JP5367490B2 - Test strip for immunochromatography - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a test strip for lateral flow type immuno-chromatography, capable of accurately detecting or quantifying not less than two types of detection targets included in a specimen at a time. <P>SOLUTION: In the test strip for lateral flow type immuno-chromatography for detecting or quantifying at least two kinds of detection targets included in a specimen, the test strip for lateral flow type immuno-chromatography includes a member for adding samples, a conjugate pad for immuno-chromatography where a labeled particle to the first detection target has been immobilized, and a membrane, a conjugate pad for immuno-chromatography where labeled particles for the second detection target have been immobilized, and a membrane including an antibody immobilization section for detecting the second detection target, and an absorption pad, where an antibody immobilization section for detecting the first detection object and an antibody immobilization section for capturing labeled particles for the first detection target are provided sequentially to a direction where the labeled particles flow by serial connection in this order. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a test strip for immunochromatography. Furthermore, the present invention relates to a method for detecting or quantifying a detection object using the immunochromatographic test strip.

The immunochromatography method is efficient because the detection target moves through the porous support by capillary action, is captured by the labeled particles, and is immobilized locally (for example, in a line) on the porous support. This is an immunoassay method for determining the presence or absence of a detection target by coloring the line on which the detection target is concentrated by contacting the target and the capture substance is immobilized.
The following three points can be mentioned as features of the immunochromatography method.
(1) The time required for determination is 20 minutes or less, and rapid inspection is possible.
(2) Since the measurement can be performed simply by dropping the specimen and the operation is simple, a plurality of specimens can be processed.
(3) Since a determination is easy without requiring a special detection device, a general consumer can inspect himself / herself.
Utilizing these characteristics, immunochromatography is used for pregnancy test drugs and influenza test drugs, and has attracted attention as a new POCT (Point Of Care Testing) technique (see, for example, Patent Document 1).
Here, POCT refers to a test for diagnosis at a place 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, since rapid and accurate treatment is possible based on examination information provided instantly, emergency examinations at hospitals and examinations during surgery are possible. Is expensive.

  In the immunochromatography method, if it is possible to detect a plurality of detection objects in one measurement, the use of the sample can be made more efficient, and the cost of the test strip for the immunochromatography method per test item can be reduced. Useful.

As a test strip for immunochromatography for detecting a plurality of detection objects, for example, a method using two test strips is known for two types of detection objects. However, this method requires twice as much sample, and cannot be used when only a small amount of sample can be used.
As a second immunochromatographic test strip for detecting a plurality of detection objects, a method in which two test lines are held by one test strip and two kinds of detection objects are detected by one test strip. It has been known. However, this method requires a small amount of sample, but since two types of lines are applied on the same membrane, the membrane processing method or type of membrane can be changed for both detection objects. However, it was difficult to optimize conditions for improving detection sensitivity. Furthermore, two types of labeled particles pass through two types of lines by capillary action, and nonspecific adsorption occurs between the labeled particles and the antibody immobilized in a line, which may cause false positives. .
As a third immunochromatographic test strip for detecting a plurality of detection objects, one test strip is used, two types of antibodies are immobilized on one test line, and two types of labeling reagents of different colors are used. A method of detecting two types of detection objects on the same line by using them is known (see, for example, Patent Document 2 and Patent Document 3). However, this method requires only a small amount of sample, but because two types of detection antibodies are applied on the same membrane, the membrane processing method or membrane type can be changed for both detection targets. It is difficult to optimize the conditions for improving the detection sensitivity. Furthermore, when two types of labeled particles react with each other on the same line, non-specific adsorption occurs between the labeled particles that are not specifically bound originally and the antibody immobilized in a line, There are cases where correct judgment cannot be made.

