JP4791867B2 - Detection method of test substance using precious metal coated magnetic particles - Google Patents

Description

The present invention relates to a method for detecting a test substance using noble metal-coated magnetic particles. More specifically,
The present invention relates to a method for detecting a test substance in a sample using noble metal-coated magnetic particles on which a substance capable of binding to the test substance is immobilized. The present invention also relates to a detection kit used in such a detection method.

  Many methods are known for quantitatively analyzing a trace amount of a test substance contained in a sample by utilizing a specific interaction such as a combination of an antibody and an antigen or a combination of a biological receptor and a ligand.

  Among them, in particular, an immunological measurement method using an “antigen-antibody reaction” in which an antigen and an antibody specifically interact with each other is widely used. One such immunological measurement method is an enzyme-linked immunosorbent assay method, that is, an ELISA method (also called “enzyme immunoassay method”). This technique is a technique for specifically and selectively capturing a test substance with an antibody, and analysis can be performed only by rough extraction. Therefore, it is not necessary to go through many complicated steps when purifying a sample, and even a trace amount of a test substance can be extracted with water, phosphate buffer, methanol, etc. in most cases. Can be used directly as a sample. Due to its simplicity, the ELISA method is most used in the field of clinical diagnosis, and has a longer history and a greater number of achievements than the PCR method and analysis using a DNA chip, which are attracting attention recently.

  One such ELISA method is the sandwich ELIZA method. In the sandwich ELIZA method, an antibody a immobilized on a plate, an antigen in a sample, and another antibody b in which an enzyme is bound in advance (“antibody b” is also referred to as “antibody-enzyme complex”), A sandwich structure consisting of immobilized antibody a-antigen-antibody b is constructed. Here, when no antigen is contained in the sample, the antibody b remains because there is no antigen to be captured and bound, and the antibody b is removed from the plate by washing the plate. That is, if an antigen is present in the sample, a sandwich structure consisting of immobilized antibody a-antigen-antibody b is constructed. On the other hand, if there is no antigen in the sample, antibody b remains and the antibody b is removed from the plate. Will be removed. When such a principle is used, a difference occurs in the amount of enzyme remaining on the plate (that is, “the amount of enzyme bound to antibody b”) due to the difference in the amount of antigen in the sample. Therefore, finally, when the enzyme staying on the plate is colored, it is possible to determine the presence or absence of the antigen or to determine the amount of the antigen.

  However, in the sandwich ELIZA method, since the enzyme that is usually used is colorless, the coloration cannot be confirmed by the enzyme alone, and a chromogenic substrate is additionally required to develop the enzyme.

  As a method similar to the sandwich ELIZA method, there is a sandwich immunoassay method using gold colloid. Such sandwich immunoassay uses gold colloid instead of the above-mentioned enzyme. That is, “antibody gold colloid complex” is used in place of the antibody-enzyme complex (ie, “antibody b”) of the sandwich ELIZA method. In this case, the antibody a ′ immobilized on the plate, the antigen in the sample, and another antibody b ′ preliminarily bound with the gold colloid (“antibody b ′” as “antibody gold colloid complex”), A sandwich structure of immobilized antibody a′-antigen-antibody b ′ will be constructed. Similar to the sandwich ELIZA method, if an antigen is present in the sample, a sandwich structure consisting of immobilized antibody a′-antigen-antibody b ′ is constructed, whereas if there is no antigen in the sample, antibody b ′ remains. This antibody b ′ will be removed from the plate. Therefore, depending on the difference in the amount of antigen in the sample, a difference occurs in the amount of colloidal gold remaining on the plate (that is, “the amount of colloidal gold bound to antibody b ′”). In the case of the above sandwich ELIZA method, a chromogenic substrate is added at the end, and the amount of antigen in the sample is quantified by color development according to the amount of enzyme, but in the case of the sandwich immunoassay method, the chromogenic substrate is not added to the plate. The amount of antigen in the sample can be quantified by the difference in coloration of gold itself depending on the amount of colloidal gold remaining. Therefore, the sandwich immunoassay method using colloidal gold can be said to be a simpler method than the sandwich ELIZA method in that the addition operation of the chromogenic substrate can be omitted.

  In the sandwich immunoassay method, depending on the size of the gold colloid, the visible color such as purple and brown can be confirmed from the gold colloid itself. Another reason why colloidal gold is used as a carrier for binding antibodies is that colloidal gold can easily prepare antibody-gold colloid complexes. Between gold and sulfur atoms, sulfur atoms are chemisorbed on gold due to metal-sulfur bonds. Therefore, it is possible to easily construct an antibody gold colloid complex due to the bond between the sulfur atom of the antibody and gold. When the antibody does not contain a sulfur atom, the sulfur atom can be introduced into the antibody by modifying the antibody with thiol or thioether.

  Although the method using colloidal gold has advantages as described above, the colloidal gold used in the sandwich immunoassay method is nonmagnetic, so it is not always advantageous in terms of preparing the “antibody colloidal gold complex”. There was no. Specifically, after the step of binding the colloidal gold and the antibody, a purification operation is required to remove excess antibody that did not bind to the colloidal gold. Such a purification operation is performed by centrifugation or filter operation. These operations themselves are not preferable in automating the entire process (see Patent Document 1).

On the other hand, a technique using magnetic fine particles instead of gold colloid has also been proposed (see Patent Document 2). In the proposed method, a test substance in a sample is quantified by moving a magnetic fine particle by applying a magnetic field. However, in this method, a substance that can specifically bind to the test substance (hereinafter also referred to as “specific binding substance”) is immobilized on the surface of the magnetic fine particles coated with the polymer substance. For this reason, specific specific binding substances such as hydrophobic antibodies can be easily immobilized on the magnetic fine particles, but most other specific binding substances are difficult to be directly immobilized on the magnetic fine particles. This is because such a specific binding substance includes a sulfide functional group such as a thiol group, a thioether group or a disulfide group or a derivative of such a functional group. It is not possible. In this case, it is necessary to modify the surface of the magnetic particle with a functional group, and this involves a complicated operation in terms of selecting such a functional group or a crosslinking reagent. In addition, when the surface of the magnetic fine particles is modified with a sulfide functional group such as a thiol group, a thioether group or a disulfide group, the thiol group, the thioether group or the disulfide is restricted in terms of the polymer substance covering the magnetic particle. In some cases, a crosslinking reagent having a functional group other than a sulfide functional group such as a group is required.
Japanese Patent No. 3108115 JP-A-9-229936

  The present invention has been made in view of the above circumstances. That is, an object of the present invention is to provide a simple detection method of a test substance that can be carried out in a relatively short time.

In order to solve the above problem, a method for detecting a test substance contained in a sample,
(I) Magnetic particles in which the central region (core region) is formed from a magnetic material and the outer shell region is formed from a noble metal material, and a substance a that can be bound to a test substance is present in the outer shell region. Preparing a fixed magnetic particle, a member on which a substance b capable of binding to a test substance is fixed, and a sample containing the test substance;
(Ii) supplying the magnetic particles and the sample to the member, and obtaining in the member a composite formed by combining the substance a of the magnetic particles and the substance b of the member through the test substance;
(Iii) measuring the state of the region where the substance b is immobilized on the member, and (iv) determining the presence or amount of the test substance based on the measurement result,
There is provided a method characterized in that the formation of the complex in step (ii) is performed under the influence of a magnetic field (or magnetic field).

  The magnetic particles used in the method of the present invention have a central region formed from a magnetic material and an outer shell region formed from a noble metal material. Therefore, since the magnetic particles have a form in which the noble metal material is coated with the magnetic material, the magnetic particles are also referred to as “noble metal-coated magnetic particles” in the present specification. Moreover, the substance a and the substance b are substances having affinity for the test substance, and are substances that specifically interact with and bind to the test substance. Therefore, in the present specification, the substance a and the substance b are also referred to as “specific binding substances”.

  In this specification, “detecting a test substance” means not only determining the presence or absence of a test substance contained in a sample, but also determining the amount of the test substance. In the method of the present invention, since the amount of the complex formed in step (ii) substantially corresponds to or is proportional to the amount of the test substance contained in the sample, it is based on the presence or absence of the complex. Thus, the presence or amount of the test substance can be examined. Specifically, the state of the region where the substance b is fixed in the member is measured. That is, the state of the region changes due to the presence or absence of the complex or the amount of the complex (for example, “the color of the region where the substance b is immobilized” changes). The presence or amount of the test substance can be examined. In the present specification, “measurement” means not only measurement by visual confirmation but also measurement by a general measuring instrument.

  For example, when the test substance contained in the sample is an antigen (or antibody), and the substance a of the magnetic particles and the substance b of the member are antibodies (or antigens) that specifically bind to the antigen, so-called A complex is formed by the “antigen-antibody reaction”. It will be understood that when such a “antigen-antibody reaction” is used to form a complex, the method for detecting a test substance of the present invention can be particularly referred to as an “immunoassay method”.

  In this specification, “immobilization” generally means “substance a capable of binding to the test substance” or “bonding to the test substance” near the surface of the magnetic particle or member. This means an embodiment in which the substance “b” is present, and means only an embodiment in which the substance a is directly attached to the surface of the magnetic particle, or only an embodiment in which the substance b is attached directly to the member. It is not a thing. Further, in this specification, “the magnetic substance“ a ”and the member“ b ”are formed by bonding through the test substance” means that both the magnetic substance “a” and the member “b” are tested. It means that it is bonded to a substance, and it means that a sandwich structure composed of magnetic particles (substance a) -test substance-member (substance b) is constructed.

  One feature of the present invention is that the magnetic particles used in step (i) contain a magnetic material in the central region and are magnetized. Therefore, when a magnetic field is applied in step (ii), the movement of the magnetic particles is promoted, and a complex can be formed in a shorter time. Specifically, the “substance b is immobilized” by placing a magnet in the vicinity of the region where the substance b of the member is immobilized, or by passing an electric current through an electromagnet arranged in such a vicinity. It promotes the movement of the magnetic particles toward the “region”.

  Another feature of the present invention is that the magnetic particles used in the method of the present invention can be easily prepared because the magnetic particles used in the step (i) have a noble metal coat. Specifically, for example, when the substance a is an antibody or an antigen containing a sulfur atom, the sulfur atom and a noble metal coat of magnetic particles (magnetic particles to which the substance a is not immobilized) are easily bonded to each other. Therefore, the magnetic particles used in step (i) can be prepared relatively easily.

