JP6605792B2 - Composite labeled particles, target substance detection method using the same, colloidal liquid and labeling reagent, and composite labeled particle manufacturing method - Google Patents

Description

  The present invention relates to composite labeled particles, a method for detecting a target substance using the same, a colloidal solution and a labeling reagent, and a method for producing composite labeled particles.

As a technique for detecting trace substances such as antigens in a living body, there is a test method using lateral flow type immunochromatography. In this method, the test substance (target substance) contained in the sample liquid is captured by the labeled particles, and the porous support is moved by capillary action. Then, the test substance is concentrated by bringing it into contact with, for example, a linearly fixed capture substance on the porous support, and a line on which the capture substance is fixed is developed. The presence or absence of the test substance can be determined by this color development. The following three points are mentioned as characteristics of such immunochromatography.
(1) The time required for determination is short and a quick inspection is possible.
(2) The measurement can be performed simply by dropping the sample and the operation is simple.
(3) Determination is easy without requiring a special detection device.
Utilizing these characteristics, immunochromatography is used for pregnancy test drugs and influenza test drugs, and is used as a new POCT (Point Of Care Testing) technique. Also, in food inspection, it is widely used as a test reagent for food allergens, and is attracting more and more attention.

  The present applicant has advanced technical development centering on the technology using fluorescent silica particles in the above-described immunochromatography (see Patent Documents 1 and 2 below).

International Publication No. 2008/018566 Pamphlet JP 2010-014631 A

The present inventors paid attention to the binding efficiency of the reagent material to the target substance in immunochromatography as a direction for further new technological development. By increasing this, it was considered that a larger amount of target substance can be captured by a certain amount of reagent or labeling material, and that accumulation of the labeling substance on the test line can be effectively promoted. Then, clear color development or fluorescence can be obtained on the test line, which can lead to accurate diagnosis and determination. In addition, if the binding efficiency of the labeling material can be increased, the amount of labeling substance (such as a biomolecule) applied can be reduced and the manufacturing cost can be reduced.
That is, the present invention provides composite labeled particles having high binding efficiency to a target substance, a target substance detection method using the same, a colloidal solution and a labeling reagent, and a method for producing composite labeled particles.

The above problem has been solved by the following means.
[1] composite labeled particles before capturing a target substance, and
A composite labeled particle for immunochromatography for detecting the target substance,
Having a linking substance and a binding substance in this order on the surface of the labeling particle, the binding substance is a composite labeling particle having a reaction site showing binding property to the target substance,
A composite label having a structure in which reaction sites of the binding substance extend in a direction radially away from the labeling particles and are connected, and the linking substance and the binding substance are linked at a linking site on the binding substance side. particle.
[2] The composite labeled particle according to [1], wherein the combination of the linking substance and the binding substance is selected from the following (i) to (viii).
――――――――――――――――――――――――――――――――――――
Binding substance / Linking substance ――――――――――――――――――――――――――――――――――――
(I) His tag introduction substance / anti-His tag antibody (ii) primary antibody / secondary antibody (iii) biotin introduction substance / avidin (iv) avidin introduction substance / biotin (v) biotin introduction substance / streptavidin (vi) strept Avidin-introduced substance / Biotin (vii) FLAG tag-introduced substance / Anti-FLAG tag antibody (viii) Fluorescein-introduced substance / Anti-fluorescein antibody ――――――――――――――――――――――― ―――――――――――――
[3] The composite labeled particle according to [2], wherein the His tag introduction substance is a fusion protein having a His tag.
[4] The composite labeled particle according to any one of [1] to [3], wherein the binding density of the binding substance on the labeled particle is 0.001 to 0.1 particles / nm ² .
[5] The composite labeling particle according to any one of [1] to [4], wherein the labeling particle has an average particle diameter of 20 to 500 nm.
[6] The composite labeling particle according to any one of [1] to [5], wherein the labeling particle is a silica particle.
[7] The composite labeled particle according to any one of [1] to [6], wherein a thiol group is present on the surface of the labeled particle, and the linking substance is introduced through the thiol group.
[8] The composite labeled particle according to any one of [1] to [7], wherein the linking substance is introduced into the labeled particle through a maleimide linker.
[9] The target substance is detected by specifically capturing the target substance using the composite label particle according to any one of [1] to [8], and detecting the composite label particle capturing the target substance. A method for detecting a target substance that performs determination and / or quantification of the target substance.
[10] A colloid solution containing the composite labeling particles according to any one of [1] to [8].
[11] A labeling reagent comprising the composite labeling particle according to any one of [1] to [8].
[12] A method for producing composite labeled particles for immunochromatography before capturing a target substance,
A step of introducing a linking substance to the surface of the labeling particles, a step of introducing a binding substance via the linking substance,
The method for producing composite-labeled particles, wherein the binding substance has a reaction site showing binding ability to a target substance.
[13] The method for producing composite labeled particles according to [12], wherein the combination of the linking substance and the binding substance is selected from the following (i) to (viii).
――――――――――――――――――――――――――――――――――――
Binding substance / Linking substance ――――――――――――――――――――――――――――――――――――
(I) His tag introduction substance / anti-His tag antibody (ii) primary antibody / secondary antibody (iii) biotin introduction substance / avidin (iv) avidin introduction substance / biotin (v) biotin introduction substance / streptavidin (vi) strept Avidin-introduced substance / Biotin (vii) FLAG tag-introduced substance / Anti-FLAG tag antibody (viii) Fluorescein-introduced substance / Anti-fluorescein antibody ――――――――――――――――――――――― ―――――――――――――
[14] The method for producing composite labeled particles according to [12] or [13], wherein the linking substance is introduced into the labeled particles at 0.001 to 0.1 particles / nm ² .
[15] The method for producing a composite labeling particle according to any one of [12] to [14], wherein the linking substance and the binding substance are linked via a binding site of the binding substance.
In this specification, having on the surface of the particle means that the object may exist in contact with the surface or may exist through other materials. Moreover, having in that order means that a plurality of objects may exist continuously or may exist through other materials. However, in the present invention, it is preferable that the connecting substance and the binding substance are connected without any other material.

The composite labeled particle of the present invention has a high binding efficiency to a target substance, and enables detection and measurement of the target substance with high accuracy in a test such as immunochromatography. Moreover, it reduces the cost by reducing the amount of labeling material applied.
The target substance detection method, colloidal solution, and labeling reagent of the present invention contain the composite labeled particles described above, and exhibit excellent effects based on their high binding efficiency with the target substance.
According to the method for producing composite labeled particles of the present invention, the above composite labeled particles can be preferably produced.

It is explanatory drawing which shows the manufacture process of the composite labeling particle which concerns on preferable embodiment of this invention. It is a disassembled perspective view which shows typically the elongate test body as preferable embodiment of this invention. It is explanatory drawing which shows typically the fixed state in the test area | region thru | or reference area | region of the labeled particle as preferable embodiment of this invention. It is explanatory drawing which shows typically the fixation state in the test area | region thru | or reference area | region of labeled particle | grains as another preferable embodiment of this invention. It is explanatory drawing which shows typically the fixed state in the test area | region thru | or reference area | region of labeled particle | grains as another preferable embodiment of this invention.

  The composite labeling particle of the present invention has a linking substance and a binding substance (sometimes referred to as “linked substance”) on its surface. Here, in the present invention, the binding substance is composed of a reaction site having a binding property to the target substance and a coupling site having a binding property to the linking substance, or a combination of the reaction site and the coupling site. . Hereinafter, the configuration of the present invention will be described in detail with reference to the preferred embodiments, with reference to the drawings.

[Composite labeled particles]
FIG. 1 is an explanatory view showing a production process of composite labeled particles according to a preferred embodiment of the present invention. As shown in the figure, the composite labeled particle 2 of this embodiment includes a labeled particle 2a, an anti-His tag antibody (linking substance) 2b applied to the surface, and a His tag-containing protein [fusion protein] introduced thereto. (Binding substance) 21. This His tag-containing protein 21 is composed of a His tag 2c (linkage site) and a protein (reaction site) 2d. The His tag 2c portion plays a role of connecting the protein 2d and the anti-His tag antibody 2b, and is interposed between the two. The protein 2d serves as a reaction site, and has a function of binding to and capturing a target substance (such as an antibody).

  The production method of the composite labeled particle 2 is not particularly limited. For example, (I) a linking substance (anti-His tag antibody 2b) is introduced into the labeled particle 2a, and then a binding substance (His tag-containing protein 21) is introduced. And (II) a linking substance (anti-His tag antibody 2b) and a binding substance (His tag-containing protein 21) are linked and bound to the labeled particles 2a. In the present invention, method (I) is preferred (see FIG. 1). Thereby, the His tag-containing protein 21 is oriented without falling down, and a form extending radially outward from the labeled particle 2a can be obtained more reliably. By such an orientation form, the protein 2d portion, which is the reaction site of the His tag-containing protein 21, protrudes further away from the particle, and the reactivity with the effective target substance is imparted. As a result, higher performance can be obtained in the binding efficiency with the target substance described later. In addition, between the step of introducing the linking substance (anti-His tag antibody) and the step of introducing the binding substance (His tag-containing protein), or the step of preparing a linking chain having these and the step of introducing it into the labeled particles Another process may be interposed between them.

  The binding substance 21, the connecting substance 2b, and the labeling particle 2a may be immobilized by a normal method. For example, as for the introduction of the linking substance, as described below, there is a method in which silica fine particles are used as labeled particles, a thiol group is introduced therein, and each antibody is introduced via a linker.

(Introduction of thiol group)
Introduction of a thiol group on the surface of silica fine particles can be obtained by a usual method. For example, in Journal of Colloid and Interface Science, 159, 150-157 (1993) and International Publication No. 2007/074722 (A1) The methods described can be employed. Specifically, silica fine particles containing a dye as a label are dispersed in a mixed solvent of water and ethanol, and a silane compound having a thiol group (3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, etc.) and A method of adding ammonia water and stirring can be mentioned. The mixed solvent of water and ethanol is preferably 1/10 to 1/1 in a volume ratio of water / ethanol. The final concentration of the functional component-containing silica fine particles is preferably 0.1 to 2% by mass, and the amount of the silane compound having a thiol group to be added is 0.2 mmol to 20 mmol with respect to 1 g of the silica fine particles. It is preferable. Moreover, it is preferable to add 28% ammonia water so that ammonia concentration may be 0.2-2 mass%.