  In the immunochromatography method, gold nanoparticles and colored latex particles are most often used as labeled particles (see, for example, Patent Document 4). The width of a test strip for immunochromatography using gold nanoparticles or colored latex particles usually needs to be about 5 mm in order to accurately determine the line. If accurate determination is possible even if the width of the strip is narrow, the amount of specimen to be used is small, so it can be used even when only a small amount of specimen can be used, and the production cost per strip Can be lowered. However, in immunochromatographic test strips using gold nanoparticles or colored latex particles, if the width of the strip is 5 mm or less, the presence or absence of a line cannot be clearly discerned and the accuracy of the determination is reduced. There is a case.

JP 2006-67979 A JP 2002-303629 A JP 2004-132892 A JP 2003-262638 A

  In view of the above problems, an object of the present invention is to provide a lateral flow type immunochromatographic test strip and a detection target capable of highly accurately detecting or quantifying two or more types of detection targets contained in a sample at one time. The present invention provides a detection method or quantification method for the above, and a detection system for immunochromatography.

  In view of the above problems, the present inventors have conducted intensive studies. As a result, it was found that a plurality of detection objects can be detected by preparing a test strip for lateral flow type immunochromatography by separating a conjugate pad and a membrane for each detection object. The present invention has been completed based on this finding.

The object of the present invention has been achieved by the following means.
(1) A test strip for a lateral flow type immunochromatography method for detecting or quantifying at least two kinds of detection objects contained in a sample at once,
Sample addition member,
A conjugate pad for immunochromatography in which labeled particles for the first detection target are immobilized;
An antibody immobilization unit for detecting the first detection object and an antibody immobilization for capturing the label particles with respect to the first detection object with respect to the direction in which the label particles flow with respect to the first detection object. A first membrane, sequentially provided with parts,
A conjugate pad for immunochromatography in which labeled particles for the second detection target are immobilized,
A second membrane having an antibody immobilization part for detecting a second detection object, and the absorbent pad possess in series connected in this order,
The immunochromatography conjugate pad on which the labeled particles for the first detection target are immobilized and the immunochromatography conjugate pad on which the labeled particles for the second detection target are immobilized are separated, and the first Second detection is performed on the downstream side of the immunochromatography conjugate pad on which the labeled particles for the detection target are immobilized and on the upstream side of the immunochromatography conjugate pad on which the labeled particles for the second detection target are immobilized. A test strip for lateral flow type immunochromatography , in which an antibody immobilization part for capturing labeled particles for a first detection target is provided immediately before a conjugate pad for immunochromatography on which a label for target is fixed .
(2) Ri Ah with silica particles of the first label particles and label particles relative to the second object to be detected with respect to the detection object, respectively it flat Hitoshitsubu径20 to 600 nm, the first object to be detected The transmittance of the labeled particles with respect to the first detection object with respect to the first membrane provided with the antibody immobilization part for detection and the antibody immobilization part for capturing the labeled particles with respect to the first detection object is 3%, wherein the der Rukoto below, wherein (1) lateral flow type immunochromatography test strip according to claim.
(3) The lateral as described in (1) or (2) above, wherein the labeled particles for the first detection target and the labeled particles for the second detection target are fluorescent silica nanoparticles, respectively. Test strip for flow type immunochromatography.
(4) The width of the test strip is 0.5 mm to 2 mm, and the volume of the sample including the detection target to be added to the sample addition member is 5 to 40 μL. The test strip for lateral flow immunochromatography according to any one of (3).
(5) The specimen is added to the sample addition member of the lateral flow type immunochromatographic test strip according to any one of (1) to (4), and the two types of detection objects contained in the specimen are A method for detecting or quantifying a detection object using a test strip for lateral flow immunochromatography , wherein the detection or quantification is performed in one test .
(6) The method for detecting a detection target using the lateral flow immunochromatographic test strip according to (5), wherein the detection target is an influenza A virus or an influenza B virus, or Quantitation method.
(7) The labeled particles for the first detection target and the label particles for the second detection target in the lateral flow type immunochromatography test strip are silica nanoparticles having an average particle diameter of 20 to 600 nm each containing a fluorescent substance. Irradiating the silica nanoparticle integrated in the antibody immobilization unit for detecting the first detection target and the antibody immobilization unit for detecting the second detection target with an excitation light source, Item (5) or (6) above, characterized in that the detection target contained in the specimen is detected or quantified by receiving the fluorescence emitted by a charge-coupled device or a photomultiplier tube or visually confirming the fluorescence. A method for detecting or quantifying a detection object using the test strip for lateral flow immunochromatography as described.
(8) A sample is added to a sample addition member of an immunochromatographic test strip using the lateral flow type immunochromatographic test strip according to any one of (1) to (4), and the sample is included in the sample. A method for detecting or quantifying a detection target using a lateral flow type immunochromatographic test strip for detecting or quantifying two types of detection targets in a single test, wherein the label for the first detection target The labeled particles for the particles and the second detection object are fluorescent silica nanoparticles, respectively, and further fluorescence at the time of fluorescence measurement in the antibody immobilization unit for detecting the first detection object and the second detection object. A method for detecting or quantifying a detection target using a test strip for lateral flow immunochromatography, which reduces background intensity .

