material to magnetic particles Example 29 In an 80 ml bottle, 30 ml of 4.3 μm 5.0% w / v carboxyl magnetic particles prepared according to the description of Example 17 was added. The particles were magnetically separated and resuspended in 50 ml phosphate buffer (0.1 M, pH 5.5). To the particle suspension was added 20 mg bovine serum albumin and 100 mg 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). The mixture was spun at room temperature for 2 hours and separated magnetically. The particles were washed once with 80 ml phosphate buffer and resuspended in phosphate buffered saline (0.1 M, pH 7.0) to make 75 ml to give a 2.0% w / v suspension. The same procedure was followed except that EDC was not used to bind bovine serum albumin to magnetic particles by passive adsorption. Example 30 To a 4 ml vial was added 1 ml of 4.3 μm 5.0% w / v carboxyl magnetic particles prepared as described in Example 7. The particles were magnetically separated, washed once with 2 ml of phosphate buffer (0.1 M, pH 5.5) and resuspended in 2 ml of the same buffer. To the particle suspension was added 1.4 mg / ml goat (Gt) anti-mouse (Ms) IgG 140 ml and 10 mg 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. The vial was rotated at room temperature for 2 hours. The particles are magnetically separated, washed once with 2 ml phosphate buffer and resuspended in 2 ml phosphate buffered saline (0.1 M, pH 7.0) to 2 ml and 2.5% w / v goat anti. Mouse IgG-coated magnetic particles were obtained. Other types of monoclonal or polyclonal antibodies can also be attached to the carboxyl magnetic particles by the same procedure. To attach goat anti-mouse IgG and other types of antibodies to magnetic particles by passive adsorption, the same procedure was followed except without the use of EDC. Example 31 To a 4 ml vial, 2.5 ml of bovine serum albumin-coated magnetic particles (4.3 μm, 2% w / v) prepared according to the description of Example 29 were added. The particles were magnetically separated and resuspended in 2 ml phosphate buffer (0.1 M, pH 5.5) to give 2 ml. To the mixture was added 10 μl of mouse anti-B erythrocyte surface antigen (20 mg / ml) and 1 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide. The mixture was rotated at room temperature for 2 hours. The particles were magnetically separated, washed once with phosphate buffer and resuspended in 2 ml phosphate buffered saline (0.1 M, pH 7.0) to give a 2.5% w / v suspension. .. Example 32 The same procedure as described in Example 31 was followed, except that 2 ml of a 2.5% w / v suspension was obtained using 40 μl of mouse anti-A erythrocyte surface antigen (5 mg / ml). Blood typing using magnetic particles Example 33 25 μl of 2.5% w / v mouse anti-A coated magnetic particles prepared according to the description of Example 32 was added to a 5 mm × 65 mm test tube labeled A. In another tube labeled B, 25 μl of 2.5% w / v mouse anti-B coated magnetic particles prepared as described in Example 31 was added. To both tubes, 50 μl of 1% packed erythrocytes prepared by diluting packed erythrocytes with isotonic buffered saline at a ratio of 1: 100 was added. The test tube was placed on the swing magnet by tapping it with a finger several times. The results are summarized below. blood type ABO AB Test tube A + --- + Test tube B-+-+ Here, + indicates a positive reaction. That is, the erythrocytes were aggregated by the magnetic particles coated with the corresponding antibody, and as a result, the supernatant of the test tube was transparent after magnetic separation. On the other hand, the supernatant of the negative reaction remains turbid even after magnetic separation because there is no agglutination between the erythrocytes and the antibody-coated magnetic particles. Immunoassay using magnetic particles Example 34 1 ml of 6% w / v 3 μm carboxyl magnetic particles were added to a 2 ml microcentrifuge tube. The particles were centrifuged at 1000 rpm for 3 minutes. The supernatant was aspirated and the particles were resuspended in acetate buffer by shaking with 1 ml of 5-100 μg / ml recombinant HBcAg. The