  Alternatively, organoalkoxysilane and tetraalkoxysilane to which functional molecules are bonded are mixed in a solvent containing ammonia water, and silica core particles containing the functional molecules are formed in the solvent to disperse the core particles. Manufactured through a step of obtaining a liquid and a step of adding a thiol-containing organoalkoxysilane and tetraethoxysilane (TEOS) to the dispersion obtained in the above step to form a shell layer on silica core particles. May be. This method is preferable because the thiol group can be continuously introduced from the preparation of the silica fine particles, so that the working time and labor can be reduced.

The amount of thiol groups on the surface of the silica fine particles (thiol introduction rate) can be measured by analyzing the amount of sulfur element by elemental analysis. The concentration of the elemental sulfur in the silica fine particles is preferably 100 ppm (mass basis) or more, more preferably 200 ppm (mass basis) or more based on the weight of the silica fine particles having a thiol group. The upper limit is preferably 10,000 ppm (mass basis) or less, more preferably 5,000 ppm (mass basis) or less, and still more preferably 2,000 ppm (mass basis) or less. When the concentration of elemental sulfur is less than the lower limit, the amount of biomolecule bound through the linker molecule is small, and it is difficult to sufficiently function as a labeling reagent. In addition, when the concentration of sulfur element exceeds the above upper limit, the amount of thiol groups present on the particle surface is too large, the hydrophobicity of the silica fine particle surface is increased, nonspecific adsorption increases due to hydrophobic interaction, and Decrease in dispersibility of particles is likely to occur.
This thiol introduction rate corresponds to the introduction rate of the connecting substance and the introduction rate of the binding substance described later. Therefore, this thiol introduction rate can be evaluated as an index of the occupancy rate of the reactive site of the binding substance.

The concentration of the above elemental sulfur can be measured by a combustion method. Specifically, the amount of sulfur element (thiol introduction rate) is measured by quantifying the amount of sulfur dioxide (SO ₂ ) produced by putting 5 to 50 μg of silica fine particles in a metal capsule such as a Ni capsule and burning it. Can do. From this result, a value (ppm) obtained by dividing the mass of sulfur element by the mass of silica fine particles is defined as the thiol introduction rate.

(Linker)
The linker molecule is preferably a molecule having a maleimide group and an amino group, carboxyl group or active ester group in the molecule, and the maleimide group and the amino group, carboxyl group or active ester group are saturated hydrocarbons, More preferably, it is a molecule linked via an unsaturated hydrocarbon and / or an aromatic hydrocarbon. Examples of the linker molecule having a maleimide group and a carboxyl group in the molecule include maleimidoacetic acid, 4-maleimidobutyric acid, 5-maleimidovaleric acid, maleimidocaproic acid, 3-maleimidobenzoic acid and the like. Examples of molecules having a maleimide and an active ester group in the molecule include N- (6-maleimidocaproyloxy) succinimide, 4- (N-maleimidomethyl) cyclohexanecarboxylic acid N-hydroxysuccinimide ester, N- (m- Maleimidobenzoyloxy) succinimide and the like. Examples of the linker having an amino group include N- (4-aminophenyl) maleimide. The molecular weight of the linker molecule is not particularly limited, but is preferably 150 to 5000 Da.

  In the production method of the present embodiment, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group introduced on the surface of the silica fine particle, thereby obtaining a silica fine particle to which the linker molecule is bonded ( See the scheme below).

  In the present embodiment, the reaction between the maleimide group and the thiol group can be performed in an aprotic solvent. When a protic solvent is used, the maleimide group may react with the solvent molecule, resulting in a decrease in reactivity with the thiol group. The aprotic solvent is preferably one in which silica fine particles can be dispersed, and examples thereof include N, N-dimethylformamide and dimethyl sulfoxide. In this reaction, the concentration of silica fine particles having a thiol group in the aprotic solvent is preferably 0.05 to 2% by mass. It is preferable to react 0.1-5 mg of linker molecules with 1 mg of silica fine particles having a thiol group. The reaction temperature is preferably 0 to 60 ° C, more preferably 0 to 40 ° C. The reaction time is preferably 5 minutes or more, and more preferably 5 to 120 minutes.

(Introduction of connected substances)
By binding the biomolecule to the linker part of the silica fine particle to which the linker molecule is bound, prepared as described above, the silica fine particle to which the biomolecule is bound is manufactured. The above scheme shows an example in which an antibody is introduced as a biomolecule. The carboxyl terminus and an amino group of a linker molecule are bonded to each other and the two are linked by an amide bond.

  In addition, although the example which introduce | transduced the thiol group and used the maleimide linker which has an amino group as surface modification (acceptor) of label | marker particle | grains was shown above, this invention is not limited to this and interpreted. Examples of other acceptors include amino groups, hydroxyl groups, carboxyl groups, maleimide groups, succinimidyl ester groups, SCN groups, epoxy groups, and CNO groups. As other linkers, the exemplified compounds shown above can be widely used.

(Linked substance)
-Anti-His tag antibody The anti-His tag antibody used as a linking substance is not particularly limited, and those generally used for this type of test and the like can be used. Commercially available products include, for example, PM032 Anti-His-tag pAb, PM032-8 Anti-His-tag pAb-Agarose, D291-3 Anti-His-tag mAb, D291-3S Anti- manufactured by Medical and Biological Laboratories Co., Ltd. His-tag mAb, D291-6 Anti-His-tag mAb-Biotin, D291-7 Anti-His-tag mAb-HRP-DirecT, D291-8 Anti-His-tag mAb-Agarose, D291-9His-Atiose tag mAb-Magnetic beads, D291-10 Anti-His-tag mAb-Magnetic Agrose, D291-A48 Anti-His-tag mAb-Alexa Fluor Trademarks) 488, D291-A59 Anti-His-tag mAb-Alexa Fluor (registered trademark) 594, D291-A64 Anti-His-tag mAb-Alexa Fluor (registered trademark) 647, M089-3 Anti-His-tag gb M136-3 Anti-His-tag mAb and the like (see http://ruo.mbl.co.jp/dtl/A/PM032/). In addition, 71184 His • Tag (registered trademark) Antibody Plate Immobilized antibody in 96-well format from Merck Millipore (Merck), Profinia anti-His antibody, 1 c (BioRad), anti-His antibody, ne4 Examples include Millipore (Millipore), His-Tag Antibody (Cell Signaling Technology), Anti-His6 (Roche), and the like.

Anti-FLAG antibody, etc. Anti-FLAG antibody includes ANTI-FLAG M1 Monoclonal Antibody, Purified IgG (F3040) manufactured by SIGMA-ALDRICH, and secondary antibodies for FLAG antibody include Goat Anti-Mouse IgG, Fab Fragment Fab Examples include Adsorbed with Human IgG (A9917), Goat Anti-Mouse IgG, Fab Fragment Peroxidase Conjugate, Adsorbed with Human IgG and Rat Serum Proteins (A3682), and the like.

Anti-fluorescein antibody As an anti-fluorescein monoclonal antibody, clone B13-DE1, mouse IgG1 (1426320) manufactured by Roche Applied Science, AP-labeled anti-fluorescein, polyclonal antibody, and Fab fragment include Anti-Fluorescein-AP manufactured by the same company Fab fragmentation sheep origin (1426338).

-Avidin and the like Avidin is, for example, neutral avidin, egg white derived from Wako Pure Chemical Industries, Ltd. 205-18263, 203-18264), Tamavidin (registered trademark) 2-REV, recombinant (203-17401, 209-19403), Tamavidin (registered trademark) 2-LPI, recombinant (202-19351, 208-19353), Tamavidin (registered trademark) 2-HOT, recombinant (209-19361, 205-19363). Streptavidin includes, for example, Streptavidin, Type II (192-11644, 194-11634, 198-11641) manufactured by Wako Pure Chemical Industries, Ltd. Other examples include 21122 ImmunoPure (registered trademark) StreptAvidin and 21125 ImmunoPure (registered trademark) StreptAvidin manufactured by Thermo Fisher Scientific.

-Secondary antibody etc. Here, the secondary antibody includes a primary antibody having binding properties with an antibody and an antibody having a different host. For example, when the primary antibody host is mouse IgG, the secondary antibody is rabbit, rat, goat, sheep, cow, human, horse, pig, chicken, guinea pig, hamster, cat, dog, etc. having binding properties with mouse IgG. Can be used.

  In the above-described embodiment, only an example in which one linking substance is used has been described. However, the structure of the reactive linking chain 22 connected in three or more stages using two or more linking substances may be used.

(Binding substance)
Biological substances (such as fusion proteins) containing His tags or FLAG tags that can be used as binding substances are appropriately prepared by applying general biochemical synthesis methods applied to this type of product. be able to. As for the His tag-containing protein, a tag consisting of 6 histidine residues (6 × His tag) is usually added to the N-terminus or C-terminus of the target antibody protein. Which terminal is to be introduced may be appropriately selected. There are mainly two methods for adding a His tag into a protein. Among these, a simple method is to insert a protein into a vector in which a His tag is added to the end. Since the His tag is short, a His tag can be added to a protein by preparing a primer including a sequence in which codons encoding histidine (CAT and CAC) are continuous and performing PCR using the primer. Primer design for adding 6 His tags includes placing 18 bases encoding 6 histidine residues immediately after the start codon or just before the stop codon. Next to it, a sequence specific to the protein of interest can be attached. As a more specific procedure, DNA encoding the protein of interest is inserted into a vector that fuses a His tag to the C-terminus of the protein. Subsequently, PCR is performed using a primer containing a tag, and the tag can be fused to the N-terminus of the target protein. Similarly, for the FLAG tag, the target protein can be obtained using a primer containing a codon sequence encoding it. As for the synthesis of His tag antibody and His tag protein, for example, International Publication No. 2003/044198 pamphlet and International Publication No. 2005/022156 pamphlet can be referred to.
In addition, a His-tagged protein can be obtained by using a commercial product. Many recombinant proteins are tagged with His tags, and these recombinant proteins can be used. For example, H1N1 / HA1 (18-344), His-tagged, Influenza A-H1N1 (Human), Recombinant (manufactured by ATGen, product code: ATGP1484), H5N1 / HA (17-338), His-tagged, Influenza A H5N1), Recombinant (manufactured by ATGen, product code: ATGP1497).