  In addition, in this preparation, since the magnetic particles are magnetized, it is one of the features of the present invention that the magnetic particles can be recovered relatively easily by a magnetic separation operation.

  Since the magnetic particles used in the method of the present invention are magnetized, when the composite is formed under the influence of a magnetic field, the composite can be obtained in a relatively short time. Therefore, the method of the present invention can detect a test substance in a relatively short time. In addition, since the magnetic particles used in the method of the present invention have a noble metal coat, it is relatively easy to prepare the magnetic particles performed prior to detection.

  Furthermore, in the method of the present invention, since the centrifugation operation and the filter operation are not performed when preparing the magnetic particles and detecting the test substance, it is easy to automate all the steps.

BEST MODE FOR CARRYING OUT THE INVENTION

  Below, the detection method of the test substance of this invention is demonstrated in detail. FIG. 1A shows a flowchart of the detection method of the present invention.

  First, in step (i), as shown in FIG. 1B, magnetic particles 1 in which a central region 2 is formed from a magnetic material and an outer shell region 3 is formed from a noble metal material, A magnetic particle 1 in which a substance a (symbol 4) that can be bonded is fixed to the outer shell region 3 is prepared. In addition, a sample 7 ′ including the test substance 7 is prepared, and a member 6 on which a substance b (reference numeral 5) capable of binding to the test substance 7 is fixed is also prepared.

About magnetic particles
The magnetic particles prepared in the step (i) have a central region made of a magnetic material and an outer shell region made of a noble metal material. The magnetic material in the central region may be any kind of material as long as it has magnetism. Examples of the magnetic material include metals such as iron, cobalt, and nickel, or alloys containing at least such metals, and also include iron oxide and yttrium iron garnet. When the magnetic particles are made of a ferromagnetic material, there is an advantage that the saturation magnetization is large and the collection property is excellent in the magnetic separation operation. Further, when the magnetic particles are made of a superparamagnetic material, the saturation magnetization is not as great as that of the ferromagnetic material, but there is an advantage that the magnetic particles do not aggregate in the absence of a magnetic field and the magnetic particles are less likely to aggregate. Therefore, in the present invention, it is preferable that the central region is formed of a magnetic material made of a ferromagnetic material or a superparamagnetic material. Examples of the ferromagnetic material include maghemite (γ-iron oxide), magnetite, nickel zinc ferrite or manganese zinc ferrite, and examples of the superparamagnetic material include maghemite (γ-iron oxide), magnetite, nickel zinc. Examples thereof include ultrafine particles such as ferrite and manganese zinc ferrite.

  The noble metal material contained in the outer shell region of the magnetic particle is not particularly limited as long as it is a general noble metal material, but gold, platinum, silver, or an alloy thereof is preferable, and a mixture thereof may be used. .

  The magnetic particles used in step (i) preferably have an average particle diameter of 5 nm to 100 μm. This is because, when the average particle diameter of the magnetic particles is smaller than 5 nm, the magnetic collection of the magnetic particles is deteriorated, whereas when the average particle diameter of the magnetic particles is larger than 100 μm, the dispersibility of the magnetic particles is deteriorated. An average particle diameter of 20 nm to 10 μm is particularly preferable. Here, the “average particle diameter” means the diameter of the magnetic particles in a state where the substance a is not immobilized. Further, in this specification, “average particle diameter” means, for example, that 100 particles are randomly selected from an image magnified by an electron microscope, an optical microscope, or the like, the diameter is read for each particle, and these are averaged. This is the particle diameter when calculated by the above. However, if the diameter is not uniform, the average of the maximum and minimum diameters is determined as the diameter of each particle. It should be understood that the preferred particle size of such magnetic particles can vary depending on the shape and type of the test substance or the magnetic particles themselves.

As magnetic characteristics of the magnetic particles used in the step (i), the coercive force has a range of 0 kA / m to 280 kA / m (0 Oersted to 3500 Oersted), and the saturation magnetization is 0.5 A · m ² / kg to 100 A. -What has the range of m < ² > / kg (0.5emu / g-100emu / g) is preferable. Thereby, the responsiveness of the magnetic particles to the magnetic field (or magnetic field) becomes practically sufficient. However, when the magnetic material in the central region of the magnetic particles is metallic iron, the coercive force has a range of 8.0 kA / m to 71.7 kA / m (100 Oersted to 900 Oersted), and the saturation magnetization is 60 A. · ^m having a range of ^{2 / kg~160A · m 2 / kg} (60emu / g~160emu / g) is preferable for practical use. If the coercive force is too large, the cohesiveness of the magnetic particles will be larger than necessary, affecting the uniform formation of the composite, while if the coercive force is too small, the magnetic particles will not be affected by the magnetic field. A preferable coercive force is 0 kA / m to 71.7 kA / m, and a more preferable coercive force is 0.8 kA / m to 15.92 kA / m.

  The substance a immobilized on the magnetic particles is preferably one of an antibody and an antigen that have specific binding properties to each other. Substance a may be either a biological receptor or a ligand having specific binding properties to each other. Further, when the test substance is a nucleotide chain, the substance a may be a nucleotide chain complementary to the nucleotide chain.

  For example, when the test substance is an antigen, the substance a is an antibody, and when the test substance is an antibody, the substance a is an antigen. Similarly, when the test substance is a biological receptor, the substance a becomes a ligand, and when the test substance is a ligand, the substance a becomes a biological receptor. Examples of typical antibodies or antigens used for the substance a include anti-influenza virus antibodies, influenza virus antigens, cardiac troponin T, and anti-cardiac troponin T antibodies. Examples of typical biological receptors or ligands used for the substance a include LDL, LDL receptor, insulin, insulin receptor and the like. Note that the terms “antibody / antigen” and “biological receptor / ligand” essentially mean a combination of substances that specifically bind to each other. It should be noted that some may belong not only to “biological receptor / ligand combinations”.

  The substance a described above is immobilized on the magnetic particles prepared in the step (i). For such immobilization, a known physical adsorption method or chemical adsorption method can be used. For example, when noble metal-coated magnetic particles (hereinafter referred to as “magnetic particles p”) in which substance a is not yet immobilized and substance a are mixed in a solution such as a buffer solution or an organic solvent for several seconds to several days, Since the substance a and the magnetic particles p cause an adsorption reaction, the substance a can be fixed to the magnetic particles p. Examples of buffers or organic solvents to be used include PBS buffer, HEPES buffer, phosphate buffer, tris (hydroxymethyl) aminomethane buffer, dimethyl sulfoxide, dimethylformamide, acetone, pyridine, toluene, ethyl acetate, hexane, Examples include ethanol, methanol, chloroform, and phenol. Further, for example, when the substance a and the magnetic particles p are brought into contact with each other in a solution such as a buffer solution or in an organic solvent as in the coacervation method, the substance a is deposited on the surface of the magnetic particles p. Can be immobilized on the magnetic particles p.

  Many antibodies (for example, proteins) have sulfide functional groups such as thiol groups, thioether groups, or disulfide groups in their molecules. Since the sulfur atom of the antibody and the noble metal material are easily bonded, the antibody can be directly immobilized on the magnetic particle p by bringing the magnetic particle p into contact with the antibody without performing any special pretreatment on the magnetic particle p. Can do.

  The magnetic particles to which the substance a is immobilized are obtained in a state where they are present in a solution such as a buffer solution or in an organic solvent, but in such a solution or solvent, an extra substance such as the substance a that has not been immobilized. Or an ingredient may be included. Therefore, as will be described in detail later, “magnetic particles having substance a immobilized” are purified using a magnetic separation operation.

  Thus, the magnetic particles used in the detection method of the present invention can be obtained. The obtained magnetic particles include tris (hydroxymethyl) aminomethane buffer, phosphate buffer, PBS buffer, HEPES buffer, It is preferably used in a state dispersed in a solvent such as a buffer solution such as MOPS buffer or TBS buffer, physiological saline, ion exchange water, sterilized water or ultrapure water.

《About members》
The substance b is immobilized on the member prepared in step (i). The member itself may have any shape and structure as long as the substance b can be fixed, and the material of the member is not particularly limited.

  One example of a preferred member is a plate member or container. Examples of such members include wells, plate-like members, spherical members, and the like. In such a case, the material of the member is glass, quartz, titanium oxide, ITO, gold, platinum, silver or other inorganic material, carbon black, glassy carbon, carbon nanotube, fullerene or other carbon material, polystyrene, polyvinyl chloride, Polymer materials such as polyacrylate, polypropylene, nylon, polyester, polycarbonate, polymethacrylate, polyvinylidene fluoride, cellulose, and nitrocellulose are preferable. If the member is a plate member or a container, the substance b can be immobilized on the surface of the member.

  Another example of a preferable member is a member made of porous or fibrous material. Examples of such a member include a nonwoven fabric, a filter paper, a polymer membrane, a porous membrane, or a plate. In such a case, the porous or fibrous material is preferably formed from a material such as silica, alumina, glass wool, dextran, polyvinylidene fluoride, cellulose, or nitrocellulose. In the member made of porous or fibrous material, since the member itself has a structure having a void, the substance b can be fixed in the internal region of the member.

  The member prepared in step (i) is provided with a flow path through which magnetic particles (specifically, “magnetic particles present in a state dispersed in a solvent”) and / or a liquid sample flow on the surface or internal region of the member. It is preferable that

  In the present invention, it is preferable that the substance b is immobilized only on a part of the surface or a part of the inner region of the member. This is because the image of the surface or internal region where the substance b is immobilized can be compared with the image of the surface or internal region where the substance b is not immobilized. This facilitates the discrimination between “positive” and “negative”, that is, the presence or absence of the test substance in the sample.

  The substance b immobilized on the member is preferably any one of an antibody and an antigen having specific binding properties like the substance a. The substance b may be any one of a biological receptor and a ligand having a specific binding property to each other like the substance a. Furthermore, when the test substance is a nucleotide chain, the substance b may be a nucleotide chain complementary to the nucleotide chain.

  Similarly to the substance a, for example, when the test substance is an antigen, the substance b is an antibody, and when the test substance is an antibody, the substance b is an antigen. Similarly, when the test substance is a biological receptor, the substance b becomes a ligand, and when the test substance is a ligand, the substance b becomes a biological receptor. As typical antibodies or antigens used for the substance b, for example, anti-influenza virus antibodies, influenza virus antigens, cardiac troponin T, anti-cardiac troponin T antibodies and the like can be mentioned. Examples of typical biological receptors or ligands used for the substance b include LDL, LDL receptor, insulin, insulin receptor and the like. It should be noted that the substance a and the substance b may be the same substance or different substances.