tube was rotated at room temperature for 2 hours and centrifuged as described above. The supernatant was aspirated and the particles were resuspended in 1 ml of recoating solution containing acetate buffer and 2-10% normal animal serum. The tube was rotated at room temperature for 2-16 hours and centrifuged as described above. The supernatant was aspirated and washed 3 times with 1 ml isotonic buffered saline (IBS) by centrifugation and resuspension. Finally, the particles were resuspended in 1 ml IBS and stored at 2-8 ° C. Example 35 To the first two rows of 96-well microtiter plates were added 20 μl of 0.25% w / v hepatitis B core antigen (HBcAg) -coated magnetic particles prepared as described in Example 34. Samples were prepared by diluting HBcAg-positive serum with negative plasma and then diluting each sample 1: 100 with sample dilution buffer (SDB). The SDB contained a phosphate buffer, a protein stabilizer, a surfactant, and an antibacterial agent. To the wells containing the particles, 50 μl of each final sample diluent was added. After incubation at 37 ° C for 30 minutes, the particles were separated by magnetic separation for 2 minutes and washed 3 times with 200 μl of wash buffer containing salts and detergent. To each well containing the particles was added 50 μl of goat anti-human IgG-BD galactosidase conjugate (0.5 μg / ml) in a diluent containing salts, protein stabilizers, glycerol, surfactants and antibacterial agents. After a 15 minute incubation at 37 ° C, the particles were separated, washed 3 times as described above and resuspended in 30 μl IBS. The particles were transferred to the first two rows of black microtiter (Dynatech). To each well containing the particles, 100 μl of a solution containing 4-methylumbelliferyl-B-galactopyranoside (MUG, Sigma) was added. Incubate the plate at 37 ° C and set the fluorescence intensity to 10x gain at 5-minute intervals using a fluorescence concentration analyzer (FCA, Pandex) equipped with filters at 365 nm on the excitation side and 450 nm on the fluorescence side. Was measured. The increase in fluorescence intensity at 5-minute intervals was recorded in an arbitrarily defined fluorescence unit (AFU) and shown in Table I.<img file="JP2589618B2_D0001.tif" />Example 36 The binding of mouse anti-HBsAg to the carboxyl magnetic particles was the same as in Example 30. To the wells of a black 96-well microtiter (Dynatech), 20 μl of 3.2 μm mouse anti-HBsAg coated carboxyl magnetic particles at 0.25% w / v were added in two rows. To the wells contained in the magnetic particles, 100 μl of untreated plasma or HBsAg-negative plasma containing various amounts of HBsAg was added. After incubating at 37 ° C for 30 minutes, the particles were separated by a magnetic separator for 2 minutes and washed once with 100 μl of a washing buffer containing salts and a surfactant. To each well containing the particles, 20 μl of mouse anti-HBsAg-B-galatatosidase conjugate in a diluent containing salts, protein stabilizers, glycerol, surfactants and antibacterial agents was added. After incubation at 37 ° C for 15 minutes, the particles were separated and washed 5 times as described above. To each well containing the particles, 50 μl of a solution containing 4-methylumbelliferyl-BD-galactopyranoside (MUG, Sigma) was added. The plate is incubated at 37 ° C, and the fluorescence intensity is measured by setting the fluorescence intensity to a 10-fold gain at 5-minute intervals using a fluorescence concentration analyzer (FCA, Pandex) equipped with filters of 365 nm on the excitation side and 450 nm on the fluorescence side. did. The increase in fluorescence intensity at 5-minute intervals was recorded in an arbitrarily defined fluorescence unit (AFU) and is shown in Table II.