In addition, when biotin or fluorescein is used as a linking site for a binding substance, it may be introduced into a reaction site (predetermined antibody or protein) by a chemical method or a biochemical method similar to the above. .
As a method of using biotin as a linking site, biotin modified with an N-hydroxysuccinimide group is bound to an amino group having a reaction site, or biotin modified with a maleimide group or an isocyanate group is used as a reaction site. There is a method of bonding to the thiol group.
As a method of using streptavidin as a linking site, for example, as described in The Journal of Biochemistry, 135, 285-288 (2004), a DNA containing a target protein and a gene encoding streptavidin is synthesized and expressed. Can be made. Alternatively, a commercially available kit, Strep-tag (registered trademark) / Strep-Tactin (registered trademark) system (manufactured by IBA) may be used.

  What can be utilized as a connection part is not restricted above, What is utilized for this kind of use can be utilized suitably. Including those described above, for example, His-tag, glutathione S transferase-tag, maltose binding protein-tag, antigen peptide-tag and the like can be mentioned. The antigen peptide-tag is a tag having a peptide in which an antibody is present, for example, His-tag, His G-tag, HA-tag, C-myc-tag, myc-tag, BPV-1-tag. , Cl-tag, Cre recombinase-tag, FLAG-tag, NSl (81) -tag, green fluorescent protein (GFP) -tag, IRS-tag, LexA-tag, Thioredoxin-tag, Polyomamedium TP medium tag , SV40 Large T Antigen-tag, Paramoxyvirus SV5-tag, Xpress-tag, GST-tag, MBP-tag, and the like.

(Introduction of binding substances)
When, for example, a His tag-containing protein is used as the binding substance, an anti-His tag antibody is introduced on the fine particle side, so that the His tag-containing protein can be introduced by bringing them into contact with each other by a conventional method. The same applies to the case where the His tag-containing protein and the anti-His tag antibody are previously bound. The contact between the two is preferably carried out in a medium, and examples thereof include an example in which the reaction is carried out in an aqueous solvent. The same applies to other binding substances, and the connection can be promoted by bringing them into contact with each other in an environment that reacts with the connection substance. Although the reaction temperature at this time is not particularly limited, an example in which the reaction is performed at room temperature (about 25 ° C.) can be mentioned.

  The composite labeled particle according to the present embodiment has reactive linking chains 22 (2b, 2c, 2d) extending radially as shown in FIG. The linking substance 2b itself is not used as the target substance capture site, but the reaction site 2d is extended from there via the binding substance (substance to be linked), so that the reactive linking chain 22 having the reaction site at the end is outward. Oriented accurately. As a result, as described above, the active site of the reaction site does not fall to the side of the labeled particle and is more reliably directed to the outside, so that it easily reacts with the target substance and is captured accurately. As a result, it is understood that, for example, higher binding efficiency with the target substance is realized as compared with the case where the linking substance 2b is used as a reaction site.

The introduction density of the binding substance is not limited, but is preferably 0.001 piece / nm ² or more, and more preferably 0.01 piece / nm ² or more. The upper limit, preferably 0.5 pieces / nm ² or less, more preferably 0.1 pieces / nm ² or less.
The introduction density of the binding substance is a value measured by the following method unless otherwise specified.
<Measurement method of introduction density>
As the introduction density of the binding substance, a protein quantification method can be used. In other words, the protein concentration of the colloid of silica particles to which the test substance is bound is measured, the number of binding substances bound to the silica particles is calculated from the molecular weight of the binding substance, and divided by the surface area of the silica particles. Density can be calculated.
The surface area of the silica particles can be calculated by the following method.
The SEM image of the silica particles is measured, the diameter of 100 particles is obtained from the SEM image, and the sum of the surface area and the volume of the particles per 100 silica particles is calculated.
Next, the amount of silica particles contained in the sample is calculated, and the number of silica particles contained in the sample is calculated from the value of the sum of the volume of silica particles obtained from the above-mentioned 100 silica particles. From the above, the surface area of the silica particles is calculated from the value of the surface area per 100 particles.
As a protein quantification method, a general method can be used. For example, a BSA method, a Bradford method, or a Lowry method can be used.

The combination of the connecting substance and the binding substance is preferably selected from the following combinations (i) to (viii).
[Table 1]
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Binding substance / Linking substance ――――――――――――――――――――――――――――――――――――
(I) His tag introduction substance / anti-His tag antibody (ii) primary antibody / secondary antibody (iii) biotin introduction substance / avidin (iv) avidin introduction substance / biotin (v) biotin introduction substance / streptavidin (vi) strept Avidin-introduced substance / Biotin (vii) FLAG tag-introduced substance / Anti-FLAG tag antibody (viii) Fluorescein-introduced substance / Anti-fluorescein antibody ――――――――――――――――――――――― ―――――――――――――
The linking substance (first substance) and the binding substance (second substance) may be reversed as the linking substance or the binding substance. However, in the present invention, it is preferable to distinguish between the connecting substance and the binding substance in the table. In the present specification, the “introduced substance” may be the substance itself or a substance introduced into another substance.

  In the present invention, it is preferable to use a binding substance having a reaction site and a linking site, or an integrated material. By doing in this way, the distance of a reaction part and a connection part can be separated, and there exists an advantage which the reactivity of a reaction part improves. From this viewpoint, among the above combinations, (i) and (iii) to (viii) are preferable.

FIG. 3 schematically shows a composite labeled particle (labeled body) according to the combination (i) as a preferred embodiment of the present invention. In this embodiment, the anti-His tag antibody used as the connection substance 2b is introduce | transduced into the labeling particle 2a. Further, a protein 2d (binding substance 21) having a His tag 2c is introduced therein, and a composite labeled particle 2 having a reactive linking chain 22 is constructed. The test substance 1 in this embodiment is an antibody.
FIG. 4 schematically shows a composite labeled particle (labeled body) according to the combination (ii) as another preferred embodiment of the present invention. In the present embodiment, a secondary antibody (anti-IgG antibody) serving as the linking substance 2b is introduced into the labeled particle 2a. Further, a primary antibody (anti-influenza A influenza nucleoprotein antibody) serving as the binding substance 21 is further introduced to constitute the composite labeled particle 2 having the reactive linking chain 22. The test substance 1 in this embodiment is a protein.
FIG. 5 schematically shows a composite labeled particle (labeled body) according to the combination (vi) as still another preferred embodiment of the present invention. In this embodiment, streptavidin serving as the linking substance 2b is introduced into the labeled particle 2a. Further, a primary antibody that becomes the binding substance 21 is further introduced, and the composite labeled particle 2 having the reactive linking chain 22 is configured. At this time, the binding substance 21 has a structure in which biotin (linkage site) 2c is introduced into anti-type A influenza nucleoprotein antibody (reaction site) 2d. The test substance 1 in this embodiment is a protein.

[Test strip]
The immunochromatographic test strip (test piece) of the present embodiment is preferably connected in series so that the following members cause capillary action.
・ Sample pad 8a
Conjugate pad 8b (member obtained by impregnating a label and drying)
・ Membrane (antibody-immobilized membrane) 8c
・ Absorption pad 8d

  In the present embodiment, as shown in FIG. 2, the test strip 10 including the membrane 8c having the above-described test region and reference region is sandwiched and included between the housing upper portion 6a and the housing lower portion 6b, and is long. A test body 100 is formed. A detection opening 61 and a sample introduction opening 62 are provided in the housing upper part 6a. In the fluorescence observation, the irradiation light can be sent to the internal test strip 10 through the detection opening 61, and the fluorescence emitted therefrom can be collected and detected and observed. On the other hand, the sample liquid S can be supplied to the test strip 10 through the sample introduction opening 62 to perform a measurement test. As described above, the immunochromatographic test strip 10 of this embodiment includes the sample pad 8a, the conjugate pad 8b, the antibody-immobilized membrane 8c, and the absorption pad 8d. Furthermore, it is preferable that each said structural member is backed by the backing sheet (not shown) with an adhesive.

(Target substance)
In the present invention, examples of the target substance 1 to be detected and quantified include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, peptides, chemical substances, and the like. In the present invention, the sample containing the target substance 1 is not particularly limited, and examples thereof include liquid samples such as urine and blood.

(Sample pad)
The sample pad 8a is a component that drops a sample containing a target substance. The material, dimensions, etc. are not particularly limited, and general materials applicable to this type of product can be used.

(Conjugate pad)
The conjugate pad 8b is a constituent member impregnated with labeling reagent silica fine particles (labeled bodies) 2 and 3. The target substance contained in the sample moved from the sample pad 8a by capillary action is captured and labeled by the labeling reagent silica fine particles (labeled body) in a specific molecular recognition reaction such as an antigen-antibody reaction. .
The content of the labeling reagent silica fine particles (labeled body) per unit area (cm ² ) in the conjugate pad 8b is not particularly limited, but is preferably 1 μg to 100 μg. Examples of the impregnation method include a method in which the dispersion liquid of the labeling reagent silica fine particles is applied, dropped or sprayed, and then dried.

(Antibody immobilized membrane)
Wherein the antibody immobilization part in the antibody immobilized membrane 8c, determine the presence or absence of the target substance, ie, providing a test line n _t of the target substance capture antibody is immobilized for determining the positive-negative. The antibody-immobilized membrane 8c preferably includes a control line _nr on which an antibody for capturing the labeling reagent silica fine particles is immobilized.
The membrane 8c is a structural member in which the target substance 1 labeled with the silica fine particles (labeled bodies) 2 and 3 moves by capillary action, and a sandwich type immune complex formation reaction comprising immobilized antibody-target substance-silica fine particles is performed. An antibody immobilization part (determination part). The shape of the antibody immobilization part (determination part) in the membrane is not particularly limited as long as the capture antibody is locally immobilized, and includes a line shape, a circular shape, a belt shape, etc. It is preferable that there is a line shape having a width of 0.5 to 1.5 mm.