  According to the test strip for lateral flow immunochromatography, the detection method or quantification method for a detection target, and the detection system for immunochromatography of the present invention, a plurality of detection targets can be detected with high accuracy at a time.

FIG. 1 (a) is a plan view of one embodiment of the immunochromatographic test strip of the present invention, and FIG. 1 (b) is a longitudinal sectional view of one embodiment of the immunochromatographic test strip of the present invention. . FIG. 2 is a diagram showing a TEM photograph image of the silica nanoparticles obtained in the example. FIG. 3 is a plan view of a comparative immunochromatographic test strip. FIG. 4 is a plan view of the immunochromatographic test strip of the present invention obtained in the examples.

First, a preferred embodiment of a test strip for lateral flow immunochromatography of the present invention will be described.
As one embodiment of the test strip for lateral flow type immunochromatography of the present invention,
(1) A member for sample addition (also referred to as “sample pad” in the present specification) and a conjugate pad for immunochromatography in which labeled particles for the first detection target are immobilized (in this specification, “first Also referred to as 1 conjugate pad)
(2) The first conjugate pad, the antibody immobilization unit for detecting the first detection object in the direction in which the labeled particles with respect to the first detection object flow, and the first detection object A membrane (also referred to as “first membrane” in the present specification) provided with an antibody immobilization part for capturing the labeled particles against
(3) The first membrane and a conjugate pad for immunochromatography (also referred to as “second conjugate pad” in the present specification) in which the labeled particles for the second detection target are immobilized, And (4) the second conjugate pad, a membrane having an antibody immobilization part for detecting the second detection target (also referred to as “second membrane” in the present specification), an absorption pad, and But,
They are connected in series so that capillarity occurs with each other.

  The shape of the test strip for lateral flow type immunochromatography is not particularly limited as long as the specimen to be added can move in the test strip by capillary action. For example, the test strip for lateral flow type immunochromatography has a sample pad, a first conjugate pad, a first membrane, a second conjugate pad, and a second membrane connected in this order in one direction. The shape, the direction in which the specimen moves in the first conjugate pad and the first membrane, and the shape in which the specimen moves in the second conjugate pad and the second antibody-immobilized membrane are reversed. Can be mentioned. In the case where the direction in which the specimen moves through the first conjugate pad and the first membrane and the direction in which the specimen moves through the second conjugate pad and the second antibody-immobilized membrane are reversed, When the antibody immobilization part of the first membrane and the control line of the second membrane are adjacent, and the antibody immobilization part of the first membrane and the antibody immobilization part of the second membrane are adjacent, When reading. Here, the “control line” refers to a part of the membrane for detecting the labeled particles that cannot detect the detection target labeled with the labeled particles but do not bind to the test line.