<img file="JP2589618B2_D0002.tif" />Example 37 The HTV-1 antigen from HTLV-IIIB / H-9 cells (Gallo strain) was bound to 3.6 μm carboxyl magnetic particles by the same procedure as described in Example 34. To the wells of the 96-well microtiter, 20 μl of 0.25% w / v HIV-coated magnetic particles were added in two rows. To the wells contained in the particles, add 50 μl of each of the positive, boundary and negative samples diluted 1: 100 to sample dilution buffer (SDB) containing phosphate buffer, protein stabilizer, surfactant and antibacterial agent. It was. After a 30 minute incubation at 37 ° C., the particles were separated by a magnetic separator for 2 minutes and washed 3 times with 100 μl of wash buffer containing salts and detergent. To each well containing the particles, 50 μl of goat anti-human-B-galactosidase (approximately 0.5 μg / ml) conjugate in a diluent containing salts, protein stabilizers, glycerol, detergents and antibacterial agents was added. After incubating at 37 ° C for 15 minutes, the particles were washed 4 times as described above. The particles were transferred to a black micro-suitable plate (Dynatech). To each well containing the particles was added 100 μl of a solution containing 4-methylumbelliferyl-BD-galactopyranoside (MUG, Sigma). Incubate the plate at 37 ° C and set the fluorescence intensity to 25x gain at 5-minute intervals using a fluorescence concentration analyzer (FCA, Pandex) equipped with filters at 365 nm on the excitation side and 450 nm on the fluorescence side. And measured. The increase in fluorescence intensity at 5-minute intervals was recorded in an arbitrarily defined fluorescence unit (AFU) and is shown in Table III.<img file="JP2589618B2_D0003.tif" />Cell separation using magnetic particles Example 38 The 4.3 μm carboxyl magnetic particles prepared according to the description of Example 7 were washed with phosphate buffered saline (PBS, pH 7.7), ultrasonically treated, sterilized with 70% ethanol for 10 minutes, and washed with PBS three times. Then, they were incubated with 0.5 mg / ml affinity purified sheep anti-mouse immunoglobulin antibody (SAM) at a ratio of 3.3 mg antibody / 100 mg particles at 4 ° C for 48 hours. Prior to use, the antibody-coated magnetic particles were washed with PBS and resuspended in PBS to the desired concentration. Human tissue culture cALLa-positive NALM-16 leukemia cells were washed with PBS and suspended. Some were not treated with antibodies (-MoAb). The other part was treated with two anti-CD10 monoclonal antibodies and one anti-CD9 monoclonal antibody at 4 ° C for 30 minutes (+ MoAb), washed with PBS and 3.5 × 10 with PBS.⁶Adjusted to pieces / ml. In two test tubes containing antibody-treated cells (+ MoAb) on one side and untreated cells (-MoAb) on the other, SAM-coated magnetic particles so that the ratio of particles to cells at the start is 45. added. The tube was rotated at 4 ° C for 30 minutes. The particles were separated by a magnetic separator. The supernatant was collected and centrifuged to collect the remaining cells. The pellet was resuspended in 100 μl trypan blue and the total cell count was counted. The results are shown in Table IV.<img file="JP2589618B2_D0004.tif" />Example 39 A mixture containing 576 ml of deionized water, 9 ml of styrene and 288 ml of 3.0% w / v magnetic metal oxide prepared as described in Example 1 was added to a sealed bottle. The bottle was aspirated and rotated at about 60 rpm in an oven at 65 ± 4 ° C for 1 hour. To the mixture was added 18 g potassium persulfate and 712 ml of 4.0 μm fluorescent Nile red polystyrene particles at 5% w / v. The bottle was resealed, aspirated and rotated for 1 hour, and 45 ml of 2.0% sodium dodecyl sulfate was added. After an additional 5 hours, 9 ml of styrene and 9 g of potassium persulfate were added to the mixture. The mixture was rotated for an additional 15 hours, filtered through a two-layer cheesecloth, magnetically separated, and washed several times with deionized water until the supernatant became clear. The obtained fluorescent magnetic particles were suspended in deionized water to make 1580 ml, and a 3.0% w / v suspension having a magnetic metal oxide content of about 11.0% and an average size of 4.4 μm was obtained. Example 40 1.580 liters of 3.0% w / v luminescent Nile red magnetic particles prepared according to the description of Example 39, 1.23 g of sodium dodecyl sulfate, 17.50 g of potassium persulfate, and 