The amount of antibody immobilization in the antibody immobilization portion (test region) n _t is not particularly limited, but when the shape is a line, 0.5 μg to 5 μg per unit length (cm) is preferable. Examples of the immobilization method include a method in which an antibody solution is applied, dropped or sprayed and then dried and immobilized by physical adsorption. After the above-described antibody immobilization, the whole antibody-immobilized membrane is preferably subjected to a so-called blocking treatment in order to prevent the influence of nonspecific adsorption on the measurement. For example, a method of immersing in a buffer solution containing a blocking agent such as albumin, casein, polyvinyl alcohol or the like for an appropriate period of time and then drying may be mentioned. Examples of the commercially available blocking agent include skim milk (manufactured by DIFCO), 4% block ace (manufactured by Meiji Dairies), and the like.
The membrane 8c further has a reference region _nr in which the labeled particle (labeled body) 3 in which the target substance is not captured is captured. Accordingly, the presence / absence and amount of the target substance can be fixed as compared with the fluorescence / absorption in the test region n _t . In order to fulfill this function, the test label 2 is composed of silica fine particles 2 a and reactive linking chains 22. The reaction site 2d of the reactive linking chain 22 has a binding property with the target substance. On the other hand, the reference label 3 is composed of silica fine particles 3a and a reference binding substance 3b. The reference capture substance has no binding property to the target substance and has a binding property to the reference capture substance (see FIG. 3).

(Absorption pad)
The absorption pad 8d is a constituent member that absorbs the specimen S (target substance) and the labeling reagent silica fine particles (labeled bodies) 2 and 3 that have moved through the membrane by capillary action, and always generates a constant flow.

There are no particular restrictions on the material of each of these constituent members, and members used for immunochromatographic test strips can be used. As sample pads and conjugate pads, glass such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) is used. A fiber pad is preferable, a nitrocellulose membrane such as Hi-Flow Plus 120 membrane (trade name, manufactured by MILLIPORE) is preferable as the membrane, and a cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) is preferable as the absorbent pad. Is preferred.
Examples of the backing sheet with an adhesive include AR9020 (trade name, manufactured by Adhesives Research).

[Meaning of technical terms]
When the meaning of the technical terms used in this specification is confirmed, the target substance 1 (shown together with the reference numerals in the figure, but is not construed as being limited thereto) is a substance to be detected by the lateral flow method. And is synonymous with the test substance in the sample. The binding substances 21 and 3b are substances having binding ability to the target substance and the capturing substance, respectively, and preferably contain biomolecules. The labeled particles 2a and 3a into which the binding substance has been introduced are referred to as labeled bodies (composite labeled particles) 2 and 3. However, in a broad sense, the term labeled particle is sometimes used to mean a label (composite labeled particle). On the other hand, the test capturing substance 4 is fixed to the membrane in the test region and captures the label 2 through the target substance 1. On the other hand, what is fixed to the membrane in the reference region is the reference capturing substance 5, and the label 3 is captured without the target substance 1 interposed therebetween.
In addition, in this specification, a substance means a compound or a chemically synthesized molecule, and also includes biomolecules (proteins, peptides, nucleic acids, etc.), and those of artificial origin or of natural origin It may be. In a broad sense, it is meant to encompass biological cells, microorganisms (bacteria, etc.), viruses, or complexes of proteins and single compounds. Bonding or linking generally refers to the continuous integration of a plurality of objects from a separated state. In addition to chemical bonds such as covalent bonds, ionic bonds, and hydrogen bonds, chemical adsorption and physical adsorption. In addition, it is meant to include fitting, screwing, biting physical connection state, and the like. Here, the term “coupled” or “coupled” means that a plurality of units may be coupled directly or indirectly through another unit.

Specimen The specimen S applied to the present embodiment is not particularly limited, but is collected from human or animal blood, plasma, serum, lymph, urine, saliva, pancreatic juice, gastric juice, sputum, nose, throat and other mucous membranes. Clinical samples typified by body fluids such as swabs and stool, food samples typified by liquid drinks, semi-solid foods, solid foods, etc., sampling samples from the natural world such as soil, rivers, seawater, etc., production in the factory Examples include environmentally sampled samples such as wiped samples from lines and clean rooms, and sampled samples by air samplers. The specimen can be used as a liquid as it is, or in the case of a semi-solid or solid substance, it can be used after being subjected to a treatment such as dilution or extraction.

-Binding substance etc. In this embodiment, the reactive connecting chain 22 (FIGS. 3-5) containing the test binding substance 21 is used integrally with the labeled particles 2a (labeled body 2). The reactive linking chain 22 is as already described in detail. 3-5, only one reactive linking chain 22 is shown, but this is shown in a simplified manner, and usually there are many linking chains on the surface of the labeled particle as shown in FIG. Is preferred.

  In the present embodiment, a reference binding substance 3b is further used. The reference binding substance 3b has binding properties with a reference capturing substance 5 described later. The label particles 3a and the reference binding substance 3b are integrated to form a reference label 3. Accordingly, the labeling body 3 is captured by the capturing substance 5 in the process of moving through the membrane. The biomolecule can be cited as an example of the binding integration of the labeling particle 3a and the binding substance 3b or the preferred kind of the binding substance 3b. However, it is preferable that the reference binding substance 3b has no binding property to the test capturing substance 4 or the target substance. On the other hand, the reactive linking chain 22 for testing may have a binding property with the reference capturing material 5, but it is more preferable not to have the binding property.

Test Capturing Substance The membrane used in the present embodiment has the test capturing substance 4 immobilized on the material of the membrane. The capture substance 4 has a binding ability to the target substance 1 so as to capture a complex containing the labeled particle 2a, the reactive linking chain 22 and the target substance 1 (see FIG. 3 and the like). Since the capturing substance 4 has the binding ability as described above, it is possible to capture a complex composed of the label 2 and the target substance 1. As a result, a line that emits fluorescence due to the label 2 or is colored by light absorption is formed in the test region n _t .

Reference capturing substance In the present embodiment, the reference capturing substance 5 is fixed to the reference region n _{r of the} membrane. This binds directly to the binding substance 3b for reference without using the target substance 1. Therefore, when the labeled body 3 mixed with the moving sample liquid S and not bound to the target substance 1 moves, it is directly captured (see FIG. 2). As a result, a line exhibiting absorption or fluorescence due to the label 3 is formed in the reference region _nr . The reference capture substance 5 is not particularly limited, and examples thereof include biomolecules having binding ability with the binding substance, specifically, antibodies and the like.

The reference capturing substance 5 and the test reactive connecting chain 22 in the reference region may have binding properties. In this case, the test label particles 2a are captured in the reference region n _r . That is, both the labeled particles 3a and the labeled particles 2a are captured in the reference region. In this case as well, the coloring / fluorescence state of the labeled particles in the reference region can be visually recognized. On the other hand, since the fluorescent label 2 with the target substance is captured in the test area, there is no problem in normal use.

-Material There is no restriction | limiting in particular as a material of each structural member employable for the plane test piece (test strip) 10 of this embodiment, The normal member used for the test strip for immunochromatography can be used. As the sample pad and the conjugate pad, for example, a glass fiber pad such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) is preferable. As the membrane, a nitrocellulose membrane such as Hi-Flow Plus 120 membrane (trade name, manufactured by MILLIPORE) is preferable. A cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) is preferable as the absorbent pad. When using the said backing sheet with an adhesive, AR9020 (A brand name, the product made by Adhesives Research) etc. are mentioned.

Introducing labeled particles into the conjugate pad In the immunochromatography of the present embodiment, it is preferable that colored particles or fluorescent fine particles bound with a binding substance are introduced into the conjugate pad as the label. . The content of the labeled particles per unit area (cm ² ) in the conjugate pad is not particularly limited, but is preferably 20 μg / cm ^{2 to 2} mg / cm ^{2, and} more preferably 20 to 200 μg / cm ² . If the content is too large, the number of analyte bindings per particle decreases, and the detection sensitivity decreases. Examples of the method of inclusion include a method in which the dispersion of the labeled particles is applied, dropped or sprayed, and then dried. At this time, colored particles or fluorescent fine particles are contained, and after drying once, fluorescent fine particles or colored particles may be contained, or colored particles and fluorescent fine particles may be mixed in advance and the mixed colloid may be contained.

[Labeled particles]
As the constituent material of the labeling particles, those applicable to this kind of test can be used as appropriate. For example, fluorescent / absorbing silica particles, fluorescent / absorbing latex particles, semiconductor fine particles, colloidal gold particles, etc. are used. Can do. In the present invention, it is particularly preferable to use fluorescent silica particles.
Silica particles have a hydrophilic surface and little protein adsorption. Therefore, when bonding the connecting substance, it can be bonded with a certain orientation by a covalent bond, and it is possible to surely direct the site that binds to the binding substance to the outside of the particle. For this reason, the binding efficiency of the binding substance is further improved. On the other hand, latex particles, semiconductor nanoparticles, and colloidal gold particles bind the linking substance by adsorption, and therefore it may be difficult to efficiently direct the binding site with the binding substance outward. Therefore, in the embodiment using silica particles, the effect obtained by the present invention can be relatively enhanced.
The fluorescent silica particles use fluorescence for detection, but fluorescence observation generally has a higher S / N ratio than visual observation of coloration. Therefore, detection can be performed without any trouble even if there is slight noise. Since this advantage appears more remarkably by the effect of improving accuracy according to the present invention, the embodiment using the fluorescent silica particles is extremely advantageous in the present invention.

-Fluorescent silica particle There is no restriction | limiting in particular in the preparation method of fluorescent silica particle, The silica particle obtained by arbitrary preparation methods may be sufficient. Examples thereof include the sol-gel method described in Journal of Colloid and Interface Science, 159, 150-157 (1993). In the present invention, it is particularly preferable to use silica particles containing a functional compound obtained according to the method for preparing fluorescent dye compound-containing colloidal silica particles described in International Publication No. 2007 / 074722A1. Specific examples of the functional compound include fluorescent dye compounds, light absorbing compounds, magnetic compounds, radiolabeled compounds, pH sensitive dye compounds and the like.

