JP5583092B2 - Immunochromatographic test kit and detection method using the same - Google Patents

Description

The present invention, immuno-chromatographic test kits, about the detection how using the same.

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 contained in the sample liquid is captured by the labeled particles, and the porous support is moved by capillary action. Then, for example, the test substance is concentrated by bringing the porous substance into contact with a capture substance fixed in a line shape, for example, and a line in 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 the immunochromatography method.
(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, the immunochromatography method 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, for example, and is attracting more and more attention.

  Here, POCT refers to a test for making a diagnosis as close as possible to the patient. Conventionally, collected blood, urine, affected tissue, and other specimens are sent to a central laboratory in a hospital or a specialized examination center to output data, so it took time to confirm the diagnosis. According to POCT, prompt and accurate treatment is possible based on examination information provided instantaneously. This makes it possible to conduct emergency inspections at hospitals and inspections during surgery, and there are particularly high needs in the medical field.

  In view of the above requirements, the present applicant has researched and developed reagents applied to membranes, and has developed a technique using fluorescent silica fine particles (see Patent Document 1, etc.). As a result, measurement and detection can be performed at a lower cost and significantly more stable than those using conventional biological materials, biological cells, or colloidal gold particles as reagents. In addition, it can respond more accurately to demands such as improved detection sensitivity and quantification, and greatly contributes to the expansion of the application area of immunochromatography.

  In addition, the present applicant has proposed a conjugate pad in which fluorescent silica fine particles and light absorbing silica fine particles are combined (see Patent Document 2). As a result, it is possible to perform detection even in a state where no fluorescence is irradiated, and it is possible to improve detection accuracy and perform quantitative measurement by detecting fluorescence as necessary.

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

The present inventors further continued research on immunochromatography using the fluorescent fine particles (hereinafter sometimes referred to as a fluorescent immunochromatographic test). As a result, more particles are adsorbed in the control line than in the test line. However, it has been found that the latter light emission intensity tends to increase. Then, when positive / negative determination is performed with high sensitivity, the visibility of a test line with a weaker light emission intensity decreases due to the light emission of a control line with a high light emission intensity.
That is, the present invention aims to solve problems peculiar to the fluorescence immunochromatography test, and uses an immunochromatography test kit capable of suppressing / preventing a decrease in the visibility of the fluorescence of the test line due to the fluorescence emission of the control line. and an object thereof is to provide a detection how.

The above problem has been solved by the following means.
[1] A planar test piece having a test area for immunochromatography and a reference area, a target substance provided with the binding property to a test capturing substance in the test area, and a test substance having a binding property to the target substance Preparing fluorescent fine particles into which a binding substance has been introduced and light-absorbing fine particles into which a reference binding substance having a binding property to a reference capturing substance in a reference region has been introduced ;
The fluorescent fine particles and the light-absorbing fine particles are previously applied to the test piece, a target substance is applied to the test piece, and the fluorescent fine particles and the light-absorbing fine particles are bonded to the fluorescent fine particles via the binding substance. In the plane test piece, the test capturing substance in the test area and the fluorescent fine particles are bonded via the target substance and the test binding substance , while the light absorbing fine particles are bonded in the reference area. The reference capture substance in the reference region of the test piece is bonded to the reference capture substance via the reference capture substance ,
A detection method by immunochromatography in which the target substance is detected from the fluorescence emission from the fluorescent fine particles in the test region and the light absorption state of the light absorbing fine particles in the reference region.
[2] The detection method according to [1], wherein one or both of the fluorescent fine particles and the light absorbing fine particles are composed of silica fine particles.
[3] The detection method according to [1] or [2], wherein one or both of the fluorescent fine particles and the light absorbing fine particles have an average particle diameter of 20 to 1000 nm.
[4] The detection method according to any one of [1] to [3], wherein a nitrocellulose membrane is used as a membrane for providing the test region and the reference region.
[5] The detection method according to any one of [1] to [4], wherein the reference region is confirmed visually and the test region is confirmed using a fluorescence detection apparatus.
[6] The detection method according to any one of [1] to [5], in which it is determined that the reference region has been used based on a color change, and whether the test region is positive or negative is determined by fluorescence emission. .
[7] The detection method according to any one of [1] to [6], wherein high sensitivity determination and / or quantitative measurement is performed by detecting fluorescence emission in the test region.
[8] The detection method according to any one of [1] to [7], wherein a distance between a test line forming the test area and a control line forming the reference area is 1 mm or more and 20 mm or less.
[9] The detection method according to any one of [1] to [8], wherein a wavelength of fluorescence emission of the fluorescent fine particles is 400 to 800 nm.
[1 0 ] A test kit comprising a combination of a planar test piece for immunochromatography, fluorescent fine particles, and light absorbing fine particles,
The flat specimen has a test area and a reference area,
The fluorescent fine particle is introduced with a test binding substance imparted with a binding property to a target substance, and the target substance has a binding property with a test capturing substance in the test region, while the light absorbing fine particle is A test kit for immunochromatography into which a reference binding substance imparted with a binding property to a reference capture substance in the reference region is introduced .
[1 1 ] The kit according to [10], wherein one or both of the fluorescent fine particles and the light absorbing fine particles are composed of silica fine particles.
[1 2] the one or both of the fluorescent particles and the absorption particles having an average particle size 20~1000nm [1 0] or serial mounting kit in [11].
[13] The kit according to any one of [10] to [12], wherein a nitrocellulose membrane is used as a membrane for providing the test region and the reference region.
[14] The kit according to any one of [10] to [13], further combined with a fluorescence detection device that detects fluorescence emission in the test region.
[15] The kit according to any one of [10] to [14], wherein a distance between the test line forming the test area and the control line forming the reference area is 1 mm or more and 20 mm or less.
[16] The kit according to any one of [10] to [15], wherein the fluorescent fine particles have a fluorescent emission wavelength of 400 to 800 nm.
[17] The kit according to any one of [10] to [16], wherein the wavelength of excitation light of the irradiator included in the fluorescence detection device is 300 to 900 nm.