1.2 ml of undecylene acid in 4.8 ml of methanol. It was carboxylated by the addition of a solution containing 0.024 ml of divinylbenzene. The mixture was added to a sealed bottle, aspirated and rotated at about 60 rpm in an oven at 55-65 ° C for 5 hours. The resulting fluorescent Nile red carboxyl magnetic particles were magnetically separated and washed several times with deionized water until the supernatant became clear. The fluorescent Nile red carboxyl magnetic particles were suspended in deionized water to make 850 ml, and a 5.0% w / v suspension having a magnetic metal oxide content of about 11.0% and an average size of 4.4 μm was obtained. Example 41 11.28% w / v 2.24 μm polystyrene particles 12.4 ml, 2.78% w / v metal oxide 65 ml, 75 ml deionized water and 0.18 g peroxidation in 6.75 ml styrene prepared as described in Example 5. A mixture containing a solution containing benzoyl, 7 mg Nile red and 0.75 ml divinylbenzene was added to a sealed bottle. The bottle was sucked and rotated at about 60 rpm in an oven at 60-70 ° C for about 15 hours. The mixture was filtered through a two-layer cheesecloth, magnetically separated and washed several times with deionized water until the supernatant became clear. The obtained fluorescent crosslinked magnetic particles were suspended in deionized water to make 170 ml, and a suspension of 5.4% w / v having a metal oxide content of about 16.5% w / v and an average size of 4.0 μm. Got Example 42 The binding of goat anti-HBsAg to fluorescent and non-fluorescent carboxyl magnetic particles (having approximately the same metal oxide content) was similar to Example 30. A complete mixture (1: 1) concentration of 4.0 μm, fluorescent and non-fluorescent carboxyl magnetic particles coated with goat anti-HBsAg in wells of a black 96-well microtiter [Pandex®]. 20 μl of 0.125% w / v, was added. To the wells containing the magnetic particles, 100 μl of untreated plasma or HBsAg-negative plasma containing various amounts of HBsAg was added. After incubation at 37 ° C for 30 minutes, the particles were separated by a magnetic separator and washed twice with 100 μl of wash buffer. To each well containing the particles, 20 μl of a combination of mouse anti-HBsAg and B-galatatosidase in dilution buffer was added. After a 15 minute incubation at 37 ° C, the particles were separated and washed 6 times as described above. To the well containing the particles, 50 μl of a solution containing 4-methylumbelliferyl-BD-galactopyranoside (MUG, Sigma) was added. The plate was incubated at 37 ° C and the fluorescence intensity was measured using a fluorescence concentration analyzer [FCA, Pandex®] equipped with filters (channel C, control channel) on the excitation side and 580 nm on the fluorescence side. , The measurement was performed by setting the gain to 25 times at 8 minute intervals. The fluorescence intensity of channel C was recorded in an arbitrarily determined fluorescence unit (AFU) and shown in Table (1). The results show that the fluorescent magnetic particles detect empty wells and also display wells that are less than the average fluorescence intensity. This is due to incorrect pipette operation or loss of particles during the assay.<img file="JP2589618B2_D0005.tif" />* 17 The AFU of one well is 19458 compared to the average AFU of 31480 in one well, which indicates the loss of particles in the well or the release of a small number of particles at the beginning of the assay. Example 43 The same procedure as described in Example 42 was followed, except that fluorescent and non-fluorescent carboxyl magnetic particles coated with goat anti-HBsAg were used simultaneously in the assay for assay capability comparison. The fluorescence intensity was measured using channel D (quantitative channel, 365 nm excitation side and 450 nm fluorescence filter) and is shown in Table (2). The results show that both fluorescent and non-fluorescent particles performed equally well in the assay. Table (2) AFU HBsAg concentration Fluorescent particles Non-fluorescent particles High 27238 30059 Moderate 5820 5976 Low 1688 1816 negative 326 403