Specifically, the silica particle containing the functional compound is a product obtained by reacting the functional compound with a silane coupling agent, and covalently bonding, ionic bonding, or other chemical bonding or adsorption. Can be prepared by condensation polymerization of one or more silane compounds to form a siloxane bond. As a result, silica particles in which the organosiloxane component and the siloxane component are bonded by siloxane are obtained.
Preferred embodiments of the method for preparing silica particles containing the functional compound include N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxy. Silane coupling having a functional group having or added to an active group such as a methyl group, an epoxy group, or a cyano group, and a substituent (for example, an amino group, a hydroxyl group, or a thiol group) that reacts corresponding to the active group. It can be prepared by reacting with an agent and covalently bonding the product obtained by condensation polymerization of one or more silane compounds to form a siloxane bond.

  The case where APS is used as the silane coupling agent and tetraethoxysilane (TEOS) is used as the silane compound is exemplified below.

  Specific examples of the functional compound having or added to the active group include 5- (and -6) -carboxytetramethylrhodamine-NHS ester (trade name, manufactured by emp Biotech GmbH), each represented by the following formula: Examples thereof include fluorescent dye compounds having an NHS ester group such as DY550-NHS ester or DY630-NHS ester (both trade names, manufactured by Dynamics GmbH).

  Specific examples of the silane coupling agent having a substituent include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane, N-2 ( Examples thereof include silane coupling agents having an amino group such as (aminoethyl) 3-aminopropylmethyldimethoxysilane and 3-aminopropyltrimethoxysilane. Of these, APS is preferable.

  The silane compound to be polycondensed is not particularly limited, but TEOS, γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thio Mention may be made of cyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane. it can. Among these, TEOS is preferable from the viewpoint of forming the siloxane component inside the silica particles, and MPS or APS is preferable from the viewpoint of forming the organosiloxane component inside the silica particles.

When prepared as described above, spherical or nearly spherical silica particles can be produced. The nearly spherical silica particles specifically have a shape in which the ratio of the major axis to the minor axis is 2 or less.
In order to obtain silica particles having a desired average particle size, ultrafiltration is performed using an ultrafiltration membrane such as YM-10 or YM-100 (both trade names, manufactured by Millipore), and the particle size is large. It is possible to remove particles that are too small or too small, or to centrifuge at an appropriate gravitational acceleration and collect only the supernatant or precipitate.

  Examples of the biomolecule (binding substance) to be adsorbed or bound to the surface of the silica particles include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, proteins, and peptides. Here, a ligand refers to a substance that specifically binds to a protein, such as a substrate that binds to an enzyme, a coenzyme, a regulatory factor, a hormone, or a neurotransmitter, as well as a low molecular weight molecule or ion. Also includes high molecular weight materials.

The average particle size (average particle size of primary particles) of the fluorescent silica particles is preferably 1 nm to 1 μm, more preferably 20 nm to 500 nm, and even more preferably 30 nm to 300 nm.
In the present invention, the average particle size (average particle size of primary particles) is a value of 100 labeling reagent silica particles randomly selected from an image of a transmission electron microscope (TEM), a scanning electron microscope (SEM), or the like. The occupying area of the labeling reagent silica particles is determined from the total projected area by an image processing apparatus, and the average of the diameters of the circles corresponding to the value obtained by dividing the total occupying area by the number (100) of the selected labeling reagent silica particles. The value (average circle equivalent diameter) was obtained.
The average particle size is the average particle size of the primary particles, unlike the “particle size by dynamic light scattering method” described later, which is a concept including secondary particles formed by aggregation of primary particles.

In the present specification, the “particle size by the dynamic light scattering method” is measured by the dynamic light scattering method, and differs from the average particle size described above in that not only the primary particles but also the primary particles are aggregated. This is a concept including the secondary particles, and serves as an index for evaluating the dispersion stability of the composite labeled particles.
Examples of the particle size measuring device by the dynamic light scattering method include Zetasizer Nano (trade name; manufactured by Malvern). This method measures the time fluctuation of the light scattering intensity by the light scatterer such as fine particles, calculates the Brownian motion velocity of the light scatterer from the autocorrelation function, and derives the particle size distribution of the light scatterer from the result. Is.

  The fluorescent silica particles are preferably monodispersed as a particulate material, and the coefficient of variation of particle size distribution, so-called CV value, is not particularly limited, but is preferably 10% or less, more preferably 8% or less.

Latex particles In the present invention, since the effect is remarkable, it is preferable to apply the silica fine particles described above, but latex particles may be used as marker particles instead of or in addition to this. Latex particles include polystyrene, styrene-sulfonic acid (salt) copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-sulfonic acid copolymer, vinyl chloride-acrylic acid ester copolymer, vinyl acetate- Examples thereof include synthetic polymer particles composed of an acrylate copolymer or the like. The latex particles can be colored by the methods described in JP-A 2000-178309, JP-A 10-48215, JP-A 8-269207, JP-A 6-306108, and the like. It should be noted that the fluorescent substance (labeling substance) can be immobilized on this kind of particles by an appropriate method. For example, reference can be made to JP 2005-534907, JP 2010-156642, JP 2010-156640, and the like. Commercially available fluorescent latex particles include Luminex's product name xMAP (registered trademark) Multi-Analyte COOH Microspheres, (http://hitachisoft.jp/products/lifescience/lineup/luxout/bout/mlhet/ /Hitasoft.jp/products/lifescience/pdf/the_luminex_labmap_system.pdf).

-Semiconductor particle etc. You may apply a semiconductor particle to a labeled body. The material of the semiconductor particles is not particularly limited, but ZnO, ZnS, ZnSe, ZnTe, CdO, CdS, CdSe, CdTe, HgS, HgSe, HgTe, InP, InAs, GaN, GaP, GaAs, TiO ₂ , WO ₃ , PbS, Or PbSe is preferably exemplified. For example, semiconductor fine particles described in Japanese Patent No. 3897285 can be used. The semiconductor fine particles can be surface-modified by replacing the —SH group of the thiol compound with atoms such as S, O, Se, Te, P, As, and N on the surface of the semiconductor fine particles. As the gold particles and the metal fine particles, gold colloid particles and metal colloid particles described in JP-A No. 2003-26638 can be used. Specific examples of the metal colloid particles include metal colloid particles such as platinum, copper, and iron oxide. Examples of the inorganic crystals include iron (III) oxide (Fe ₂ O ₃ ), silver (I) oxide (Ag ₂ O), tin oxide (IV) (SnO ₂ ), titanium oxide (IV) (TiO ₂ ), and indium. Examples include tin oxide (ITO). For example, inorganic crystals described in JP-A-2005-76064 can be used.

-Absorption coefficient When the labeling agent has light-absorbing fine particles, it is preferable that the labeling agent absorbs visible light and is visible. The molar absorption coefficient ε is preferably a particle having 5 × 10 ⁶ M ⁻¹ cm ⁻¹ or more, and the molar absorption coefficient ε is 5 × 10 ⁷ M ⁻¹ cm ⁻¹ to 1 × 10 ¹⁰ M ⁻¹ cm. More preferably, it is ^-1 .

Here, the molar extinction coefficient ε can be calculated from the following Lambert-Bale equation.
A = Log ₁₀ (I ₀ / I) = εbp = a _s bp ′
[A: absorbance, I: intensity of transmitted light, I0: intensity of incident light, ε: molar extinction coefficient (M-1 cm-1), b: optical path length (cm), p: labeled particles (colored particles and fluorescent fine particles Concentration (M (mol / l)), a _s : specific absorbance, p ′: concentration (including mixed dispersion of colored particles and fluorescent fine particles) (g / l) ]]

The concentration p ′ (g / l) is a value obtained by determining the mass obtained by collecting only the labeled particles from a fixed amount (for example, 1 ml) of the labeled particle dispersion and drying it. On the other hand, for the concentration p (mol / l), the size of the labeled particle is obtained from a TEM photograph, the volume of one particle is determined, and the particle density (for example, 2.3 g / cm ^{3 in} the case of silica particles) is It is a value obtained by determining the mass of particles, determining the number of moles from the mass of labeled particles obtained by collecting only the labeled particles from a fixed amount (for example, 1 ml) of the labeled particle dispersion and drying them. In the present invention, “molar extinction coefficient ε of labeled particles” means the molarity of labeled particles in the dispersion obtained by measuring the absorbance of the labeled particle dispersion and applying it to the Lambert-Beer equation. It refers to the extinction coefficient ε. The absorbance, absorbance spectrum, and ε of the labeled particles can be measured as a dispersion such as an aqueous dispersion, an ethanol dispersion, and an N, N-dimethylformamide dispersion using an arbitrary absorptiometer or a plate reader.

  The embodiment relating to the surface modification of the light-absorbing fine particles and the introduction of the binding substance (linking chain) is the same as that of the fluorescent fine particles.

[Detection method]
Immunochromatography is a detection method in which the determination is performed by accumulating the particles in a determination unit by using labeling reagent silica fine particles (labeled bodies) 2 and 3 that normally move using capillary action or the like. For example, it is preferable to use immunochromatography, a microchannel chip, or the like. At this time, the labeling reagent silica fine particles can be suitably used as a label for lateral flow. Furthermore, in the method for detecting a target substance of the present invention, it is preferable to detect the target substance using lateral flow type immunochromatography.

  In order to make it easy to cause capillary action between the members in the order of the sample pad, the conjugate pad, the antibody-immobilized membrane, and the absorption pad, the test strip is produced by adjoining both ends of each member. And 1 to 5 mm on top of each other (preferably on a backing sheet).

The fluorescence detection system for immunochromatography preferably comprises a label having a fluorescent dye, a test strip having an antibody-immobilized membrane, and an excitation light source.
In the fluorescence detection system, it is preferable that the excitation light source emits excitation light having a wavelength of 200 nm to 400 nm from the viewpoint of visually detecting fluorescence emitted from the label. Examples of the excitation light source include a mercury lamp, a halogen lamp, and a xenon lamp. In the present invention, it is particularly preferable to use excitation light irradiated from a laser diode or a light emitting diode.
In addition, the fluorescence detection system preferably includes a filter that transmits only light of a specific wavelength from the excitation light source. Further, from the viewpoint of detecting only fluorescence by visual observation or the like, the excitation light is More preferably, a filter that removes and transmits only fluorescence is provided.
It is particularly preferable that the fluorescence detection system includes a photomultiplier tube or a CCD detector that receives the fluorescence, thereby detecting fluorescence of intensity or wavelength that cannot be visually confirmed and further measuring the fluorescence intensity. Substances can also be quantified, enabling highly sensitive detection and quantification.