  The above-mentioned substance b is immobilized on the member prepared in step (i). For such immobilization, a known physical adsorption method, chemical adsorption method, chemical bonding method using a crosslinking reagent, or these A combination of these methods can also be used.

  For example, if the member b in which the substance b is not yet immobilized (hereinafter referred to as “unfixed member”) and the substance b are immersed in a solution such as a buffer solution or an organic solvent for several seconds to several days, Since the adsorption reaction occurs with the immobilization member, the substance b can be immobilized on the non-immobilization member. After immobilization, the member b is preferably washed with a PBS buffer solution, a phosphate buffer solution, a buffer solution such as acetone or ethanol, or an organic solvent.

  Note that “a member in which the substance b is immobilized only on a part of the surface or a part of the internal region” is obtained by dropping the solution, suspension, or dispersed substance b onto the member using a spotter or the like. Obtainable.

  Hereinafter, “immobilization of the substance b on the non-immobilized member” will be described in detail with specific substances / functional groups.

<Immobilization using heterobivalent cross-linking reagent>
By using a heterobivalent cross-linking reagent, the substance b can be immobilized on the non-immobilized member by a chemical bonding method. As "heterobivalent cross-linking reagent"
Sulfide functional groups such as thiol groups, thioether groups or disulfide groups, aldehyde groups, epoxy groups, tosyl groups, azide groups, succinimide groups, maleimide groups, hydrazide groups, primary amino groups, secondary amino groups, tertiary amino groups, A functional group such as a carboxyl group, an imide ester group, a carbodiimide group, an isocyanate group, an iodoacetyl group, an acid chloride, a double bond, or the functional group derivative or an inorganic substance;
Sulfide functional groups such as thiol groups, thioether groups or disulfide groups, aldehyde groups, epoxy groups, tosyl groups, azide groups, succinimide groups, maleimide groups, hydrazide groups, primary amino groups, secondary amino groups, tertiary amino groups, Examples thereof include heterobivalent crosslinking reagents comprising a functional group such as a carboxyl group, an imide ester group, a carbodiimide group, an isocyanate group, an iodoacetyl group, an acid chloride, a double bond, or a combination with the functional group derivative or an inorganic substance.
In order to immobilize the substance b on an unimmobilized member using such a heterobivalent crosslinking reagent, the “unimmobilized member” itself is a sulfide such as a thiol group, a thioether group, or a disulfide group. Functional group, aldehyde group, epoxy group, tosyl group, azide group, succinimide group, maleimide group, hydrazide group, primary amino group, secondary amino group, tertiary amino group, carboxyl group, imide ester group, carbodiimide group, isocyanate group It preferably has a functional group such as an iodoacetyl group, an acid chloride, a double bond, the functional group derivative, or an inorganic substance.
Similarly, “substance b” includes a sulfide functional group such as thiol group, thioether group or disulfide group, aldehyde group, epoxy group, tosyl group, azide group, succinimide group, maleimide group, hydrazide group, primary amino group, Includes functional groups such as secondary amino groups, tertiary amino groups, carboxyl groups, imide ester groups, carbodiimide groups, isocyanate groups, iodoacetyl groups, acid chlorides, double bonds, or functional group derivatives or inorganic substances. Preferably it is.

  By contacting and mixing the non-immobilized member and the substance b with the heterobivalent crosslinking reagent as described above, the substance b can be immobilized on the non-immobilized member via the heterobivalent crosslinking reagent. For example, first, by immersing the heterobivalent cross-linking reagent and the above-described non-immobilized member in a solution such as a buffer solution or in an organic solvent, by the reaction between the heterobivalent cross-linking reagent and the non-immobilized member, A heterobivalent cross-linking reagent is bound to the non-immobilized member. Substance b is then added. Thus, the substance b is immobilized on the non-immobilized member through the heterobivalent crosslinking reagent by the reaction between the heterobivalent crosslinking reagent and the substance b. Finally, the member on which the substance b is immobilized may be washed with a buffer solution or an organic solvent.

<Immobilization using homobivalent cross-linking reagent>
Similarly to the heterobivalent crosslinking reagent, the substance b can be immobilized on the non-immobilized member by a chemical bonding method by using a homobivalent crosslinking reagent. “Homobivalent cross-linking reagents” include thiol groups, thioether groups, disulfide groups and other sulfide functional groups, aldehyde groups, epoxy groups, tosyl groups, azide groups, succinimide groups, maleimide groups, hydrazide groups, and primary amino groups. , Secondary amino groups, tertiary amino groups, carboxyl groups, imide ester groups, carbodiimide groups, isocyanate groups, iodoacetyl groups, acid chlorides or double bonds, or the like Valent crosslinking reagent. The “non-immobilized member” and “substance b” are the same as in the case of the heterobivalent crosslinking reagent. The method of immobilizing the substance b on the non-immobilized member using a homobivalent crosslinking reagent is also the same as the above-described heterobivalent crosslinking reagent. That is, the homobivalent cross-linking reagent and the above-described non-immobilized member are immersed in a solution such as a buffer solution or an organic solvent, thereby binding the homo-bivalent cross-linking reagent to the non-immobilized member. Next, the substance b is added, and the substance b is immobilized on the non-immobilized member via the homobivalent crosslinking reagent. Finally, the member on which the substance b is immobilized is washed with a buffer solution or an organic solvent.

  In addition, in the above-mentioned “immobilization using a heterobivalent cross-linking reagent” and “immobilization using a homobivalent cross-linking reagent”, it is possible to suppress non-specific reactions to members as necessary. preferable. Therefore, blocking that suppresses non-specific adsorption to the member may be performed by a known method such as bringing normal serum, bovine serum albumin, human serum albumin, casein, skim milk powder or the like into contact with the member.

About the sample
The sample prepared in step (i) contains a test substance. Any sample may be used as long as there is a possibility that a test substance to be detected exists. For example, body fluids such as human or animal urine, blood, serum, plasma, semen, saliva, sweat, tears, ascites, amniotic fluid; human or animal organs, hair, skin, nails, bones, muscles or nerve tissues Suspension or extract; Suspension or extract of cultured cells or tissue; Suspension or extract of virus; Culture of bacterial cells; Soil suspension or extract; Examples of the sample include turbid liquids or extracts; suspensions or extracts of foods and processed foods;

  The sample may be diluted with a liquid that does not affect detection. For example, tris (hydroxymethyl) aminomethane buffer, phosphate buffer, PBS buffer, HEPES buffer, MOPS buffer or TBS buffer, buffer solution, physiological saline, ion exchange water, sterilized water or ultrapure The sample may be diluted with water or the like.

  The test substances contained in the sample include, for example, proteins, glycoproteins, peptides, nucleic acids, sugars, lipids, glycolipids, high molecular compounds, low molecular compounds, complexes, inorganic substances, vectors, bacteria, fungi, yeast, viruses and Examples include biological substances such as cells, food-related substances, and environment-related substances.

More specific test substances can include the following substances:
HA antibody, IgG type HA antibody, IgM type HA antibody, HBc antibody, HBe antigen, HBe antibody, HBs antigen, HBs antibody, HCV core antigen, HCV core antibody, HCV antigen, HCV antibody, HIV I antigen, HIV I antibody, Virus-related antigens or antibodies such as HIV II antigen, HIV II antibody, influenza virus antigen, influenza A antibody, influenza B antibody, rubella virus antibody, herpes simplex type I or herpes simplex type II antibody;
Mycoplasma antigen, anti-Mycoplasma antibody, Mycobacterium tuberculosis antigen, anti-M. Tuberculosis antibody, Clostridium difficile antigen, Aspergillus antigen, Aspergillus antibody, ASO, Candida antigen, anti-Candida antibody, Ohm disease antibody, Chlamydia trachomatis antibody IgA, Chlamydia trachoma Chis antibody IgM, Chlamydia trachomatis antibody IgG, Chlamydia pneumoniae antibody IgA, Chlamydia pneumoniae antibody IgG, anti-acid-fast bacteria antibody, anti-streptokinase antibody, endotoxin, anti-endotoxin antibody, Legionella antigen, pertussis antibody, Helicobacter pylori Antigens, Helicobacter pylori antibodies, syphilis treponema antigens, syphilis treponema antibodies, gonococcal antigens, anti-gonococcal antibodies, leptospiral antigens, anti-leptospiral antibodies and other bacterial related antigens or antibodies;
Microorganism-related antigens or antibodies such as Tsutsugamushi Gilliam IgG, Tsutsugamushi Gilliam IgM, Tsutsugamushi Karp IgG, Tsutsugamushi Karp IgM, Tsutsugamushi Kato IgG, Tsutsugamushi Kato IgM, Toxoplasma antibody, Toxoplasma antibody IgG or Toxoplasma antibody IgM;
Blood group-related antigens or antibodies such as A antigen, anti-A antibody, B antigen, anti-B antibody, RhoD antigen, anti-RhoD antibody or irregular antibody;
An immunoglobulin or immunoglobulin superfamily such as immunoglobulin A (IgA), immunoglobulin G (IgG), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE);
Granulocyte elastase, eosinophil basic protein, CRP, ferritin, corticotropin releasing factor, α1 acid glycoprotein, α1 microglobulin, α2 microglobulin, β2 microglobulin, α1 antitrypsin, type IV collagen, amyloid A, albumin, myocardium Troponin T, ventricular myosin light chain I, pulmonary surfactant protein D, human heart-derived fatty acid binding protein, procollagen III peptide, myoglobin, IgG rheumatoid factor, LE factor, rheumatoid factor, platelet surface IgG, anti-DNA antibody, anti-dsDNA Antibody IgG, anti-dsDNA antibody IgM, anti-Jo-1 antibody, anti-LKM-1 antibody, anti-RNP antibody, anti-Scl-70 antibody, anti-Sm antibody, anti-SS-A antibody, anti-ssDNA antibody IgG, anti-ssDNA antibody IgM, Anti-acetylcholine receptor binding antibody, anti-acetylcholine receptor blocking antibody, anti-gastric wall cell antibody, antinuclear antibody group, anti-galactose Deficient IgG antibody, anti-cardiolipin β2GPI complex antibody, anti-cardiolipin antibody, anti-platelet antibody, anti-neutrophil cytoplasmic antibody, anti-neutrophil cytoplasmic myeloperoxidase antibody, anti-glomerular basement membrane antibody, anti-centromere antibody, anti-intrinsic factor antibody Anti-adrenocortical antibody, anti-smooth muscle antibody, anti-mitochondrial M2 antibody, anti-mitochondrial antibody, anti-thyroglobulin antibody, anti-microsomal antibody, myelin basic protein, immune complex, anti-C3d antibody, complement C3 or complement C4, etc. Inflammation / clinical laboratory markers; or
Type I collagen-C-telopeptide, type I collagen cross-linked N-telopeptide, α-fetoprotein, BCA225, CA15-3, CA19-9, CA54 / 61, CA72-4, CA125, CA130, CA602, DUPAN2, HER2, NCC -Tumor markers such as ST-439, PIVKA-II, PSA-ACT, PSA, SCC antigen, Span-1 antigen, γ-seminoprotein, elastase 1 or basic fetoprotein.
In addition, various hormones; various allergy-related antigens or antibodies; various blood coagulation-related substances; various viral DNAs or RNAs; various fungal / bacterial DNAs or RNAs; various animals such as humans or DNA or RNA of plants; DNA or RNA of various microorganisms; various food allergens; various environmental hormones, etc.
PRTR-regulated substances, Chemical Substances Control Law-regulated substances, Occupational Safety and Health Law-regulated substances, Fire and Fire Law-regulated substances, Poisonous and Deleterious Substances Control Law-regulated substances, High-Pressure Gas Control Law-regulated substances, Explosives Regulation Law-regulated substances, Air Pollution Control Law regulations Substances, Water Pollution Control Law Control Substances, Odor Control Law Control Substances, Marine Pollution Control Law Control Substances, Pharmaceutical Law Control Substances, Narcotics Control Law Control Substances, Hazard Control Substances, Aviation Law Control Substances, Port Law Control Substances, Chemical Various controlled substances such as substances regulated by the Weapons Prohibition Law, Agricultural Chemicals Regulation Law, ozone layer protection law, waste sweep law or import / export trade control regulations;
Various carbon materials; Various inorganic materials; Various cells; Various growth factors; Various viruses; Various bacteria; Various archaea; Various fungi; Various actinomycetes; Various filamentous fungi; Etc. can be cited as test substances.