Immunochromatographic test kit of the present invention, according to the detection how using the same, can suppress or prevent lowering of visibility of the fluorescence of the test line by fluorescence emission control lines. Further, since the fluorescent fine particles and the light absorbing fine particles are applied in combination, it can be visually confirmed that they are used.

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.

The immunochromatographic test kit of the present invention is a combination of a planar test piece for immunochromatography having a reference region and a test region, a fluorescent fine particle having a specific property, and a light absorbing fine particle having a specific property. It has. Hereinafter, the configuration of the present invention will be described in detail focusing on preferred embodiments thereof.
[Labeling reagent]
(Fluorescent fine particles)
This onset bright fluorescent particles are contained in the labeled reagent are surface modified target substance (analyte) recognizing binding substance.

-Average particle diameter Although the average particle diameter of the said label | marker particle | grain is not specifically limited, It is preferable that it is 20-1000 nm, It is more preferable that it is 20-600 nm, It is more preferable that it is 60-300 nm. If the particle size is too small, the detection sensitivity decreases, and if the particle size is too large, the porous support (membrane) used in the immunochromatography method becomes clogged.

  In the present invention, the average particle size is determined from the total projected area of 50 labeled particles randomly selected from an image of a transmission electron microscope (TEM), a scanning electron microscope (SEM), or the like, Is obtained by an image processing apparatus, and an average value (average circle equivalent diameter) of the diameters of the circles corresponding to a value obtained by dividing the total occupied area by the number (50) of the selected labeled particles is obtained. The coefficient of variation of the particle size distribution, the so-called CV value, is not particularly limited, but is preferably 15% or less, more preferably 10% or less, and particularly preferably 8% or less.

-Constituent material of fine particles Although there is no restriction | limiting in particular in the material of fluorescent fine particles, For example, a silica particle, latex particle, etc. can be used. The fluorescent fine particles of this embodiment are preferably surface-modified with a substance that recognizes the target substance of the specimen as described above. As a method for surface modification, those usually applied to this kind of material may be appropriately used.

Silica particles In the present invention, the fluorescent fine particles are preferably composed of silica particles. There is no restriction | limiting in particular as a silica particle, The silica particle obtained by arbitrary arbitrary preparation methods may be sufficient. For example, it is particularly preferable to use silica particles containing a labeling substance obtained according to the method for preparing fluorescent dye compound-containing colloidal silica particles described in International Publication No. 2007 / 074722A1. Specifically, the silica particles containing the labeling substance are 1 or 2 in a product obtained by reacting the labeling substance and a silane compound and covalently bonding, ionic bonding, or other chemical bonding or adsorption. It can be prepared by polymerizing two or more silane compounds. Preferred embodiments of the silica particles containing the labeling substance include N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl. The labeling substance having an active group such as a group, an epoxy group or a cyano group is reacted with a silane coupling agent having a substituent (for example, an amino group, a hydroxyl group or a thiol group) that reacts with the active group. The product obtained by covalent bonding can be prepared by polymerizing one or more silane compounds.

  Specific examples of the labeling substance (fluorescent substance) having the active group have an NHS ester group such as 5- (and -6) -carboxytetramethylrhodamine succinimidyl ester (trade name, manufactured by emp Biotech GmbH). Things can be mentioned.

  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 polymerized is not particularly limited, but includes tetraethoxysilane (TEOS), γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS). ), 3-thiocyanatopropyltriethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxy Mention may be made of silane. Among these, TEOS, MPS, or APS is preferable. In the present embodiment, the labeling substance is in a state of being immobilized in 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, 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.