  A test strip for lateral flow immunochromatography can be prepared by connecting a conjugate pad and a membrane in series according to the type of detection target (also referred to as target substance) to be detected or quantified. For example, when there are two types of objects to be detected, a test strip is formed by serially connecting a sample pad, a first conjugate pad, a first membrane, a second conjugate pad, and a second membrane in this order. Can be created. When there are three types of detection objects, a test strip can be created by further connecting the third conjugate pad and the third membrane in addition to the above-described test strip components. That is, the test strip of the present invention can be appropriately created according to the type of detection object. In this way, when there are three or more types of detection objects, a set of membranes and conjugate pads for each detection object is provided to detect the third or more types of detection objects for each detection object. be able to.

In lateral flow type immunochromatography test strip for methods of the present invention, the first membrane having an antibody immobilization part for detecting the first detection object to capture labeled particles to the first object to be detected The antibody immobilization part (also referred to as “labeled particle capturing part” in the present specification) downstream of the immunochromatographic conjugate pad on which the labeled particles for the first detection object are immobilized and the second detection object The immunochromatographic conjugate pad on which the labeled particles for the second detection target are immobilized upstream of the immunochromatographic conjugate pad on which the labeled particles are immobilized . By providing the first membrane with the antibody immobilization part for capturing the labeled particles in this way, the labeled particles for the first detection target are prevented from moving to the second membrane on the downstream side, and false positives are prevented. It can be prevented from occurring. The position of the labeled particle capturing unit on the first membrane is such that the labeled particles on the downstream side of the immunochromatography conjugate pad on which the labeled particles for the first detection target are immobilized and the second detection target are immobilized. The detection target is placed upstream of the immunochromatographic conjugate pad, immediately before the immunochromatographic conjugate pad on which the labeled particles for the second detection target are immobilized, and in the direction in which the labeled particles flow. Provided downstream from the antibody immobilization section for detection.

The antibody immobilization part for capturing the labeled particles of the present invention is formed by immobilizing an antibody that binds only the labeled particles. For example, when mouse IgG is bound to the surface of the labeled particle, an antibody immobilization part on which a goat anti-mouse IgG antibody is immobilized can be used. Examples of antibodies that can be used in the present invention include mouse IgG, goat IgG, rabbit IgG, chicken IgY, mouse IgE, human IgG, and human IgE. The shape of the antibody immobilization part (labeled particle capturing part) is not particularly limited as long as the capturing antibody is locally immobilized, and includes a line shape, a circular shape, a belt shape, etc. Is preferable, and a strip shape having a width of 1.0 to 3.0 mm is more preferable. Further, a fixed amount of antibody of the antibody immobilization part to the label particles sufficiently capture is preferably 1-100 [mu] g / cm ^2, more preferably 10-100 [mu] g / cm ^2.
Examples of the antibody immobilization method include a method in which an antibody solution is applied, dropped or sprayed and then dried and immobilized by physical adsorption.

  Next, each member of the lateral flow type immunochromatographic test strip of the present invention will be described with reference to FIGS. 1 (a) and 1 (b). However, the present invention is not limited to this.

1) Sample addition member (sample pad) 2
The sample pad 2 is a constituent member that drops a sample containing a detection target.

2) Labeled particle impregnated members (conjugate pads) 3 and 5
The conjugate pads 3 and 5 are constituent members impregnated with labeled particles for the first detection target and labeled particles for the second detection target, respectively, and are included in the sample that has moved from the sample pad 2 by capillary action. A detection target to be detected is a portion that is captured and labeled by the labeled particles in a molecular recognition reaction such as an antigen-antibody reaction.
The content of the labeled particles per unit area (cm ² ) in the conjugate pad is not particularly limited, but is preferably 50 μg to 2 mg. Examples of the impregnation method include a method of applying, dropping or spraying the dispersion of the labeled particles and then drying.