  In step (i), after preparing the magnetic particles, members, and sample as described above, step (ii) is subsequently performed. In step (ii), magnetic particles and a sample are supplied to the member. Since both the magnetic particle substance a and the member substance b have binding properties to the test substance, when the magnetic particles and the sample come into contact with the member, the substance a and the substance b change the sample test substance. Thus, a composite body (ie, “a sandwich structure composed of a magnetic particle substance a, a test substance, and a member substance b”) is obtained. In forming the complex, not only the mode in which the substance a or the substance b is directly bonded to the test substance but also the mode in which the substance a or the substance b is bonded to the test substance via a linker may be used.

  The magnetic particles supplied in step (ii) have been described above in connection with step (i), but tris (hydroxymethyl) aminomethane buffer, phosphate buffer, PBS buffer, HEPES buffer, MOPS buffer or It is preferably used in a state dispersed in a solvent such as a buffer solution such as TBS buffer, physiological saline, ion exchange water, sterilized water or ultrapure water. Similarly, the sample supplied in step (ii) may be tris (hydroxymethyl) aminomethane buffer, phosphate buffer, PBS buffer, HEPES buffer, MOPS buffer, TBS buffer, etc., if necessary. It is preferably used in a state diluted with a buffer solution, physiological saline, ion exchange water, sterilized water or ultrapure water.

  In the step (ii), when a test substance is present in the sample, a composite having a sandwich structure is formed in a region where the material b of the member is fixed, while there is no test substance in the sample. And a composite of sandwich structure is not formed. Thus, when the test substance is present in the sample, the state of the member region where the substance b is immobilized changes due to the complex formed. Therefore, in the present invention, the presence / absence of the test substance or the amount of the test substance is determined by measuring the distribution state of the magnetic particles accompanying the formation of the complex using the change in physical characteristics or chemical characteristics. can do. For example, when the test substance is present, the color of the member area where the substance b is immobilized differs from the color of the member area where the substance b is not immobilized due to the complex formed. . Therefore, in the step (iii), the presence or absence of the test substance in the sample can be determined by visually confirming such a region (step (iv)). When the test substance is present, the color of the member region where the substance b is immobilized is caused by, for example, the coloration of the magnetic particles constituting the complex.

  When it is desired to determine the amount (or concentration) of the test substance contained in the sample, the color change, film thickness (or “thickness”) in the member region where the substance b is immobilized in step (iii). ) Change, weight change, conductivity change, magnetic change, or light scattering property change, etc. may be measured. In this case, it is preferable to obtain in advance a calibration curve representing the relationship between the change amount of the parameter and the amount of the test substance (that is, “correlation between the change amount and the test substance amount”). Thereby, in the step (iv), the amount of the test substance can be obtained based on the calibration curve and the parameter change amount measured in the step (iii). In addition, when the amount of change of the parameter is 0 in the step (iii), the amount of the test substance is also 0, and therefore the presence or absence of the test substance can be determined by measuring the parameter change amount.

  Next, various embodiments of the present invention will be described with reference to the drawings. In addition, about the same component, the same code | symbol may be attached | subjected and description may be abbreviate | omitted.

(First embodiment)
The first embodiment mainly shows the principle of the method of the present invention. FIG. 2 shows the formation process of the composite according to the first embodiment. An example will be described in which the test substance contained in the sample is an antigen, and substance a and substance b are antibodies that can specifically bind to the antigen.

  First, as shown in FIG. 2A, a member 6 on which an antibody 5 is immobilized is prepared. Next, by supplying a sample containing the test substance 7 (ie, “antigen”) to the member 6, the antibody 5 of the member 6 and the test substance 7 react with each other as shown in FIG. Connect with each other. Next, as shown in FIG. 2C, the magnetic particles 1 are supplied to the member 6 and a magnetic field is applied from the back side of the member 6. Specifically, the magnet 11 is disposed below the surface opposite to the surface on which the antibody 5 is immobilized. As a result, the magnetic particles 1 are placed under the influence of the magnetic field, and the magnetic particles 1 move in the direction of the antibody 5 of the member 6. Finally, the antibody 8 of the magnetic particle 1 binds to the test substance 7 to form a composite 9 having a sandwich structure as shown in FIG. After the complex 9 is formed, it is preferable to remove the magnetic particles 1 that have not contributed to the complex formation by stopping the application of the magnetic field and washing the member surface on which the antibody 5 is immobilized. .

  In such a method of the present invention, when the test substance 7 is present in the sample, the complex 9 is formed in the region a (see FIG. 2A) of the member 6 to which the antibody 5 is immobilized. The presence or absence of the test substance 7 (that is, “positive” or “negative”) can be determined from the state change of the region a caused by the complex 9. In addition, when the state change in the region a is measured, the amount of the test substance 7 can also be obtained.

  In the method of the present invention, since the complex 9 is formed under the influence of a magnetic field, the complex 9 can be formed in a relatively short time. As a result, the test can be performed in a relatively short time. There is an advantage that the substance 7 can be detected.

(Second Embodiment)
In the second embodiment, the member prepared in the step (i) is a plate member or a container, and the substance b is immobilized on at least a part of the surface a of the surface, and the step (ii) 2, after supplying the sample to the surface a, the magnetic particles are supplied to the surface b of the member other than the surface a, and a magnetic field is applied from the vicinity of the surface a. As the “form in which a magnetic field is applied from the vicinity of the surface a”, for example, a form in which a magnet is arranged in the vicinity of the surface a, or a form in which a current is passed through an electromagnet arranged in advance in the vicinity of the surface a can be considered. FIG. 3A shows a process of forming a complex according to the second embodiment. As in the first embodiment, a case where the test substance contained in the sample is an antigen and the substance a and the substance b are antibodies that can specifically bind to the antigen will be described as an example.

  First, as shown in FIG. 3A (a), the antibody 5 is immobilized on a part of the surface a of the member 10 such as a plate member. Next, by supplying a sample containing the test substance 7 to the surface a, as shown in FIG. 3A (b), the antibody 5 of the member 10 and the test substance 7 are bonded to each other by an antigen-antibody reaction. Next, as shown in FIG. 3A (c), the magnetic particles 1 are supplied to the surface b of the member other than the surface a. Then, as shown in FIG. 3A (d), a magnetic field is applied from the vicinity of the surface a to place the magnetic particles 1 under the influence of the magnetic field. Specifically, the magnet 11 is arranged on the back surface or side surface of the member 10 so that the supplied magnetic particle 1 and the magnet 11 sandwich the surface a so that the magnetic particle 1 passes through the surface a. Thereby, the magnetic particle 1 moves from the surface b to the surface a. Eventually, since the antibody 8 of the magnetic particle 1 binds to the test substance 7, as shown in FIG. 3A (e), a complex 9 is formed on the surface a on which the antibody 5 of the member 10 is immobilized. Is done. Therefore, the presence or absence of the test substance 7 can be determined from the state change of the region a caused by the complex 9, and the amount of the test substance 7 can be obtained from the change amount. Also in this embodiment, since the complex 9 is formed under the influence of a magnetic field, the movement of the magnetic particles 1 is promoted, and the complex 9 can be formed in a relatively short time. Therefore, the test substance 7 can be detected in a relatively short time. A groove may be provided in the region of the member 10 to which the magnetic particles 1 move so that the magnetic particles can move easily.

  As can be seen from this embodiment, in this specification, “applying a magnetic field from the vicinity of the surface a” substantially means “applying a magnetic field so as to promote the movement of the magnetic particles to the surface a”. It should be noted that the present invention is not limited to a mode in which a magnetic field is applied only from a position close to the surface a. In such an embodiment, the magnetic particles that did not contribute to the formation of the complex are finally attracted to the vicinity of the magnet through the surface a, so that the operation of removing excess magnetic particles is performed. There is also an advantage in terms of saving.

(Third embodiment)
In the third embodiment, the member prepared in the step (i) is a plate member or a container, and the substance b is immobilized on at least a part of the surface a of the surface, and the step (ii) 2, after supplying a sample and magnetic particles to the surface b of the member other than the surface a, a magnetic field is applied from the vicinity of the surface a. FIG. 3B shows the formation process of the complex according to the third embodiment. As in the first embodiment and the second embodiment, the case where the test substance contained in the sample is an antigen and the substance a and the substance b are antibodies that can specifically bind to the antigen is taken as an example. explain.