Latex particles In the present invention, the latex particles include polystyrene, styrene-sulfonic acid (salt) copolymer, styrene-methacrylic acid copolymer, acrylonitrile-butadiene-sulfonic acid copolymer, vinyl chloride-acrylic acid. Examples thereof include synthetic polymer particles composed of an ester copolymer, a vinyl acetate-acrylic acid ester copolymer, and 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 type of particles by a conventional method as appropriate. 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 product name xMAP (registered trademark) Multi-Analyte COOH Microspheres, (http://hitachisoft.jp/products/lifescience/lineup/luminex/about/bead.htmlhttp:/ /hitachisoft.jp/products/lifescience/pdf/the_luminex_labmap_system.pdf).

-Surface modification The method for surface-modifying the substance that recognizes the specimen on the labeled particles is not particularly limited, and the substance that recognizes the specimen by electrostatic attraction, van der Waals force, hydrophobic interaction, etc. is labeled. You may make it adsorb | suck to particle | grains and may make it couple | bond with a chemical bond with a crosslinking agent or a condensing agent. Further, when the labeled particle surface has a thiol group, it may be bonded to the thiol group of a substance that recognizes the specimen by an SS bond.
In the case where particles aggregate when binding a substance that recognizes the specimen, such as biomolecules (for example, antibodies, antigens, DNA, RNA) on the surface of the labeled particles, an alternate adsorption method is used on the surface of the labeled particles in advance. May be subjected to surface treatment. The alternating adsorption method is a method of forming a polymer thin film on the surface of a substrate or particle by adsorbing a charged polymer to the surface of the substrate or particle having a charge by electrostatic attraction. By carrying out the alternate adsorption treatment on the surface of the labeled particle, a charge can be imparted to the particle surface, so that an electrostatic repulsive force is generated between the particles and the dispersibility is improved. Further, since the polymer bonded to the particles has an excluded volume, the dispersibility is improved also by the effect of the steric repulsive force.

(Absorbing fine particles)
Absorption fine particle for use in the present invention that have been surface-modified with biomolecules such as capable of binding to a reference for capturing material. The kind and shape are not particularly limited. Preferable examples of the average particle diameter and constituent materials of the light absorbing fine particles are the same as those of the fluorescent fine particles. As the labeling substance, there is no need to use it as long as the constituent material of the fine particles is light-absorbing. When applying a labeling substance (light-absorbing substance), organic pigments such as polycyclic pigments and azo pigments, and inorganic pigments such as carbon black and ultramarine blue can be used. For example, the light-absorbing substance may be included in the silica particles or latex particles. It is also preferable to use the following semiconductor fine particles as the light absorbing fine particles.

Semiconductor particles and the like 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 nanoparticles described in Japanese Patent No. 3897285 can be used. The semiconductor nanoparticles 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 nanoparticles. As the gold particles and the metal nanoparticles, colloidal gold particles and metal colloidal particles described in JP-A-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 crystal 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 thereof include tin oxide (ITO). For example, inorganic crystals described in JP-A-2005-76064 can be used.

-Absorption coefficient It is preferable that the light-absorbing fine particles absorb visible light and can be colored and visually recognized. The molar extinction coefficient ε is preferably a particle having 5 × 10 ⁶ M ⁻¹ cm ⁻¹ or more, and the molar extinction 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, or an N, N-dimethylformamide dispersion using an arbitrary absorptiometer or plate reader.

  The embodiment of the surface modification of the light absorbing fine particles is the same as that of the fluorescent fine particles. However, it is preferable that the light-absorbing fine particles have a binding property to the reference capturing substance and are modified with a biomolecule or the like having a binding property to the reference capturing substance.

(Long specimen)
FIG. 1 is an exploded perspective view schematically showing a long test body as a preferred embodiment of the present invention. The planar test piece (test strip) 80 of the present embodiment includes a sample pad 8a that is a member to which a sample (specimen) s containing a test substance (target substance) 1 is added from the right side of FIG. Next, a conjugate pad 8b is disposed next to it. Here, a fluorescent label 2 having fluorescent fine particles into which a binding substance that specifically binds to a target substance is introduced, and a light-absorbing fine particle into which a binding substance that specifically binds to a reference capturing substance 5 is introduced The light-absorbing label 3 having
Furthermore, a membrane 8c made of a porous support is disposed. Here, a test region n _t in which a test capturing substance 4 for capturing fluorescent fine particles via a binding substance and a target substance is locally fixed is provided. The membrane 8c, there is a further reference area n _r in front of the flow direction L of the test area n _t. A control line for capturing a light-absorbing label having light-absorbing fine particles is formed by the reference capturing material 5 fixed thereto. Thereafter, in order to flow the sample liquid in the lateral flow direction (L), an absorption pad 8d for sucking up the sample liquid from the membrane is arranged and connected in the above order.
In summary, the flat test piece 80 has a structure in which the sample pad 8a and the conjugate pad 8b, the conjugate pad 8b and the membrane 8c, and the membrane 8c and the absorbent pad 8d are connected so as to partially overlap each other. . When the sample liquid is dropped on the sample pad 8a, the sample liquid sequentially moves to the conjugate pad 8b, the membrane 8c, and the absorption pad 8d by capillary action (capillary force).