  The wavelength of the excitation light is preferably 300 nm to 700 nm. The wavelength of the fluorescence is preferably a wavelength that can be visually recognized, and is preferably 350 nm to 800 nm. Moreover, since high visibility is obtained when it observes visually, it is more preferable that it is 530 nm-580 nm. At this time, the wavelength of the excitation light is preferably 500 nm to 550 nm in order to efficiently generate fluorescence in the above wavelength band.

The test strip according to the preferred embodiment of the present invention is easy to operate even for general consumers who are not skilled in the technique, and from the viewpoint of POCT (Point Of Care Testing), the test strip detection line is visually observed. The observation window is preferably made of a housing (casing) made of a plastic material or the like. For example, the housing etc. which are described in Unexamined-Japanese-Patent No. 2000-356638 etc. are mentioned.
Here, POCT refers to a test for diagnosis as close as possible to the patient. Conventionally, collected blood, urine, affected tissue, and other specimens are sent to the hospital's central laboratory or specialized examination center, and data is output, so it took time to confirm the diagnosis (for example, more than one day) . According to POCT, prompt and accurate treatment can be performed based on examination information provided instantaneously. Because of this, emergency inspections at hospitals and inspections during surgery are possible, and there is a great need in the medical field recently.

As described above, the immunochromatography method has been described, but the present invention is not limited to the immunochromatography method, and can be applied to particles that have a binding substance via a linking substance on the particle surface and use the binding property to the target substance, It can be used for various immunological tests (immunoassays). For example, it can be used for labeled particles such as ELISA, EIA (enzyme immunoassay), FIA (fluorescence immunoassay), RIA (radioimmunoassay), which are general immunological test methods.
In addition, immunoagglutination, which is a test method that agglutinates particles by antigen-antibody reaction with the antigen or antibody contained in the sample by mixing the antibody or antigen-bound particle and the sample, and the antibody or antigen-bound particle and the sample The mixture is mixed to form an antigen-antibody complex precipitate generated by an antigen-antibody reaction with the antigen or antibody contained in the specimen, the aggregate is irradiated with light, and the attenuation of the irradiation light due to scattering is measured with a spectrophotometer. Anti-turbidimetry, which is a test method for measuring and measuring the amount of antigen contained in a sample, antigen-generated by antigen-antibody reaction with an antigen or antibody contained in a sample by mixing antibody or antigen-bound particles and the sample By forming an antibody complex and measuring the light scattered by applying light to the dispersion of the complex, an immunoratio method for quantifying the antigen or antibody contained in the specimen is combined with the antibody or antigen. The detected particles and enzyme-labeled antibody are reacted with a measurement substance to form a particle-measurement substance-enzyme-labeled antibody complex. After removing unreacted substances, a luminescent reagent is added, and the amount of luminescence is measured and detected. It can be used for the CLEIA method (chemiluminescence enzyme immunoassay), which is a test method for quantifying substances.
In addition, it can also be used as labeled particles in flow cytometry, labeled particles in biochips and the like.

  Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.

(Example 1)
(Preparation of silica fine particles)
3.1 mg of 5- (and -6) -carboxyrhodamine 6G.succinimidyl ester (trade name, manufactured by emp Biotech GmbH) was dissolved in 1 mL of dimethylformamide (DMF). 1.2 μL of APS (manufactured by Shin-Etsu Silicone) was added thereto and reacted at room temperature (23 ° C.) for 1 hour to obtain a 5- (and-6) -carboxyrhodamine 6G-APS complex (5 mM). 600 μL of the obtained 5- (and -6) -carboxyrhodamine 6G-APS complex solution, ethanol 140 mL, tetraethoxysilane (TEOS) (manufactured by Shin-Etsu Silicone) 6.5 mL, distilled water 20 mL, and 28% by mass ammonia 15 mL of water was mixed and reacted at room temperature for 24 hours. The reaction solution was centrifuged at a gravity acceleration of 18000 × g for 30 minutes, and the supernatant was removed. 4 mL of distilled water was added to the precipitated silica fine particles, the particles were dispersed, and centrifuged again at a gravity acceleration of 18000 × g for 30 minutes. This washing operation was further repeated twice to remove unreacted TEOS, ammonia, and the like contained in the dispersion of fluorescent molecule-containing silica fine particles to obtain 1.65 g of silica fine particles containing fluorescent molecules having an average particle diameter of about 270 nm. (Yield about 94%).

(Introduction of thiol group)
20 mg of the particles obtained above were dispersed in 1 mL of a mixture of water / ethanol = 1/4. 20 μL of mercaptopropyltrimethoxysilane (MPS) (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto. Subsequently, 50 μL of 28% aqueous ammonia was added and mixed at room temperature for 4 hours. The reaction solution was centrifuged at a gravity acceleration of 18000 × g for 30 minutes, and the supernatant was removed. 1 mL of distilled water was added to the precipitated silica fine particles to disperse the particles, and the mixture was again centrifuged for 30 minutes at a gravitational acceleration of 18000 × g. This washing operation is further repeated twice to remove unreacted MPS, ammonia, etc. contained in the dispersion of the thiol group-containing fluorescent molecule-containing silica fine particles, and the silica fine particles (silica containing the thiol group-introduced fluorescent molecules). Fine particles A) were obtained.

(Elemental analysis of sulfur element)
Elemental analysis was performed using 20 mg of the above silica fine particles A. As a result, the content of sulfur element was 374 ppm (mass basis) with respect to the total mass of the particles.

[Example 1] Production of labeled protein (binding using anti-His tag antibody)
・ Introduction of linking substance (anti-His tag antibody) To 40 μl of a dispersion of thiol group-introduced fluorescent silica nanoparticles A (concentration 25 mg / ml, dispersion medium: distilled water), add DMF 460 μl, and centrifuge for 10 minutes at a gravitational acceleration of 15000 × g. separated. The supernatant was removed, 500 μl of DMF was added and centrifuged, and the supernatant was removed. Again, 500 μl of DMF was added to disperse the thiol group-introduced fluorescent silica nanoparticles A. By adding 1 mg of N- (4-aminophenyl) maleimide as a linker molecule and mixing for 30 minutes, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticles. It was.
The reaction solution was centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Then, 74.6 μL of distilled water was added to disperse the particles. Subsequently, 0.5 μM MES (2-morpholinoethanesulfonic acid) (pH 6.0) 100 μl, 50 mg / ml NHS (N-hydroxysuccinimide) 230.4 μl, 19.2 mg / ml EDC (1-ethyl-3- ( 75 μl of 3-dimethylaminopropyl) carbodiimide) was added and mixed. To this, 20.0 μl of anti-His tag antibody (1.0 mg / ml, manufactured by MBL) was added and mixed at room temperature for 1 hour.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. After further washing twice by the same operation, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, and the particles were dispersed to obtain a colloid.
Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the protein binding amount per 1 g of fluorescent molecule-containing silica nanoparticles was 9.7 mg.

-Introduction of binding substance (influenza A recombinant protein) Subsequently, 20.0 μl of influenza A recombinant protein (1 mg / ml, manufactured by Imgenex) into which the His tag was introduced into the colloid was added and mixed at room temperature for 1 hour. . Subsequently, 10 μl of 10% BSA was added and mixed for 10 minutes. Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 500 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Again, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, and the colloid in which the functional molecule-containing silica nanoparticles A bound to the His-tag-containing influenza A recombinant protein were dispersed (hereinafter referred to as colloid 1 of the present invention). Called). See the schematic diagram in FIG. 3 for the structure of the reactive linking chain on the particle.

Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the protein binding amount per 1 g of the fluorescent molecule-containing silica nanoparticles was 18.8 mg. Since the binding amount of influenza A recombinant protein was a value obtained by subtracting the binding amount of the antibody from the binding amount obtained in this measurement, it was estimated to be 9.1 mg per 1 g of the fluorescent molecule-containing silica nanoparticles.
The introduction density of the binding substance estimated from the measurement result by the protein quantification method and the measurement result of the surface area of the silica nanoparticles used in this example was 0.0064 / nm ² .

[Comparative Example 1] Production of labeled protein (binding without using anti-His tag antibody)
-Direct introduction of binding substance (influenza A recombinant protein) To a dispersion of thiol group-introduced fluorescent silica nanoparticles A (concentration 25 mg / ml, dispersion medium: distilled water), add DMF 460 μL, and with a gravitational acceleration of 15000 × g Centrifuge for 10 minutes. The supernatant was removed, 500 μL of DMF was added and centrifuged, and the supernatant was removed. Again, 500 μl of DMF was added to disperse the thiol group-introduced fluorescent silica nanoparticles A. By adding 1 mg of N- (4-aminophenyl) maleimide as a linker molecule and mixing for 30 minutes, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticles. It was.
This reaction solution was centrifuged for 10 minutes at a gravitational acceleration of 15000 × g, and after removing the supernatant, 74.6 μl of distilled water was added to disperse the particles. Subsequently, 0.5 μM MES (2-morpholinoethanesulfonic acid) (pH 6.0) 100 μl, 50 mg / ml NHS (N-hydroxysuccinimide) 230.4 μl, 19.2 mg / ml EDC (1-ethyl-3- ( 75 μl of 3-dimethylaminopropyl) carbodiimide) was added and mixed. 20.0 μl of influenza A recombinant protein (1.0 mg / ml, manufactured by Imgenex) into which a His tag was introduced was added thereto, and mixed at room temperature for 1 hour.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. After further washing twice by the same operation, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, and the particles were dispersed to obtain a colloid.
Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the binding amount of the antibody per 1 g of the fluorescent molecule-containing silica nanoparticles was 9.6 mg.

Subsequently, 10 μl of 10% BSA was added to the colloid and mixed for 10 minutes. Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 500 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Again, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles, and colloid in which the functional molecule-containing silica nanoparticles A bound with anti-influenza A nucleoprotein antibody were dispersed (hereinafter referred to as colloid 1 of Comparative Example) Obtained.)

  Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the protein binding amount per 1 g of the fluorescent molecule-containing silica nanoparticles was 18.8 mg. Since the binding amount of influenza A nucleoprotein recombinant was a value obtained by subtracting the binding amount of the antibody from the binding amount obtained in this measurement, it was estimated to be 9.2 mg per 1 g of the fluorescent molecule-containing silica nanoparticles.

[Test Example 1] Immunochromatographic test (Preparation of test strip for immunochromatography)
An antibody-immobilized membrane was prepared by the following method.
Goat-derived anti-mouse IgG antibody (polyclonal antibody, Meridian Inc.) as a test line for detection of anti-influenza A influenza antibody at a position about 6 mm from the end of the membrane (length 25 mm, trade name: Hi-Flow Plus120 membrane, manufactured by MILLIPORE) Manufactured solution) ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) was applied at a coating amount of 0.75 μl / cm with a width of about 1 mm.

Subsequently, as a control line having a width of about 1 mm, 1 mg / ml of an anti-influenza A influenza nucleoprotein antibody (Product No. InA245, derived from mouse, manufactured by HyTest) was applied at a coating amount of 0.75 μl / cm, and 30 minutes at 50 ° C. Dried. The distance between the test line and the control line was 4 mm.
The antibody-immobilized membrane, sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), and absorption pad (Cellulose Fiber Sample Pad (CFSP), manufactured by MILLIPORE) are used as a backing sheet (trade name AR9020, manufactured by Adhesives Research). ) Assembled above. The membrane was configured in such a direction that the test line for influenza A influenza was on the sample pad side and the control line was on the absorption pad side.

(Detection device)
A detection unit including a light source, an optical filter, and a photomultiplier tube (PMT), the detection unit having a mechanism that linearly moves at a constant speed by a motor, and a recording mechanism that records the received light intensity of the PMT every 50 μsec. A detection device was prepared. The detection unit is a laser diode having a light source of 532 nm. The sample is irradiated with the laser diode, and the reflected light is transmitted through an optical filter that transmits only light having a wavelength of 550 nm or more, and then a photomultiplier tube (PMT). It has a mechanism to receive light.

(Rapid determination of anti-influenza A nucleoprotein antibody)
With the composition shown in Table 2, a solution of anti-influenza A nucleoprotein antibody (product number InA108, derived from mouse, manufactured by HyTest) was prepared. Subsequently, 100 μl of the same mixed solution and 1 μl of the colloid 1 of the present invention (2.5 mg / ml) were dropped onto the sample pad portion of the test strip. The test was similarly performed on colloid 1 of the comparative example. After 15 minutes, the light emission intensity of the line was measured by the above detector and digitized. The results are shown in Table 2.

＊１ 抗Ａ型インフルエンザ核タンパク質抗体

* 1 Anti-influenza A nucleoprotein antibody

  As shown in Table 2, when the particles modified with the influenza A nucleoprotein recombinant were compared by the method of Example 1 and the method of Comparative Example 1, a sample containing no anti-A influenza nucleoprotein antibody (0 μg / ml) Then, compared with the colloid of Comparative Example 1, it was found that the colloid of Example 1 has reduced test line strength and less nonspecific adsorption. This is because the portion other than the reaction site contributes to non-specific adsorption, and in Example 1, the reaction site is directed radially away from the particles, so that the exposure of the portion other than the reaction site is suppressed, and the adsorption is performed. This is thought to be due to suppression. On the other hand, in the samples containing anti-influenza A nucleoprotein antibodies (5 μg / ml and 20 μg / ml), the test line strength of the colloid of Example 1 was significantly increased compared to the colloid of Comparative Example 1. From this result, it can be seen that the binding efficiency of the test substance (anti-influenza A influenza nucleoprotein antibody) is increased in the composite labeled particles of the present invention (Example).

[Example 2] Production of labeled antibody (binding using secondary antibody)
-Introduction of linking substance (secondary antibody: goat-derived anti-mouse IgG antibody) To 40 µL of a dispersion of thiol group-introduced fluorescent silica nanoparticles A (concentration 25 mg / ml, dispersion medium: distilled water), add 460 µl of DMF, and 15000 x Centrifuge for 10 minutes at the gravitational acceleration of g. The supernatant was removed, 500 μL of DMF was added and centrifuged, and the supernatant was removed. Again, 500 μl of DMF was added to disperse the thiol group-introduced fluorescent silica nanoparticles A. By adding 1 mg of N- (4-aminophenyl) maleimide as a linker molecule and mixing for 30 minutes, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticles. It was.
The reaction solution was centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Then, 74.6 μL of distilled water was added to disperse the particles. Subsequently, 0.5 μM MES (2-morpholinoethanesulfonic acid) (pH 6.0) 100 μl, 50 mg / ml NHS (N-hydroxysuccinimide) 230.4 μl, 19.2 mg / ml EDC (1-ethyl-3- ( 75 μl of 3-dimethylaminopropyl) carbodiimide) was added and mixed. 20.0 μl of goat-derived anti-mouse IgG antibody (1 mg / ml, polyclonal antibody, manufactured by Meridian) was added thereto, and mixed at room temperature for 1 hour.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. After further washing twice by the same operation, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, and the particles were dispersed to obtain a colloid.
Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the binding amount of the antibody per 1 g of the fluorescent molecule-containing silica nanoparticles was 10.3 mg.

-Introduction of binding substance (primary antibody: anti-influenza A nucleoprotein antibody) Subsequently, 1 mg / ml anti-influenza A nucleoprotein antibody (product number InA108, derived from mouse, manufactured by HyTest) was added to the colloid as described above. 0 μl was added and mixed for 1 hour at room temperature. Subsequently, 10 μl of 10% BSA was added and mixed for 10 minutes. Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 500 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Add 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) again, disperse the particles, and disperse silica nanoparticles A containing functional molecules to which anti-influenza A nucleoprotein antibody (product number InA108, derived from mouse, manufactured by HyTest) was bound. Thus obtained colloid (hereinafter referred to as colloid 2 of the present invention) was obtained. See the schematic diagram in FIG. 4 for the structure of the reactive linking chain on the particle.

Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the protein binding amount per 1 g of fluorescent molecule-containing silica nanoparticles was 20.4 mg. Since the binding amount of the influenza nucleoprotein antibody was a value obtained by subtracting the binding amount of the antibody from the binding amount obtained in this measurement, it was estimated to be 10.1 mg per 1 g of fluorescent molecule-containing silica nanoparticles.
The introduction density of the binding substance estimated from the measurement result by the protein quantification method and the measurement result of the surface area of the silica nanoparticles used in this example was 0.029 / nm ² .

[Comparative Example 2] Production of labeled antibody (binding without using secondary antibody)
-Direct introduction of binding substance (primary antibody ... anti-influenza A nucleoprotein antibody) To 40 μl of a dispersion of thiol group-introduced fluorescent silica nanoparticles A (concentration 25 mg / ml, dispersion medium: distilled water), add 460 μl of DMF, Centrifugation was performed for 10 minutes at a gravitational acceleration of 15000 × g. The supernatant was removed, 500 μL of DMF was added and centrifuged, and the supernatant was removed. Again, 500 μL of DMF was added to disperse the thiol group-introduced fluorescent silica nanoparticles A. By adding 1 mg of N- (4-aminophenyl) maleimide as a linker molecule and mixing for 30 minutes, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticles. It was.
This reaction solution was centrifuged for 10 minutes at a gravitational acceleration of 15000 × g, and after removing the supernatant, 90.6 μL of distilled water was added to disperse the particles. Subsequently, 0.5 μM MES (2-morpholinoethanesulfonic acid) (pH 6.0) 100 μl, 50 mg / ml NHS (N-hydroxysuccinimide) 230.4 μL, 19.2 mg / ml EDC (1-ethyl-3- ( 75 μl of 3-dimethylaminopropyl) carbodiimide) was added and mixed. To this was added 20.0 μl of 1 mg / mL anti-influenza A influenza nucleoprotein antibody (product number InA108, mouse-derived, HyTest) and mixed at room temperature for 1 hour.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. After further washing twice by the same operation, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, and the particles were dispersed to obtain a colloid.
Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the binding amount of the antibody per 1 g of the fluorescent molecule-containing silica nanoparticles was 10.3 mg.

Subsequently, 10 μl of 10% BSA was added to the colloid and mixed for 10 minutes. Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 500 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Again, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, and a colloid in which the functional molecule-containing silica nanoparticles A bound with anti-influenza A nucleoprotein antibody were dispersed (hereinafter referred to as colloid 2 of Comparative Example) Obtained.)

[Test Example 2] Immunochromatographic test (Preparation of test strip for immunochromatography)
An antibody-immobilized membrane was prepared by the following method.
Anti-influenza A nucleoprotein antibody (Product No. InA245, mouse) is used as a test line for detecting influenza A nucleoprotein at a position approximately 6 mm from the end of the membrane (length 25 mm, trade name: Hi-Flow Plus120 membrane, manufactured by MILLIPORE). A solution ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) containing 1 mg / ml derived from HyTest was applied at a coating amount of 0.75 μl / cm with a width of about 1 mm.

Subsequently, as a control line having a width of about 1 mm, 1 mg / ml of goat-derived anti-mouse IgG antibody (polyclonal antibody, manufactured by Meridian) was applied at a coating amount of 0.75 μl / cm and dried at 50 ° C. for 30 minutes. The distance between the test line and the control line was 4 mm.
The antibody-immobilized membrane, sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), and absorption pad (Cellulose Fiber Sample Pad (CFSP), manufactured by MILLIPORE) are used as a backing sheet (trade name AR9020, manufactured by Adhesives Research). ) Assembled above. The membrane was configured in such a direction that the test line for influenza A influenza was on the sample pad side and the control line was on the absorption pad side.

(Detection device)
A detection unit including a light source, an optical filter, and a photomultiplier tube (PMT), the detection unit having a mechanism that linearly moves at a constant speed by a motor, and a recording mechanism that records the received light intensity of the PMT every 50 μsec. A detection device was prepared. The detection unit is a laser diode having a light source of 532 nm. The sample is irradiated with the laser diode, and the reflected light is transmitted through an optical filter that transmits only light having a wavelength of 550 nm or more, and then a photomultiplier tube (PMT). It has a mechanism to receive light.