  First, as shown in FIG. 3B (a), the antibody 5 is immobilized on a part of the surface a of the member 10 such as a plate member. Next, as shown in FIG. 3B (b), the sample containing the test substance 7 and the magnetic particles 1 are supplied to the surface b of the member other than the surface a. Next, as shown in FIG. 3B (c), a magnetic field is applied from the vicinity of the surface a to place the magnetic particles under the influence of the magnetic field. Specifically, the magnet 11 is arranged on the back surface or side surface of the member 10 so that the supplied magnetic particles 1 and the magnet 11 sandwich the surface a so that the magnetic particles pass through the surface a. As a result, as shown in FIG. 3B (c), the magnetic particles 1 move from the surface b toward the surface a. At this time, the magnetic particle 1 and the test substance 7 are bonded to each other by an antigen-antibody reaction between the antibody 8 of the magnetic particle 1 and the test substance 7. Finally, since the test substance 7 binds to the antibody 5 of the member 10, as shown in FIG. 3B (d), the complex 9 is formed on the surface a on which the antibody 5 of the member 10 is immobilized. Will be. Therefore, the presence or absence of the test substance 7 can be determined from the state change of the region a caused by the complex 9, and the amount of the test substance 7 can be obtained from the change amount. Also in this embodiment, since the complex 9 is formed under the influence of a magnetic field, the movement of the magnetic particles 1 is promoted, and the complex 9 can be formed in a relatively short time. Therefore, the test substance 7 can be detected in a relatively short time. As in the second embodiment, a groove may be provided in the region of the member 10 to which the magnetic particles 1 move so that the magnetic particles 1 can move easily.

(Fourth embodiment)
In the fourth embodiment, the member prepared in step (i) is a member made of porous or fibrous material, and the substance b is fixed to at least a part of the internal region a. Yes, in step (ii), the sample and the magnetic particles are provided to the region b of the member other than the internal region a, and then a magnetic field is applied from the vicinity of the internal region a (immunochromatography method). As the “form in which a magnetic field is applied from the vicinity of the internal area a”, for example, a form in which a magnet is arranged in the vicinity of the internal area a, or a form in which a current is passed through an electromagnet arranged in advance in the vicinity of the internal area a is conceivable. . FIG. 4A shows a process of forming a complex according to the fourth embodiment. As in the first to third embodiments, the case where the test substance contained in the sample is an antigen and the substance a and the substance b are antibodies that can specifically bind to the antigen is taken as an example. explain.

  First, a member 12 in which the antibody 5 is immobilized in at least a part of the internal region a is prepared, and the magnetic particles 1 and the sample are provided in a region b other than the internal region a of the member 12. Specifically, as shown in FIG. 4A (a), a magnetic particle-impregnated member (for example, a member made of porous or fibrous material) 13 impregnated with a dispersion of magnetic particles 1 is disposed in a region b, and a sample A pad (for example, a porous or fibrous sample pad) 14 is disposed in the region b, and a sample is supplied to the sample pad 14. It is preferable that the magnetic particle impregnated member 13 and the sample pad 14 are arranged so as to partially overlap each other. When the sample is supplied to the sample pad 14, the test substance 7 contained in the sample is included in the sample pad 14 as shown in FIG. 4A (b). Next, since the test substance 7 in the sample moves from the sample pad 14 into the magnetic particle impregnated member 13 due to so-called capillary phenomenon, as shown in FIG. The magnetic particles 1 and the test substance 7 are bound to each other by an antigen-antibody reaction between the antibody 8 of the particle 1 and the test substance 7. Next, as shown in FIG. 4A (d), a magnetic field is applied from the vicinity of the inner region a to place the magnetic particles 1 under the influence of the magnetic field. Specifically, the magnet 11 is arranged on the back surface or the side surface of the member 12 so that the provided magnetic particles 1 and the magnet 11 sandwich the internal region a so that the magnetic particles pass through the internal region a. As a result, the magnetic particles 1 move together with the test substance 7 in the direction from the region b toward the internal region a. Finally, since the test substance 7 and the antibody 5 of the member 12 are bound, as shown in FIG. 4A (e), the complex 9 is formed in the internal region a where the antibody 5 of the member 12 is immobilized. Will be formed. Therefore, the presence or absence of the test substance 7 can be determined from the change in the state of the internal region a caused by the complex 9, and the amount of the test substance 7 can also be obtained from the change amount. Also in this embodiment, the complex 9 is formed under the influence of a magnetic field, and the complex 9 can be formed in a relatively short time. As a result, the test substance 7 can be formed in a relatively short time. It can be detected. In the fourth embodiment, the magnetic particles 1 and the test substance 7 can move in the member 12 due to capillary action even when no magnetic field is applied, but are placed under the influence of the magnetic field. Note that this facilitates such movement.

  Further, as can be seen from this embodiment, in this specification, “applying a magnetic field from the vicinity of the internal region a” means “applying a magnetic field so as to promote the movement of the magnetic particles to the internal region a”. It should be noted that this means substantially, and the present invention is not limited to the form in which the magnetic field is applied only from the position close to the inner region a. In addition, “a member in which an antibody is immobilized in at least a part of the internal region a” does not require that all of the antibody is immobilized in the internal region, and a part of the antibody is solidified on the surface. Note also that it may be in the form. Even in such an embodiment, as in the second embodiment, the magnetic particles that have not contributed to the formation of the complex will eventually be attracted to the vicinity of the magnet through the internal region a. There is an advantage in that the operation of removing excess magnetic particles can be omitted.

(Fifth embodiment)
In the fifth embodiment, the member prepared in step (i) is a member made of porous or fibrous material, and the substance b is fixed to at least a part of the inner region a. Yes, in step (ii), after supplying the sample to the internal region a, the magnetic particles are provided to the region b of the member other than the internal region a, and then a magnetic field is applied from the vicinity of the internal region a. FIG. 4B shows a formation process of the complex according to the fifth embodiment. As in the first to fourth embodiments, the case where the test substance contained in the sample is an antigen and the substance a and the substance b are antibodies that can specifically bind to the antigen is taken as an example. explain.

  First, as shown in FIG. 4B (a), a member 12 in which the antibody 5 is immobilized in at least a part of the internal region a is prepared. Next, by subjecting the sample containing the test substance 7 to the internal region a, as shown in FIG. 4B (b), the antibody 5 of the member 12 and the test substance 7 are bonded to each other by an antigen-antibody reaction. Next, as shown in FIG. 4B (c), the magnetic particles 1 are provided in the region b of the member other than the internal region a. At this time, similarly to the fourth embodiment, a magnetic particle impregnated member (for example, a member made of porous or fibrous material) 13 impregnated with a dispersion of magnetic particles 1 is preferably disposed in the region b. Next, as shown in FIG. 4B (d), a magnetic field is applied from the vicinity of the inner region a to place the magnetic particles 1 under the influence of the magnetic field. Specifically, the magnet 11 is arranged on the back surface or side surface of the member 12 so that the provided magnetic particle 1 and the magnet 11 sandwich the internal region a so that the magnetic particle 1 passes through the internal region a. As a result, the magnetic particles 1 move from the region b toward the inner region a. Eventually, the test substance 7 in the inner region a and the antibody 8 of the magnetic particle 1 bind to each other, so that a complex 9 is formed in the inner region a. Therefore, the presence or absence of the test substance 7 can be determined from the state change of the region a caused by the complex 9, and the amount of the test substance 7 can be obtained from the change amount. Also in this embodiment, since the complex 9 is formed under the influence of a magnetic field, the complex 9 can be formed in a relatively short time, and the test substance 7 can be detected in a relatively short time. can do. In this embodiment as well, as in the fourth embodiment, the magnetic particles 1 can move inside the member 12 due to capillary action even when no magnetic field is applied, but under the influence of the magnetic field. Note that such movement is facilitated by being placed.

(Sixth embodiment)
In the sixth embodiment, a substance b is immobilized in a partial region a of the member prepared in the step (i). In the step (ii), a sample is placed in the region b of the member other than the region a. The magnetic particles are supplied to move the magnet from the vicinity of the region b to the vicinity of the region a. FIG. 5A shows a process of forming a complex according to the sixth embodiment. As in the first to fifth embodiments, the case where the test substance contained in the sample is an antigen and the substance a and the substance b are antibodies that can specifically bind to the antigen is taken as an example. explain. Further, the region a and the region b of the member will be described as the surface a and the surface b of the member.

  First, as shown in FIG. 5A (a), the antibody 5 is immobilized on a part of the surface a of the member 10. Next, as shown in FIG. 5A (b), the sample containing the test substance 7 and the magnetic particles 1 are supplied to the surface b of the member other than the surface a. Next, as shown in FIG. 5A (c), the magnet 11 is moved in the direction of arrow A from the vicinity of the surface b to the vicinity of the surface a. As a result, the magnetic particles 7 move from the surface b toward the surface a. At this time, the magnetic particle 1 and the test substance 7 are bonded to each other by an antigen-antibody reaction between the antibody 8 of the magnetic particle 1 and the test substance 7. Eventually, as shown in FIG. 5A (d), the complex 9 is formed on the surface a on which the antibody 5 of the member 10 is immobilized. Therefore, the presence or absence of the test substance 7 can be determined from the change in the state of the surface a caused by the complex 9, and the amount of the test substance 7 can also be obtained from the change amount. Also in this embodiment, since the complex 9 is formed under the influence of a magnetic field, the movement of the magnetic particles 1 is promoted, and the complex 9 can be formed in a relatively short time. Therefore, as a result, the test substance 7 can be detected in a relatively short time. As in the second or third embodiment, a groove may be provided in the region of the member 10 to which the magnetic particles 1 move so that the magnetic particles 1 can move easily.

  In the sixth embodiment, the sample is supplied to the surface b. However, similarly to the second embodiment (that is, the form shown in FIG. 3A), the sample is supplied to the surface a and is preliminarily bound to the antibody 5 of the member 10. (This form is referred to as “seventh embodiment”). In the case of the seventh embodiment, as shown in FIG. 5B, the substance b is immobilized in a partial region a (“surface a” in the illustrated form) of the member prepared in step (i). In the step (ii), after supplying the sample to the region a, the magnetic particles are supplied to the region b (“surface b” in the illustrated embodiment) of the member other than the region a, and then in the vicinity of the region b In this mode, the magnet is moved to the vicinity of the region a. The specific formation process of the complex of the seventh embodiment is not described because it overlaps with the second embodiment and the sixth embodiment.