  In the present embodiment, the above-described flat test piece 80 is sandwiched and enclosed between the upper part 6a and the lower part 6b of the casing, and forms a long test body 100. A detection opening 61 and a sample introduction opening 62 are provided in the housing upper part 6a. Through this detection opening 61, the irradiation light can be sent to the internal test piece 80, and the fluorescence emitted there can be collected and detected and observed. On the other hand, the sample liquid s can be supplied to the test piece 80 through the sample introduction opening 62 to perform a measurement test.

・ Definition of terms When the definition of terms is confirmed here, target substance 1 (shown together with the symbol in the figure, but is not construed as being limited thereby) is a substance to be detected by the lateral flow method. And is synonymous with the test substance in the sample. The binding substances 2b and 3b (FIG. 2) are substances having binding ability to the target substance and the capturing substance, respectively, and are preferably biomolecules. Labeled particles 2a and 3a (FIG. 2) into which a labeling substance (not shown) has been introduced are referred to as labeled bodies 2 and 3. However, in a broad sense, the term labeled particle is sometimes used in the sense of including a label. The labeled particle is a general term for fluorescent fine particles and light absorbing fine particles. 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.). It may be. In a broad sense, it is meant to include living cells, microorganisms (such as bacteria), and viruses. Bonding generally refers to continuous integration from the state in which multiple objects are separated. In addition to chemical bonds such as covalent bonds, ionic bonds, and hydrogen bonds, chemical adsorption, physical adsorption, etc. The meaning includes fitting, screwing, and biting physical connection state. Here, the term “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. If the sample is liquid, it can be used as it is, and if it is semi-solid or solid, it can be used after being subjected to treatment such as dilution or extraction.

-Binding substance In this embodiment, the binding substance 2b for test (FIG. 2) is used integrally with the labeled particles 2a (labeled body 2). Although the specific example of the binding substance 2b is not specifically limited, The biomolecule which has the ability to bind with the said target particle is mentioned, Specifically, an antibody is mentioned. The binding substance may be directly bonded and integrated with the labeling particle, or may be indirectly bonded through another substance. The bond between the labeled particle and the binding substance is via a functional group, such as a physical adsorption method such as a hydrophobic interaction, a bond between a succinimide group and an amino group, or a bond between a maleimide group and a thiol group. It can be performed by a conventional method such as a chemical bonding method. When the label particles are nanoparticles, a plurality of binding substances can be bound to the surface of one label. In addition, for an embodiment of a labeled body integrated with a binding substance using fluorescent silica fine particles as the labeled particles, for example, International Publication No. 2008/018566 can be referred to.

  In the present embodiment, a reference binding substance 3b is used separately from the test binding substance 2a. The reference binding substance 3b has binding properties with a reference capturing substance 5 described later. The labeled particles (light-absorbing fine particles) 3a and the reference binding substance 3b are integrated to form a reference labeled body 3. Accordingly, the labeling body 3 is captured by the capturing substance 5 in the process of moving through the membrane. The mode of integration of the labeled particles 3a and the binding substance 3b or the preferred kind of the binding substance 3b is the same as the binding substance 2b for the test. 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 test binding substance 2b may have binding properties with the reference capturing substance 5, but it is more preferable not to have the binding properties.

Test Capturing Substance The membrane used in the present embodiment has the test capturing substance 4 immobilized on the material of the membrane. This capturing substance 4 has a binding ability to the target substance 1 so as to capture a complex containing the labeled particles 2a, the binding substance 2b, and the target substance 1 (see FIG. 2). 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 generates fluorescence by the label 2 is formed in the test region n _t . As an example of the combination of “binding substance” — “target substance” — “capture substance”, antibody (B) —antigen (C) of antibody (B) —antibody (D) of antigen (C), antigen ( E) -antigen (E) antibody (F) -antibody (F) antibody (G), nucleic acid (H) -nucleic acid having a sequence complementary to nucleic acid (H) -complementary to nucleic acid (I) Nucleic acid (J) having a different sequence from that of nucleic acid (H), receptor (K) -receptor (K) ligand (L) -antibody against ligand (L) (M), Aptamer (N) -Aptamer (P), Aptamer (P), Aptamer (N) -Aptamer (P), which specifically binds at a site different from the aptamer (N) Protein (R) that specifically binds to (Q) -antibody (S) against protein (R) It is, but the present invention is not limited thereto.