(Rapid determination of anti-influenza A nucleoprotein antibody)
With the composition shown in Table 2, a solution of anti-influenza A nucleoprotein antibody (product number InA108, derived from mouse, manufactured by HyTest) was prepared. Subsequently, 100 μl of the same mixed solution and 1 μl of the colloid 2 of the present invention (2.5 mg / ml) were dropped onto the sample pad portion of the test strip. The test was similarly performed on the colloid 3 of the present invention and the colloid 2 of the comparative example. After 15 minutes, the light emission intensity of the line was measured by the above detector and digitized. The results are shown in Table 3.

＊１ Ａ型インフルエンザ核タンパク質

* 1 Influenza A nuclear protein

  As shown in Table 3, when the particles modified with the anti-influenza A nucleoprotein antibody by the method of Example 2 and Example 3 and the method of Comparative Example 2 were compared, a sample containing no influenza A nucleoprotein (0 μg) / Ml), the colloids of Example 2 and Example 3 were found to have less test line strength and less non-specific adsorption than the colloid of Comparative Example 2. On the other hand, in the samples containing influenza A nucleoprotein (20 μg / ml and 50 μg / ml), the test line strength of the colloids of Example 2 and Example 3 was significantly increased compared to the colloid of Comparative Example 2. . From this result, it can be seen that in the composite labeled particles of the present invention (Examples 2 and 3), the binding efficiency of the test substance (type A influenza nucleoprotein) is increased.

  From the above results, it can be seen that the composite labeled particles of the present invention have a high binding efficiency to the target substance, and enable detection and measurement of the target substance with high accuracy in tests such as immunochromatography.

[Example 3] Production of labeled antibody (binding using streptavidin)
-Introduction of linking substance (streptavidin) To 40 µl of a dispersion of thiol group-introduced fluorescent silica nanoparticles A (concentration 25 mg / ml, dispersion medium: distilled water), 460 µl of DMF was added and centrifuged at a gravitational acceleration of 15000 xg for 10 minutes. . The supernatant was removed, 500 μl of DMF was added and centrifuged, and the supernatant was removed. Again, 500 μl of DMF was added to disperse the thiol group-introduced fluorescent silica nanoparticles A. By adding 1 mg of N- (4-aminophenyl) maleimide as a linker molecule and mixing for 30 minutes, a thioether bond is formed between the maleimide group of the linker molecule and the thiol group of the thiol group-introduced fluorescent silica nanoparticles. It was.
The reaction solution was centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Then, 74.6 μL of distilled water was added to disperse the particles. Subsequently, 0.5 μM MES (2-morpholinoethanesulfonic acid) (pH 6.0) 100 μl, 50 mg / ml NHS (N-hydroxysuccinimide) 230.4 μl, 19.2 mg / ml EDC (1-ethyl-3- ( 75 μl of 3-dimethylaminopropyl) carbodiimide) was added and mixed. 20.0 μl of streptavidin (1.0 mg / ml, manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto and mixed at room temperature for 1 hour.
Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added to disperse the particles. Subsequently, centrifugation was performed at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. After further washing twice by the same operation, 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) was added, and the particles were dispersed to obtain a colloid.
Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the protein binding amount per 1 g of fluorescent molecule-containing silica nanoparticles was 5.1 mg.

Introduction of biotin 5 mg / mL anti-influenza A nucleoprotein antibody (product number InA108, derived from mouse, HyTest) 200.0 μl, phosphate buffer 250 μL, 1 mg / mL NHS-Biotin (Thermo Scientific) Was added and reacted at 25 ° C. for 30 minutes. Thereafter, unreacted NHS-Biotin was removed by ultrafiltration to obtain an anti-influenza A influenza nucleoprotein antibody bound to biotin (hereinafter, biotinylated anti-influenza A nucleoprotein antibody).

・ Introduction of binding substance (biotin: anti-influenza A nucleoprotein antibody) Subsequently, 20.0 μl of 1 mg / ml biotinylated anti-influenza A nucleoprotein antibody was added to the particle colloid bound with the above streptavidin, Mix for 1 hour at room temperature. Subsequently, 10 μl of 10% BSA was added and mixed for 10 minutes. Centrifugation was carried out at a gravitational acceleration of 15000 × g for 10 minutes, and the supernatant was removed. 500 μL of 10 mM KH ₂ PO ₄ (pH 7.5) was added, the particles were dispersed, centrifuged at 15000 × g for 10 minutes, and the supernatant was removed. Add 400 μl of 10 mM KH ₂ PO ₄ (pH 7.5) again, disperse the particles, and disperse silica nanoparticles A containing functional molecules to which anti-influenza A nucleoprotein antibody (product number InA108, derived from mouse, manufactured by HyTest) was bound. Thus obtained colloid (hereinafter referred to as colloid 3 of the present invention) was obtained. See the schematic diagram of FIG. 5 for the structure of the reactive linking chain on the particle.

Using this colloid as a sample, protein quantification was performed. For protein quantification, Pierce BCA Protein Assay Kit (manufactured by Thermo Fisher Scientific) was used. As a result, the protein binding amount per 1 g of the fluorescent molecule-containing silica nanoparticles was 11.8 mg. Since the binding amount of the influenza nucleoprotein antibody was a value obtained by subtracting the binding amount of the antibody from the binding amount obtained in this measurement, it was estimated to be 6.7 mg per 1 g of fluorescent molecule-containing silica nanoparticles.
The introduction density of the binding substance estimated from the measurement result by the protein quantification method and the measurement result of the surface area of the silica nanoparticles used in this example was 0.019 / nm ² .

  The obtained fluorescent molecule-containing silica nanoparticles (composite label) were used in the same manner as in Example 2 to detect influenza A nucleoprotein. As a result, it was confirmed that good detection ability (binding efficiency) was exhibited as in Example 2. From these results, it can be seen that in the composite-labeled particle of the present invention, it is possible to form a suitable linked chain using various types of materials.

1 Target substance (test substance)
2 Labeled body (composite labeled particle)
2a Labeled particle 2b Linked substance 2c Linked site 2d Reaction site 21 Binding substance 22 Binding linked chain 3 Labeled body 3a Labeled particle 3b Reference binding substance 4 Test capturing substance 5 Reference capturing substance 6 Housing 61 Detection Opening 62 Specimen introduction opening 6a Case upper part 6b Case lower part 8a Sample pad 8b Conjugate pad 8c Membrane 8d Absorption pad 10 Test strip 100 Long test specimen n _r Reference area n _t Test area L Lateral flow direction S Specimen

Claims (15)

Complex labeled particles before capturing the target substance, and
A composite labeled particle for immunochromatography for detecting the target substance,
Having a linking substance and a binding substance in this order on the surface of the labeling particle, the binding substance is a composite labeling particle having a reaction site showing binding property to the target substance,
A composite label having a structure in which reaction sites of the binding substance extend in a direction radially away from the labeling particles and are connected, and the linking substance and the binding substance are linked at a linking site on the binding substance side. particle. The composite labeling particle according to claim 1, wherein the combination of the linking substance and the binding substance is selected from the following (i) to (viii).
――――――――――――――――――――――――――――――――――――
Binding substance / Linking substance ――――――――――――――――――――――――――――――――――――
(I) His tag introduction substance / anti-His tag antibody (ii) primary antibody / secondary antibody (iii) biotin introduction substance / avidin (iv) avidin introduction substance / biotin (v) biotin introduction substance / streptavidin (vi) strept Avidin-introduced substance / Biotin (vii) FLAG tag-introduced substance / Anti-FLAG tag antibody (viii) Fluorescein-introduced substance / Anti-fluorescein antibody ――――――――――――――――――――――― ――――――――――――― The composite labeling particle according to claim 2, wherein the His tag introduction substance is a fusion protein having a His tag. Composite label particles according to any one of claims 1 to 3 applied density on label particles is 0.001 to 0.1 pieces / nm ² of the binding agent.   The composite labeling particle according to any one of claims 1 to 4, wherein the labeling particle has an average particle diameter of 20 to 500 nm.   The composite labeling particle according to any one of claims 1 to 5, wherein the labeling particle is a silica particle.   The composite labeled particle according to any one of claims 1 to 6, wherein a thiol group is present on the surface of the labeled particle, and the linking substance is introduced through the thiol group.   The composite labeling particle according to claim 1, wherein the linking substance is introduced into the labeling particle via a maleimide linker.   A target substance is specifically captured using the composite label particles according to any one of claims 1 to 8, and the target substance detection determination and / or target is detected by detecting the composite label particles capturing the target substance. A target substance detection method for quantifying substances.   A colloidal solution containing the composite labeling particles according to any one of claims 1 to 8.   A labeling reagent comprising the composite labeling particle according to any one of claims 1 to 8. A method for producing composite labeled particles for immunochromatography before capturing a target substance,
A step of introducing a linking substance to the surface of the labeling particles, a step of introducing a binding substance via the linking substance,
The method for producing composite-labeled particles, wherein the binding substance has a reaction site showing binding ability to a target substance. The method for producing composite labeled particles according to claim 12, wherein the combination of the linking substance and the binding substance is selected from the following (i) to (viii).
――――――――――――――――――――――――――――――――――――
Binding substance / Linking substance ――――――――――――――――――――――――――――――――――――
(I) His tag introduction substance / anti-His tag antibody (ii) primary antibody / secondary antibody (iii) biotin introduction substance / avidin (iv) avidin introduction substance / biotin (v) biotin introduction substance / streptavidin (vi) strept Avidin-introduced substance / Biotin (vii) FLAG tag-introduced substance / Anti-FLAG tag antibody (viii) Fluorescein-introduced substance / Anti-fluorescein antibody ――――――――――――――――――――――― ――――――――――――― The method of producing a composite label particles according to claim 12 or 13 is introduced in the 0.001 to 0.1 pieces of the connecting material against the label particles / nm ^2. The method for producing a composite labeling particle according to any one of claims 12 to 14, wherein the linking substance and the binding substance are linked via a binding site of the binding substance.

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