  The members used in the sixth embodiment and the seventh embodiment are not only plate members or containers as in the second embodiment or the third embodiment, but also porous members as in the fourth embodiment or the fifth embodiment. It may be a member made of fibrous material. When the member is made of porous or fibrous material, the substance b is fixed to a part of the inner region of the member. Furthermore, in the sixth embodiment and the seventh embodiment, the magnet to be moved may be an electromagnet. In the case of using an electromagnet, an electromagnet is disposed in the vicinity of the region b in advance, and a current is passed through the electromagnet after the magnetic particles are supplied, and the electromagnet is moved in the direction of the region a.

  As mentioned above, although various forms regarding the detection method of the present invention have been described, those skilled in the art will understand that the detection method of the present invention is not limited to this and various modifications can be made.

  For example, an avidin-biotin reaction may be used to form a complex. In this case, it becomes the following modes. Incidentally, avidin and biotin have the property of easily binding to each other. Avidin may be streptavidin or neutravidin.

(Case 1)
Case 1 will be described with reference to FIG. 6A. First, as shown in FIG. 6A (a), the specific binding substance 20 (ie, “substance b”) is immobilized on the member 21. Next, a sample is supplied, and the test substance 22 is bound to the specific binding substance 20 as shown in FIG. 6A (b). Next, as shown in (c) and (d) of FIG. 6A, the specific binding substance 24 modified with avidin 23 is bound to the test substance 22. Next, as shown in FIG. 6A (e), the noble metal-coated magnetic particles 26 to which biotin 25 is immobilized are bound to the avidin 23 via an avidin-biotin reaction. Thereby, a composite 27 having a sandwich structure as shown in FIG. 6A (f) can be obtained.

(Case 2)
Case 2 will be described with reference to FIG. 6B. First, as shown in FIG. 6B (a), the specific binding substance 20 (ie, “substance b”) is immobilized on the member 21. Next, a sample is supplied, and the test substance 22 is bound to the specific binding substance 20 as shown in FIG. 6B (b). Next, as shown in (c) and (d) of FIG. 6B, the specific binding substance 24 modified with biotin 25 is bound to the test substance 22. Next, as shown in FIG. 6B (e), the noble metal-coated magnetic particles 26 to which the avidin 23 is immobilized are bound to the biotin 25 through an avidin-biotin reaction. Thereby, a composite 27 having a sandwich structure as shown in FIG. 6B (f) can be obtained.

(Case 3)
Case 3 will be described with reference to FIG. 6C. First, as shown in FIG. 6C (a), the specific binding substance 20 (ie, “substance b”) is immobilized on the member 21. Next, a sample is supplied, and the test substance 22 is bound to the specific binding substance 20 as shown in FIG. 6C (b). Next, as shown in FIG. 6C (c), the specific binding substance 24 modified with avidin 23 is preliminarily bound to the noble metal-coated magnetic particles 26 on which biotin 25 is immobilized, via an avidin-biotin reaction. Next, by binding the specific binding substance 24 to the test substance 22 on the specific binding substance 20, a composite 27 having a sandwich structure as shown in FIG. 6C (d) can be obtained.

(Case 4)
Case 4 will be described with reference to FIG. 6D. First, as shown in FIG. 6D (a), the specific binding substance 20 (ie, “substance b”) is immobilized on the member 21. Next, a sample is supplied, and the test substance 22 is bound to the specific binding substance 20 as shown in FIG. 6D (b). Next, as shown in FIG. 6D (c), the specific binding substance 24 modified with biotin 25 is preliminarily bound to the noble metal-coated magnetic particles 26 on which the avidin 23 is immobilized, through an avidin-biotin reaction. Next, by binding the specific binding substance 24 to the test substance 22 on the specific binding substance 20, a complex 27 having a sandwich structure as shown in FIG. 6D (d) can be obtained.

  The description so far is based on the sandwich method for forming a complex composed of the substance b of the member, the substance to be detected and the substance a of the magnetic particles, but is not necessarily limited thereto. For example, instead of using a combination of an enzyme and a chromogenic substrate in a competitive method, immunoblotting method, Western blotting method, Southern blotting method, Northern blotting method, etc., when using magnetic particles prepared in step (i) of the present invention, The detection of the test substance can be suitably performed. Further, for example, in the latex agglutination turbidimetry, latex near-infrared turbidimetry, particle agglutination, etc., the detection of the test substance is preferably performed by using the magnetic particles prepared in step (i) of the present invention. Can do.

  Next, the method for preparing magnetic particles prepared in step (i) of the detection method of the present invention will be described in more detail below.

The magnetic particles used in step (i) are preferably prepared by a method comprising the following steps (1) to (3):
(1) preparing a magnetic particle p having a central region formed of a magnetic material and an outer shell region formed of a noble metal material, and a substance a capable of binding to a test substance;
(2) contacting the magnetic particles p with the substance a to obtain a mixture comprising the magnetic particles p ′ having the substance a immobilized thereon (that is, “magnetic particles used in the step (i)”); 3) A step of applying a magnetic field to the obtained mixture to separate the magnetic particles p ′ from the mixture (ie, “magnetic separation step”).

  Step (2) and Step (3) are carried out in a solution such as a buffer (eg, tris (hydroxymethyl) aminomethane buffer, phosphate buffer, PBS buffer, HEPES buffer or MOPS buffer) or in an organic solvent. It is preferable to carry out.

  In such a preparation method, since the magnetic particles p ′ are separated from the mixture under the influence of a magnetic field, the magnetic particles used in the step (i) can be obtained in a relatively short time. Further, since the separation operation and the filter operation are not performed when the magnetic particles p ′ are separated, there is an advantage that the whole process can be easily automated. Further, when the substance a contains a sulfur atom, the sulfur atom and the noble metal material of the magnetic particle p are easily bonded to each other, so that the substance a can be fixed to the magnetic particle p relatively easily.

  In step (3), as shown in FIG. 7, by applying a magnetic field, the mixture obtained in step (2) is divided into phase A having more magnetic particles p ′ and phase B having fewer magnetic particles p ′. After that, it is preferred to remove phase B from the mixture.

  Further, in the step (2), the “linker containing a functional group capable of binding to the substance a and a sulfur atom” is bonded to the magnetic particle p, and then the substance a is bonded to the linker, thereby magnetically Particle p ′ may be obtained. Similarly, in step (2), a “linker comprising a functional group capable of binding to substance a and a sulfur atom” is bonded to substance a in advance, and then the linker is bonded to magnetic particles p, thereby forming magnetic properties. Particle p ′ may be obtained. The reason why the linker contains a sulfur atom is that the sulfur atom has a property of being easily bonded to a noble metal contained in the magnetic particle p, and the linker is easily bonded to the magnetic particle by utilizing the property. Because. Examples of the “functional group capable of binding to substance a” include sulfide functional groups such as thiol groups, thioether groups or disulfide groups, aldehyde groups, epoxy groups, tosyl groups, azide groups, succinimide groups, maleimide groups, hydrazide groups, amino groups. A functional group such as a group, a carboxyl group, an imide ester group, a carbodiimide group, an isocyanate group, an iodoacetyl group or a double bond, or a functional group derivative thereof.

As the linker, for example, “heterobivalent crosslinking reagent” or “homobivalent crosslinking reagent” can be used.
“Heterobivalent cross-linking reagents” include, for example,
A sulfide functional group such as a thiol group, a thioether group or a disulfide group or the functional group derivative;
Sulfide functional groups such as thiol groups, thioether groups or disulfide groups, aldehyde groups, epoxy groups, tosyl groups, azide groups, succinimide groups, maleimide groups, hydrazide groups, primary amino groups, secondary amino groups, tertiary amino groups, Mention may be made of heterobivalent crosslinking reagents comprising a functional group such as a carboxyl group, an imide ester group, a carbodiimide group, an isocyanate group, an iodoacetyl group, an acid chloride or a double bond, or a combination with the functional group derivative.
Examples of the “homobivalent crosslinking reagent” include a homobivalent crosslinking reagent comprising a sulfide functional group such as thiol, thioether, disulfide or the like and a functional group derivative thereof.

  As an example, a mode using a “heterobivalent crosslinking reagent” as a linker will be described. First, the substance a and the heterobivalent cross-linking reagent are added and stirred in a buffer solution or an organic solvent. Thereby, both react and the conjugate | bonded_body of the substance a and heterobivalent crosslinking reagent is obtained. Next, this conjugate is extracted and brought into contact with the magnetic particle p, whereby the substance a is immobilized on the magnetic particle p ′ through the intervention of the heterobivalent crosslinking reagent. Finally, it is preferable to magnetically separate the magnetic particles p ′ and then wash with a buffer solution or an organic solvent.

  When the substance a is bound to the magnetic particle p using a linker such as the above-mentioned “heterobivalent crosslinking reagent” and “homobivalent crosslinking reagent”, a non-specific reaction to the magnetic particle p is performed as necessary. Is preferably suppressed. Therefore, blocking that suppresses nonspecific adsorption to the magnetic particles p may be performed by a known method such as bringing normal serum, bovine serum albumin, human serum albumin, casein, skim milk powder, etc. into contact with the magnetic particles p. .

  As described above, the detection method of the test substance using the noble metal-coated magnetic particles and the preparation method of the noble metal-coated magnetic particles have been described. In the present invention, not only such a detection method but also a test substance detection kit that can be used for the detection method is provided.

  The test substance detection kit of the present invention comprises magnetic particles and members. The “magnetic particles” and the “member” are respectively prepared as “the central region is formed from a magnetic material and the outer shell region is formed from a noble metal material, prepared in the step (i) of the test substance of the present invention. Magnetic particles in which a substance a capable of binding to a test substance is immobilized in the outer shell region "and" a substance b capable of binding to a test substance are immobilized. Means a member. Therefore, since the “magnetic particles” and “members” included in the test substance detection kit have already been described in detail above, no further description will be given to avoid duplication. The detection kit of the present invention may include a magnet or an electromagnet used for applying a magnetic field.

  In order to confirm the effect of the detection method of the present invention, the following Examples 1 to 3 and Comparative Examples 1 to 3 were carried out. Examples 1 to 3 are examples using noble metal coated magnetic particles, and Comparative Examples 1 to 3 are examples using gold colloid.