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 that is 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 light absorption by the label 3 is formed in the reference region _nr . The reference capture substance 5 is not particularly limited, and examples thereof include biomolecules capable of binding to the binding substance, and specific examples include antibodies.

The reference capturing substance 5 and the test binding substance 2b in the reference region may have binding properties. In this case, the test fluorescent fine particles 2a are captured in the reference region _nr . That is, both the light-absorbing fine particles 3a and the fluorescent fine particles 2a are captured in the reference region. In this case, the colored state of the light-absorbing fine 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. The ratio of the fluorescent fine particles trapped in the reference region is not particularly limited, but a smaller one is preferable, and it is preferably 30 to 0 several percent, more preferably 15 to 0 several percent of the whole fluorescent fine particles.

-Material There is no restriction | limiting in particular as a material of each structural member employable for the plane test piece 80 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 fluorescent immunochromatographic test, it is preferable that fluorescent particles similar to colored particles to which a binding substance is bound 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.

[Detection method]
An inspection method using the immunochromatographic labeling reagent according to a preferred embodiment of the present invention will be described. An inspection method using an immunochromatographic test strip can be performed by first dropping a sample liquid (sample) containing a specimen onto a sample pad. At this time, the color development of the control line can be visually confirmed, and positive or negative can be determined.

On the other hand, in this embodiment, the color development of the test line is not visually confirmed, but it is possible to make a positive or negative determination with high sensitivity by observing the strip using an immunochromatographic fluorescence detection apparatus. On the other hand, in the inspection method using the immunochromatographic test strip of the present embodiment, the control line is colored with light-absorbing fine particles, so that it can be visually confirmed that it has been used. And it is possible to determine the fluorescence of the test line with high sensitivity without being inhibited by the fluorescence of the control line. The inspection method using the immunochromatographic test strip preferably performs high-sensitivity determination and / or quantitative measurement using a fluorescence detection apparatus after visual determination.
In the present specification, “detection” is a concept including not only qualitative detection but also quantitative detection, and various other measurements, identification, analysis, evaluation, and the like.

From the standpoint that the effects of the present invention are remarkably exhibited, the following settings are preferred.
(1) The distance (d in FIG. 1) between the test line and the control line is preferably 1 mm or more, and more preferably 2 mm or more. Although there is no particular upper limit, for example, 20 mm or less to 10 mm or less is practical.
(2) The wavelength of fluorescent emission on the test line for fluorescent fine particles is generally 400 to 800 nm as described below, and preferably 500 to 700 nm. On the other hand, the absorption wavelength of the light-absorbing fine particles is preferably 400 to 800, and more preferably 450 to 650.

[Detection device]
The detection apparatus for immunochromatography according to a preferred embodiment of the present invention comprises an excitation light source and a filter. Examples of the excitation light source include a mercury lamp, a halogen lamp, a xenon lamp, a laser diode, and a light emitting diode. The filter is a filter for transmitting only light of a specific wavelength from an excitation light source, and a filter that removes the excitation light and transmits only fluorescence from the viewpoint of detecting only fluorescence. It selects suitably from a fluorescence wavelength and a fluorescence wavelength. The fluorescence detection apparatus may include a photomultiplier tube or a CCD detector that receives the fluorescence. As a result, fluorescence having an intensity or wavelength that cannot be visually confirmed can be detected. Further, since the fluorescence intensity can be measured, the specimen can be quantified, and highly sensitive detection and quantification are possible.

  Although the wavelength of the excitation light to irradiate is not specifically limited, It is preferable that it is 300-900 nm, and it is more preferable that it is 400-800 nm. The wavelength of the fluorescence emitted from the fluorescent fine particles is generally about 400 to 800 nm, although it depends on the wavelength of the excitation light irradiated and the type of fluorescent dye.

  Detection of the light-absorbing fine particles may be performed by a conventional method, and detection may be performed visually without using an apparatus.

  As still another embodiment, an example in which collected fluorescence is converted into a signal using a light receiver is given. Then, an image may be displayed by a liquid crystal display device connected to the end. Thus, the inspection record may be stored by fetching the signal into the storage device. Alternatively, predetermined image processing may be performed to make it easier to visually recognize the state of fluorescence emission, or a mechanism that performs automatic determination may be used. Furthermore, it is good also as a structure which calculates the intensity | strength of the obtained detection signal, and measures automatically the quantitative measurement of a to-be-tested substance.

  Examples of the light receiver in the fluorescence measuring device include a photodiode (PD), an avalanche photodiode (APD), a photomultiplier tube (PMT), or a CCD. PD is preferable from the viewpoint of manufacturing cost, and is complicated. It is more preferable that it is a CAN mounting type PD which does not require a manufacturing process. The lens on the light receiving surface of the CAN-mounted PD is not particularly limited as long as the light from the condenser lens is condensed on the PD. For example, a CAN mounting type PD of a rotationally symmetric biconvex lens having a diameter of 1.5 mm, a condensing lens side numerical aperture (NA) of 0.2, and a PD side NA of 0.4 is commercially available. In addition, JP, 2010-197248, A can be referred to for the device composition provided with the detector, or the quantification method using the same.

[Modification]
The immunochromatographic test does not necessarily consist of two lines (one test line and one control line). In multi-item diagnostic immunochromatography, there are multiple test lines. Influenza detection kit (Quick Navi ^TM- Flu [trade name]) manufactured by Denka Seiken makes it possible to determine influenza A and influenza B in a single flow test. If positive in such a case, either line + control line will develop color (emit light). In particular, it is predicted that kits for simultaneously detecting various items as well as influenza will be produced. For example, if multiple lines that simultaneously detect adenovirus, influenza, RS virus, etc. exist on one test strip, multiple test lines + control lines can develop and emit light if multiple infections are present. There is sex.
Even in such a case, according to the present invention, by appropriately combining the fluorescent fine particles and the light absorbing fine particles, the problem of detection sensitivity when only the fluorescent fine particles are used can be solved, and the effect is exhibited.

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

[Manufacturing Example 1] Preparation of Labeling Particles Fluorescent dye-containing silica as core particles was prepared by producing rhodamine 6G-containing silica particles having a particle size of about 300 nm by the method described in JP2009-221059. Specifically, it was produced by the following method. 5- (and-6) -carboxyrhodamine 6G succinimidyl ester (trade name, manufactured by HiLyte Biosciences) dimethylformamide (DMF) was added, APS was added, and the reaction was performed at room temperature (23 ° C.) for 1 hour. A DMF solution of carboxyrhodamine 6G-APS was obtained. A DMF solution of TEOS and the carboxyrhodamine 6G-APS was added to a mixed solvent of ammonia water and ethanol, and the mixture was stirred at 40 ° C. for 2 hours. After completion of the reaction, the mixture was centrifuged to settle the particles, and the supernatant was immediately removed. The obtained precipitate was redispersed in ethanol and centrifuged again to settle the particles and remove unreacted TEOS and the like. Furthermore, rhoamine 6G-containing silica particles having a particle size of about 300 nm were prepared by performing the same washing operation four times except that distilled water was used in place of ethanol to remove free dye and the like. The maximum absorption wavelength of rhodamine 6G, which is a fluorescent dye in the particles, is 535 nm.

[Production Example 2] Antibody modification of fluorescent fine particles To the colloid of the particles prepared in Production Example 1, MES (2-Morpholinoethanesulfonic acid) buffer (pH 6.0), NHS, EDC (1-ethyl-3- (3- ( Dimethylamino) propyl) carbodiimide) and distilled water were added and mixed for 30 minutes.
The reaction solution was centrifuged, the supernatant was removed, and distilled water was added to disperse the particles. Similarly, centrifugation and dispersion in distilled water were repeated to wash the particles, and finally, the particles were dispersed in 1 mL of 50 mM KH ₂ PO ₄ buffer (pH 8.0). An anti-hCG antibody (Anti-hCG mouse IgG1, manufactured by Medix Biochemica) dissolved in KH ₂ PO ₄ buffer (pH 8.0) was added thereto and mixed for 2 hours. Subsequently, the BSA solution was added and further mixed for 1 hour.
The reaction solution was centrifuged and the supernatant was removed, and then KH ₂ PO ₄ buffer (pH 8.0) was added to disperse the particles. Further, centrifugation and dispersion in KH ₂ PO ₄ buffer (pH 8.0) are repeated to wash the particles. Finally, the particles are dispersed in 1 mL of KH ₂ PO ₄ buffer (pH 8.0) and labeled with fluorescent fine particles. Anti-hCG antibody colloids were obtained.

[Production Example 3] Modification of anti-hcG antibody to colored particles Au colloidal particles having a particle size of about 40 nm (manufactured by BBI), MES (2-Morpholinoethanesulfonic acid) buffer (pH 6.0), NHS, EDC (1- Ethyl-3- (3- (dimethylamino) propyl) carbodiimide) and distilled water were added and mixed for 30 minutes.
The reaction solution was centrifuged, the supernatant was removed, and distilled water was added to disperse the particles. Further, the particles were washed by repeating centrifugation and dispersion in distilled water, and finally dispersed in 1 mL of KH ₂ PO ₄ buffer (pH 8.0). A goat IgG antibody (goat IgG2b, manufactured by Takara Bio Inc.) dissolved in KH ₂ PO ₄ buffer (pH 8.0) was added thereto and mixed for 2 hours. Subsequently, 100 μL of BSA solution was added and further mixed for 1 hour.
The reaction solution was centrifuged and the supernatant was removed, and then KH ₂ PO ₄ buffer (pH 8.0) was added to disperse the particles. Further, centrifugation and dispersion in KH ₂ PO ₄ buffer (pH 8.0) are repeated to wash the particles. Finally, the particles are dispersed in 1 mL of KH ₂ PO ₄ buffer (pH 8.0) and labeled with fluorescent fine particles. A colloid of goat IgG antibody was obtained.