Example 1
(Production and preparation of precious metal coated magnetic particles)
In Example 1, magnetic particles prepared in step (i) of the detection method of the present invention were produced and prepared. Fe ₅₀ Pt ₅₀ alloy (hereinafter simply referred to as “Fe ₅₀ Pt ₅₀ ”) is used as the magnetic material, Au is used as the noble metal material, and egg white albumin is used as “substance a capable of binding to the test substance”. Antibody was used.

In the production of noble metal-coated magnetic particles, Fe ₅₀ Pt ₅₀ magnetic particles were used as the material of the central region, and the surface thereof was covered with an Au layer by a displacement plating method. The obtained particles were embedded with an acrylic resin, an ultrathin section was prepared using a microtome, and observed with an electron microscope. As a result, it was found that the diameter of the noble metal-coated magnetic particles was 50 nm, and the outer periphery thereof was covered with Au having a thickness of 20 nm. Moreover, as a result of measuring the magnetic characteristics of the obtained particles using a sample vibration type magnetometer (manufactured by Toei Kogyo, model VSM-5), the saturation magnetization is 60 emu / g, and the coercive force is 800 Oersted. I understood that.

First, ovalbumin antibody was dissolved in PBS to prepare a solution of ovalbumin antibody having a concentration of 1 mg / ml. Next, Au-coated Fe ₅₀ Pt ₅₀ magnetic particles were dispersed in a PBS buffer solution to obtain an Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion. Next, an ovalbumin antibody solution in which a thiol group was introduced with S-acetylacetic acid active ester was added to 5 mg / ml Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion, and 1 ml of Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion. After adding at a rate of 10 μg per 0.1 ml, 0.1 ml of BSA having a concentration of 10 mg / ml was added to 1 ml thereof. And it attached to magnetic separation as shown in FIG. 7, and removed the supernatant liquid. By using PBS containing BSA at 1 mg / ml, it was washed three times by magnetic separation. Through the above operation, a dispersion of Au-coated Fe ₅₀ Pt ₅₀ magnetic particles on which the ovalbumin antibody was immobilized was obtained.

<< Comparative Example 1 >>
(Manufacture and preparation of colloidal gold)
In order to compare with “Au coated Fe ₅₀ Pt ₅₀ magnetic particles with immobilized ovalbumin antibody” in Example 1, “gold colloid with immobilized ovalbumin antibody” was prepared and prepared.

  First, 200 ml of 0.01% by weight gold chloride aqueous solution is boiled, 1% by weight sodium citrate aqueous solution is added, and the mixture is heated and boiled until the color of the solution changes from pale yellow to purple or red, thereby dispersing the gold colloid. A liquid was prepared (average particle diameter of colloidal gold: 0.04 μm).

  Ovalbumin antibody was dissolved in PBS to prepare a solution of ovalbumin antibody having a concentration of 1 mg / ml. Next, to the 5 mg / ml colloidal gold dispersion, an ovalbumin antibody solution into which a thiol group was introduced with S-acetylacetic acid activated ester was added at a rate of 10 μg per 1 ml of colloidal gold dispersion, and then 1 ml 0.1 ml of BSA having a concentration of 10 mg / ml was added. Subsequently, the supernatant was removed by centrifugation. By using PBS containing BSA at 1 mg / ml, it was washed three times by centrifugation. Through the above operation, a colloidal gold dispersion in which the ovalbumin antibody was immobilized was obtained.

Example 2
(Detection of ovalbumin by immunochromatography using precious metal-coated magnetic particles)
The detection method of the present invention was performed by immunochromatography as shown in FIG. 4A. As the noble metal-coated magnetic particles, the “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion” obtained in Example 1 was used. The test substance is ovalbumin.

First, ovalbumin antibody was dissolved in PBS to prepare a solution of ovalbumin antibody having a concentration of 1 mg / ml. Next, an ovalbumin antibody solution in which a thiol group was introduced with S-acetylacetic acid active ester was added to 5 mg / ml of “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion”, and Au-coated Fe ₅₀ Pt ₅₀ magnetic particles After adding 10 μg per 1 ml of the dispersion, 0.1 ml of BSA having a concentration of 10 mg / ml was added to 1 ml of the dispersion. Subsequently, the supernatant was removed by magnetic separation. After washing three times by magnetic separation using PBS containing BSA at 1 mg / ml, Au-coated Fe ₅₀ Pt ₅₀ magnetic particles were redispersed in 1 ml of the PBS. Next, the “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle-labeled antibody-containing nonwoven fabric” was prepared by impregnating the Au-coated Fe ₅₀ Pt ₅₀ magnetic particles into a non-woven fabric and lyophilization.

  A 10 mm × 50 mm chromatographic strip is cut from commercially available nitrocellulose (Millipore), and 2 μl of ovalbumin antibody solution is applied in a 10 mm length in a direction perpendicular to the development direction at a position 2 cm from the end a. did.

The end of the short side of the chromatographic strip was aligned and overlapped on a 10 mm × 80 mm adhesive resin, and a filter paper of 10 mm × 60 mm was further stacked thereon and fixed. “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle-labeled antibody-containing non-woven fabric” was disposed in a region near the end b different from the end a of the chromatographic strip. Furthermore, a 10 mm × 20 mm non-woven fabric was placed as a sample pad so as to overlap the “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle-labeled antibody-containing non-woven fabric”.

  Commercially available ovalbumin (CALBIOCHEM) was diluted with PBS buffer containing BSA at 1 mg / ml to prepare a 20 ng / ml ovalbumin sample.

  200 μl of the ovalbumin sample was dropped onto a sample pad and developed. After 10 minutes, “a chromatographic strip region coated with an ovalbumin antibody solution” was observed. As a result, a line indicating the presence of ovalbumin (ie, “color change”) could be confirmed.

Next, the same immunochromatography method was implemented in the aspect which arrange | positions a magnet in the position as shown to FIG. 4A. That is, immunochromatography was performed in a state where Au-coated Fe ₅₀ Pt ₅₀ magnetic particles were placed under the influence of a magnetic field. Even in this case, a line indicating the presence of ovalbumin could be confirmed in the "chromatographic strip region coated with an ovalbumin antibody solution".

<< Comparative Example 2 >>
(Detection of ovalbumin by immunochromatography using colloidal gold)
By using gold colloid instead of noble metal-coated magnetic particles, an immunochromatography method as shown in FIG. 4A was performed. As the gold colloid, the gold colloid dispersion obtained in Comparative Example 1 was used. The test substance is ovalbumin as in Example 2.

  First, ovalbumin antibody was dissolved in PBS to prepare a solution of ovalbumin antibody having a concentration of 1 mg / ml. Next, after adding a solution of ovalbumin antibody introduced with a thiol group with S-acetylacetic acid activated ester to a 5 mg / ml colloidal gold dispersion at a rate of 10 μg per 1 ml of colloidal gold dispersion, 0.1 ml of BSA having a concentration of 10 mg / ml was added to 1 ml. Subsequently, the supernatant was removed by centrifugation. After washing with PBS containing BSA at 1 mg / ml three times by centrifugation, the gold colloid was redispersed in 1 ml of the PBS. Subsequently, a colloidal gold colloid was impregnated into a non-woven cloth and subjected to lyophilization to prepare a “gold colloid-labeled antibody-containing nonwoven fabric”.

  A 10 mm × 50 mm chromatographic strip was cut from commercially available nitrocellulose (manufactured by Millipore), and 2 μl of ovalbumin antibody solution was applied in a length of 10 mm perpendicular to the development direction at a position 2 cm from the end a. .

  The end of the short side of the chromatographic strip was aligned and overlapped on a 10 mm × 80 mm adhesive resin, and a filter paper of 10 mm × 60 mm was further stacked thereon and fixed. A “golden colloid-labeled antibody-containing non-woven fabric” was arranged in a region near the end b different from the end a of the chromatographic strip. Further, a 10 mm × 20 mm non-woven fabric was placed as a sample pad so as to overlap the “gold colloid-labeled antibody-containing non-woven fabric”.

  Commercially available ovalbumin (CALBIOCHEM) was diluted with PBS buffer containing BSA at 1 mg / ml to prepare a 20 ng / ml ovalbumin sample.

  200 μl of the ovalbumin sample was dropped onto a sample pad and developed. After 10 minutes, “a chromatographic strip region coated with an ovalbumin antibody solution” was observed. As a result, a line indicating the presence of ovalbumin (ie, “color change”) could be confirmed.

Example 3
(Detection of C-reactive protein (CRP) by sandwich immunoassay with magnetic induction of precious metal-coated magnetic particles)
The detection method of the present invention was performed by a sandwich immunoassay method using magnetic induction as shown in FIG. 3A. As the noble metal-coated magnetic particles, the “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion” obtained in Example 1 was used. The test substance is protein (CRP).

First, CRP antibody was dissolved in PBS to prepare a CRP antibody solution having a concentration of 1 mg / ml. For 5 mg / ml of “Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion”, a solution of a CRP antibody into which a thiol group was introduced with S-acetylacetic acid activated ester was added to 1 ml of Au-coated Fe ₅₀ Pt ₅₀ magnetic particle dispersion. After adding at a rate of 10 μg per 0.1 ml, 0.1 ml of BSA having a concentration of 10 mg / ml was added to 1 ml thereof. Subsequently, the supernatant was removed by magnetic separation. Using PBS containing BSA at 1 mg / ml, it was washed three times by magnetic separation. Through the above operation, a dispersion of noble metal-coated magnetic particles having a CRP antibody immobilized thereon was obtained.

  A commercially available glass slide (manufactured by Matsunami Glass) was subjected to silane coupling treatment, and the surface of the glass slide was aminated. Next, glutaraldehyde was added to the surface of the slide glass, reacted, and then washed. Next, a solution in which CRP antibody was dissolved in PBS buffer solution was applied to a part of the surface of the slide glass, then allowed to stand overnight, and washed with PBS. By such an operation, a plate substrate having a CRP antibody immobilized on a part of the surface was obtained. The plate region where the CRP antibody is immobilized is referred to as “CRP antibody immobilization site”, and the plate region where the CRP antibody is not immobilized is referred to as “CRP antibody non-immobilization site”.

  A commercially available CRP (manufactured by CHEMICON) was diluted with a PBS buffer containing BSA at 1 mg / ml to prepare a 200 ng / ml CRP sample.