Example 1 (Preparation of a conjugate pad containing both colored latex particles and fluorescent silica particles)
0.25 ml of fluorescent silica particle colloid (10 mg / ml) modified with anti-hcG antibody prepared in Production Example 2 and Au colloid particles (10 mg / ml) modified with goat IgG antibody prepared in Production Example 3 05 ml, 50 mg / ml sucrose 3 ml, and distilled water 1.7 ml were mixed.
Subsequently, 0.8 ml of the mixed particle colloid was added to one Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE, 8 × 150 mm) (Pad 101).
Glass Fiber Conjugate Pad 101 containing mixed particles of colored Au colloid particles and fluorescent latex particles was light red, and it was confirmed by visual observation that particles were contained in the pad.

Comparative Example 1 (Preparation of conjugate pad containing fluorescent silica particles)
0.3 ml of fluorescent silica particles (10 mg / ml) modified with anti-hCG antibody prepared in Production Example 2, 3 ml of 50 mg / ml sucrose, and 1.7 ml of distilled water were mixed. 0.8 ml of the obtained fluorescent silica particle colloid was added to one Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE, 8 × 150 mm). Glass Fiber Conjugate Pad c01 containing particles was light red, and it was confirmed visually that the particles were contained in the pad.

Example 2 (Detection of recombinant hCG using immunochromatographic test strip prepared using conjugate pad 101 containing mixed particles of colored Au colloid particles and fluorescent silica particles)
(1) Preparation of antibody-immobilized membrane Anti-hCG antibody (alpha subunit of of the test) with a width of about 1 mm around the center (about 12 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 of FSH (LH), clone code / 6601, manufactured by Medix Biochemica was applied at a coating amount of 0.75 μL / cm.
Subsequently, as a control line having a width of about 1 mm, a solution ((50 mM KH ₂ PO ₄ , pH 7.0) sugar free) containing 1 mg / mL of goat IgG antibody (goat IgG2b, manufactured by Takara Bio Inc.) is 0.75 μL / cm. The coating amount was applied and dried at 50 ° C. for 30 minutes.
Next, the entire membrane was immersed in a blocking buffer for 30 minutes at room temperature as a blocking treatment.
The membrane was transferred to a membrane washing / stable buffer and allowed to stand at room temperature for 30 minutes. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature to prepare an antibody-immobilized membrane.
The obtained membrane, a conjugate pad containing a mixed particle of colored Au colloid particles and fluorescent silica particles obtained in Example 1, a sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), an absorption pad ( Cellulose Fiber Sample Pad (CFSP) (manufactured by MILLIPORE) is assembled on a backing sheet (trade name AR9020, manufactured by Adhesives Research) and cut into a strip of 5 mm width and length 60 mm, and the test shown in FIG. The strip 101 was obtained as described above with reference to Fig. 1. However, the backing sheet is not shown.
In addition, as shown in FIG. 1, each component member was pasted by superimposing about 2 mm on both ends with adjacent members (the same applies hereinafter). The distance d between the test line and the control line was 3 mm.

(2) Detection of recombinant hCG 100 μL of 100, 50, 20, 10, 5, 2, 0.2, 0.1 IU / L of recombinant hCG (manufactured by Rohto Pharmaceutical Co., Ltd.) was dropped on the sample pad portion of the strip, 100 μL. A line where the anti-hCG antibody (alpha subunit unit of FSH (LH), clone code / 6601, manufactured by Medix Biochemica) was applied visually (test line) and a line where the goat IgG antibody was applied (control line) As a result, it was confirmed that the control line was colored. On the other hand, the color development of the test line was not confirmed visually.
Subsequently, FF01-482 (trade name, manufactured by Semrock) is used as a filter on the excitation light source side, and FF01-536 (trade name, manufactured by Semrock) is used as a filter on the detector side, and the recombinant hCG is 100, 50, 20 , 10, 5, 0.2, 0.1 IU / L of the test strip was irradiated with a mercury lamp (103W) and a CCD detector (C2741-35A (trade name, manufactured by Hamamatsu Photonics)) was used as the detector. ) Was used to image fluorescence. As a result, the fluorescent color development of the test line and the control line was confirmed for any sample having the recombinant hCG of 100, 50, 20, 10, 5, 0.2, and 0.1 IU / L.
From the above results, the test strip using the mixed particles of the colored Au colloid particles and the fluorescent silica particles can be determined by the fluorescence detection device even when the recombinant hCG is 100 to 0.1 IU / L. I understand.

Comparative Example 2 (Detection of recombinant hCG using an immunochromatographic test strip prepared using a conjugate pad containing fluorescent silica particles)
The test strip c01 produced in the same manner as in Example 2 (1) was used in the same manner as in Example 2 (2), except that the conjugate pad c01 containing fluorescent silica particles was used as the conjugate pad. Recombinant hCG was detected. As a result, when the color development of the test strip lines left for 5 minutes was confirmed, no color development was confirmed in any of the lines.
In the evaluation using the fluorescence detector, the fluorescence development of the test line and the control line was confirmed only for the samples having the recombinant hCG of 100, 50, 20, 10, 5, 2, 0.2 IU / L. When the recombinant hCG was 0.1 IU / L, no color development on the test line was confirmed.
From the above results, it can be seen that the test strip using the fluorescent silica particles can be determined by the fluorescence detection apparatus when the recombinant hCG is 0.2 IU / L or more.

1 Target substance (test substance)
2 Fluorescent Label 2a Fluorescent Fine Particles 2b Test Binding Substance 3 Absorbent Label 3a Absorbent Fine Particles 3b Reference Binding Substance 4 Test Capture Substance 5 Reference Capture Substance 6 Case 61 Detection Opening 62 Sample Introduction Opening 6a Case upper part 6b Case lower part 80 Flat test piece (test strip)
8a Sample pad 8b Conjugate pad 8c Membrane 8d Absorption pad 100 Long specimen _rn Reference area (control line)
n _t test area (test line)
L Lateral flow direction S

Claims (17)

A planar test piece having a test region for immunochromatography and a reference region, a target material to which binding property to a test capturing substance in the test region is given, and a test binding material having binding property to the target material And a fluorescent fine particle into which a reference binding substance having a binding property to a reference capturing substance in a reference region is introduced ,
The fluorescent particles and the pre-Symbol absorption fine advance imparted to the test piece advance, by applying a target substance through the binding substance is bound to the fluorescent particles to the specimen, fluorescent microparticles and a light absorbing particles preparative is shifted in a plane specimen, its a test capture material on the test area and the fluorescent fine particles are bonded through the target substance and binding substance for testing, while before the reference region SL extinction microparticles is bound via a reference trapping substance and the capture substance for the reference in the reference area of the test piece,
A detection method by immunochromatography in which the target substance is detected from fluorescence emission from the fluorescent fine particles in the test region and the light absorption state of the light absorbing fine particles in the reference region.   The detection method according to claim 1, wherein one or both of the fluorescent fine particles and the light absorbing fine particles comprise silica fine particles.   The detection method according to claim 1 or 2, wherein one or both of the fluorescent fine particles and the light absorbing fine particles have an average particle diameter of 20 to 1000 nm.   The detection method according to any one of claims 1 to 3, wherein a nitrocellulose membrane is used as a membrane for providing the test region and the reference region.   The detection method according to claim 1, wherein the reference region is confirmed visually, and the test region is confirmed using a fluorescence detection device.   The detection method according to claim 1, wherein the detection method is determined based on a change in color of the reference region, and positive or negative is determined based on fluorescence emission of the test region.   The detection method according to claim 1, wherein high-sensitivity determination and / or quantitative measurement is performed by detecting fluorescence emission in the test region.   The detection method according to any one of claims 1 to 7, wherein a distance between a test line forming the test area and a control line forming the reference area is 1 mm or more and 20 mm or less.   The detection method according to any one of claims 1 to 8, wherein a wavelength of fluorescence emission of the fluorescent fine particles is 400 to 800 nm. A test kit comprising a combination of a planar test piece for immunochromatography, fluorescent fine particles, and light absorbing fine particles,
The planar specimen has a test area and a reference area,
The fluorescent fine particle is introduced with a test binding substance imparted with a binding property to a target substance, and the target substance has a binding property with a test capturing substance in the test region, while the light absorbing fine particle is A test kit for immunochromatography into which a reference binding substance imparted with a binding property to a reference capture substance in the reference region is introduced . The fluorescent fine particles and kit according to claim 1 0 either or both of absorption fine particles comprising silica fine particles. The kit according to claim 10 or 11 , wherein one or both of the fluorescent fine particles and the light absorbing fine particles have an average particle diameter of 20 to 1000 nm.   The kit according to any one of claims 10 to 12, wherein a nitrocellulose membrane is used as a membrane for providing the test region and the reference region.   The kit according to any one of claims 10 to 13, further combined with a fluorescence detection device for detecting fluorescence emission in the test region.   The kit according to any one of claims 10 to 14, wherein a distance between a test line forming the test area and a control line forming the reference area is 1 mm or more and 20 mm or less.   The kit according to any one of claims 10 to 15, wherein a wavelength of fluorescence emission of the fluorescent fine particles is 400 to 800 nm.   The kit according to any one of claims 10 to 16, wherein a wavelength of excitation light of an irradiator included in the fluorescence detection device is 300 to 900 nm.

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