  200 μl of the CRP sample was dropped onto the plate substrate, allowed to stand, washed, and then a “dispersion of precious metal-coated magnetic particles with the CRP antibody immobilized” was further dropped onto the CRP antibody non-immobilized site. did. Next, the supplied noble metal-coated magnetic particles and magnets sandwich the CRP antibody immobilization site so that the “noble metal-coated magnetic particles with CRP antibody immobilized” pass through the CRP antibody immobilization site. A magnet was placed on the side. As a result, the “noble metal coated magnetic particles with the CRP antibody immobilized” were subjected to magnetic induction under the influence of a magnetic field. Thereafter, the CRP antibody immobilization site was observed. As a result, a change in color indicating the presence of CRP could be confirmed.

<< Comparative Example 3 >>
(Detection of C-reactive protein (CRP) by sandwich immunoassay using colloidal gold)
Sandwich immunoassay was performed by using gold colloid rather than precious metal coated magnetic particles. The gold colloid was prepared in the same manner as in Comparative Example 1. The test substance is protein (CRP) as in Example 3.

  First, a gold colloid dispersion was prepared by the method described in Comparative Example 1 (average particle diameter of gold colloid: 0.04 μm).

  Next, CRP antibody was dissolved in PBS to prepare a CRP antibody solution having a concentration of 1 mg / ml. Next, a solution of CRP antibody into which thiol group was introduced with S-acetylacetic acid active ester was added to 5 mg / ml of gold colloid at a rate of 10 μg per 1 ml of colloidal gold dispersion, and then 10 mg concentration in 1 ml 0.1 ml of / ml BSA was added. Subsequently, the supernatant was removed by centrifugation. By using PBS containing BSA at 1 mg / ml, it was washed three times by centrifugation. Through the operations described above, “a dispersion of colloidal gold in which the CRP antibody was immobilized” was obtained.

  A commercially available glass slide (manufactured by Matsunami Glass) was subjected to silane coupling treatment, and the surface of the glass slide was aminated. Next, glutaraldehyde was added to the surface of the slide glass, reacted, and then washed. Next, a solution in which CRP antibody was dissolved in PBS buffer solution was applied to a part of the surface of the slide glass, then allowed to stand overnight, and further washed with PBS. By such an operation, a plate substrate having a CRP antibody immobilized on a part of the surface was obtained. As in Example 3, the plate region where the CRP antibody is immobilized is called “CRP antibody immobilization site”, and the plate region where the CRP antibody is not immobilized is called “CRP antibody non-immobilization site”. .

  A commercially available CRP (manufactured by CHEMICON) was diluted with a PBS buffer containing BSA at 1 mg / ml to prepare a 200 ng / ml CRP sample.

  After 200 μl of the CRP sample was dropped on the plate substrate, allowed to stand, washed, and further, a “colloidal gold colloid immobilized with CRP antibody” was placed on the CRP antibody non-immobilized site on the substrate. It was dripped. Next, the plate substrate was tilted so that “the gold colloid on which the CRP antibody was immobilized” flows to the CRP antibody immobilization site. Thereafter, the CRP antibody immobilization site was observed. As a result, a change in color indicating the presence of CRP could be confirmed.

(Summary)
(1) The cleaning time was compared between Example 1 in which Au-coated Fe ₅₀ Pt ₅₀ magnetic particles were prepared and Comparative Example 1 in which a gold colloid was prepared. The results are shown in Table 1. Example 1 is an example purified by a magnetic separation operation, and a comparative example is an example purified by a centrifugal separation operation.

As can be seen from Table 1, the preparation time of “Au coated Fe ₅₀ Pt ₅₀ magnetic particles with immobilized ovalbumin antibody” is shorter than the preparation time of “gold colloid with immobilized ovalbumin antibody”. Thus, it was found that the magnetic particles used in the method of the present invention can be prepared in a relatively short time.

(2) In the case of Example 2 subjected to magnetic transport using “Au-coated Fe ₅₀ Pt ₅₀ magnetic particles on which ovalbumin antibody is immobilized” and “gold colloid on which ovalbumin antibody is immobilized” The detection time of the test substance was compared with the case of Comparative Example 2 in which magnetic transport was not performed. The results are shown in Table 2.

  As can be seen from Table 2, the detection time of Example 2 was shorter than the detection time of Comparative Example 2, and it was found that the detection method of the present invention can detect a test substance in a short time.

  The method for detecting a test substance of the present invention is useful not only in the life science field such as a clinical laboratory field, but also in the food safety field such as a food allergen or BSE test, as well as environmental measurements such as soil and water pollution tests. It is also useful in fields.

FIG. 1A shows a flowchart of the detection method of the present invention. FIG. 1B is a cross-sectional view schematically showing magnetic particles, a member, and a sample used in the detection method of the present invention. FIG. 2 is a cross-sectional view schematically showing the detection method of the present invention according to the first embodiment. FIG. 3A is a cross-sectional view schematically showing the detection method of the present invention according to the second embodiment. FIG. 3B is a cross-sectional view schematically showing the detection method of the present invention according to the third embodiment. FIG. 4A is a cross-sectional view schematically showing the detection method of the present invention according to the fourth embodiment. FIG. 4B is a cross-sectional view schematically showing the detection method of the present invention according to the fifth embodiment. FIG. 5A is a cross-sectional view schematically showing the detection method of the present invention according to the sixth embodiment. FIG. 5B is a cross-sectional view schematically showing the detection method of the present invention according to the seventh embodiment. FIG. 6A is a cross-sectional view schematically showing an embodiment in which an avidin-biotin reaction is used for formation of a complex (case 1). FIG. 6B is a cross-sectional view schematically showing an embodiment in which an avidin-biotin reaction is used for formation of a complex (case 2). FIG. 6C is a cross-sectional view schematically showing an embodiment in which an avidin-biotin reaction is used for formation of a complex (Case 3). FIG. 6D is a cross-sectional view schematically showing an embodiment in which an avidin-biotin reaction is used for formation of a complex (case 4). FIG. 7 is a cross-sectional view schematically showing a cleaning operation when preparing magnetic particles.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Precious metal coat magnetic particle in which substance a was immobilized, 2 ... Central region, 3 ... Outer shell region, 4 ... Substance a, 5 ... Antibody immobilized on member, 6 ... Member, 7 ... Test substance, 7 '... sample, 8 ... antibody immobilized on precious metal-coated magnetic particles, 9 ... composite, 10 ... member (plate member or container), 11 ... magnet or electromagnet, 12 ... member (porous or fibrous) Member), 13 ... magnetic particle impregnated member, 14 ... sample pad, 20 ... specific binding substance (substance b), 21 ... member, 22 ... test substance, 23 ... avidin, 24 ... specific modified with avidin or biotin Binding substance, 25 ... biotin, 26 ... noble metal-coated magnetic particles, 27 ... complex, 30 ... magnetic particles p ', 31 ... substance a not immobilized on the magnetic particles p.

Claims (15)

A method for detecting a test substance contained in a sample,
(I) A magnetic particle in which a central region is formed from a magnetic material and an outer shell region is formed from a noble metal material, and a substance a capable of binding to the test substance is immobilized on the outer shell region. Preparing the magnetic particles prepared, a member on which the substance b capable of binding to the test substance is immobilized, and a sample comprising the test substance,
(Ii) Supplying the magnetic particles and the sample to the member, and forming a complex formed by binding the substance a of the magnetic particles and the substance b of the member via the test substance in the member Obtaining step,
(Iii) measuring the state of the region where the substance b is immobilized on the member; and (iv) determining the presence or amount of the test substance based on the measurement result. Consisting of
The formation of the complex in the step (ii) is performed under the influence of a magnetic field,
The member prepared in the step (i) is a member made of porous or fibrous material, and the substance b is immobilized in at least a part of the inner region a of the inner region, and the step (ii) ), After providing the sample and the magnetic particles to the region b of the member other than the internal region a, the magnetic field is applied from the vicinity of the internal region a, or the sample is applied to the internal region a. And supplying the magnetic particles to the region b of the member other than the internal region a and applying the magnetic field from the vicinity of the internal region a.   The application of the magnetic field is performed by arranging a magnet in the vicinity of the surface a or the inner region a, or by passing a current through an electromagnet arranged in the vicinity of the surface a or the inner region a. The method of claim 1, characterized in that   The method according to claim 1 or 2, wherein the magnetic material of the magnetic particles prepared in the step (i) is a ferromagnetic material or a superparamagnetic material.   The noble metal material of the magnetic particles prepared in the step (i) is a material selected from the group consisting of gold, platinum, silver, and alloys thereof. The method in any one of.   The method according to any one of claims 1 to 4, wherein an average particle diameter of the magnetic particles prepared in the step (i) is 5 nm to 100 µm. Wherein the saturation magnetization of the magnetic particles is prepared in the step (i) is ^{0.5A · m 2 / kg~100A · m} 2 / kg, according to claim 1 Method.   The method according to claim 1, wherein the magnetic particles have a coercive force of 0 kA / m to 280 kA / m.   The method according to claim 1, wherein the substance a is any one of an antibody and an antigen having specific binding properties to each other.   The method according to any one of claims 1 to 8, wherein the substance b is one of an antibody and an antigen having specific binding properties to each other.   The method according to claim 1, wherein the state of the region in step (iii) is a color change of the region.   The method according to claim 10, wherein the color is caused by coloration of the magnetic particles constituting the complex. The magnetic particles used in the step (i) are:
(1) preparing a magnetic particle p having a central region formed of the magnetic material and an outer shell region formed of the noble metal material, and the substance a capable of binding to the test substance;
(2) contacting the magnetic particles p with the substance a to obtain a mixture comprising the magnetic particles p ′ on which the substance a is immobilized; and (3) applying a magnetic field to the mixture, The method according to any one of claims 1 to 11, characterized in that it is prepared by a method comprising the step of separating the magnetic particles p 'from the mixture.   In the step (3), by applying the magnetic field, the mixture is divided into a phase A having more magnetic particles p ′ and a phase B having fewer magnetic particles p ′, and then the phase B is separated from the mixture. 13. A method according to claim 12, characterized in that it is removed.   In the step (2), a linker comprising a functional group capable of binding to the substance a and a sulfur atom is bonded to the magnetic particle p, and then the substance a is bonded to the linker. 14. Method according to claim 12 or 13, characterized in that magnetic particles p 'are obtained.   In the step (2), after the linker comprising a functional group capable of binding to the substance a and a sulfur atom is bound to the substance a, the linker is bound to the magnetic particle p, whereby the magnetic particles 14. Method according to claim 12 or 13, characterized in that p 'is obtained.

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