3) Antibody-immobilized membrane (first membrane 4 and second membrane 6 )
The first membrane 4 and the second membrane 6 are components in which the detection target labeled with the labeled particles moves by capillary action, and forms a sandwich-type immune complex composed of immobilized antibody-analyte-labeled particles. The antibody immobilization unit (first detection target determination unit) (41) for the first detection target and the antibody immobilization unit (second detection target for the second detection target) in which the reaction is performed Each has a determination unit) (61).
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 the shape is a line, and the width may be 0.5 to 2.0 mm.

  The detection target labeled with the labeled particles is captured and formed in the antibody immobilization part (determination part) (41 and 61) by a sandwich-type immune complex formation reaction consisting of immobilized antibody-detection target-labeled particles. The presence or absence of a detection target can be determined based on the degree of labeling such as color development or fluorescence derived from the labeled particles in the complex, that is, positive / negative. That is, the labeled particles are concentrated in the antibody immobilization unit (determination unit) (41 and 61), and can be detected and determined visually as a colored signal or using a detection device.

  In order to sufficiently complete the sandwich-type immune complex formation reaction, or to avoid the influence on the measurement by the colored substance in the sample and the influence on the measurement by the labeled particles not bound to the detection target, judgment on the membrane It is preferable that the portion is provided at a position (for example, the middle of the membrane) at some distance from the connection end with the conjugate pad and the connection end with the absorption pad.

  The amount of antibody immobilization in the antibody immobilization part (determination part) (41 and 61) is not particularly limited. However, when the shape is a line, it is preferably 0.1 μg to 5 μg per unit length (cm). Examples of the immobilization method include a method in which an antibody solution is applied, dripped or sprayed and then dried and immobilized by physical adsorption.

  After the above-described antibody immobilization, the entire membrane may be subjected to a so-called blocking treatment in order to prevent the influence of nonspecific adsorption on the measurement. Examples of the blocking treatment include a method of immersing in a buffer solution containing a blocking agent such as albumin, casein, polyvinyl alcohol or the like for a suitable time and then drying. Examples of the commercially available blocking agent include skim milk (manufactured by DIFCO), 4% block ace (manufactured by Meiji Dairies), and the like.

  In the present invention, a labeled particle capturing unit 43 is provided to capture only the labeled particles for the first detection target and prevent the labeled particles for the first detection target from entering the second membrane 6.

In the present invention, it is preferable to provide a control unit 42 for the first detection object and a control unit 62 for the second detection object. The control unit 42 for the first detection object is provided to confirm that the labeled particles for the first detection object are moving on the first membrane. The control unit 62 for the second detection object is provided for confirming that the labeled particles for the second detection object are moving on the second membrane.

Here, the control unit 42 of the first detection target and the control unit of the second detection target are surface-modified to the label particles for the first detection target and the label particles for the second detection target, respectively. An antibody that binds to a substance that recognizes a detection target contained in a specimen is immobilized. For example, when mouse IgG is surface-modified on the labeled particles, an antibody immobilization part on which a goat anti-mouse IgG antibody is immobilized can be used as a control part.
The labeled particles that move through the membrane by capillary action and reach the control part are captured by the antibody immobilized on the membrane in the control part. Therefore, if a label such as color development or fluorescence derived from the labeled particles is detected from the control unit, it can be confirmed that the labeled particles have moved to the control unit. On the contrary, when a label such as color development or fluorescence derived from the labeling particles is not detected from the control unit, it means that the labeling particles have not moved through the membrane for some reason. In such a case, even if the detection unit is not labeled with the labeled particles, it is not determined as negative but is evaluated as being indeterminate. By evaluating in this way, it is possible to prevent an erroneous determination that the negative determination is made when the determination unit is not labeled because the labeled particle does not move well through the membrane even though it is a positive sample.

  The shape of the control part is not particularly limited as long as the antibody that locally captures the labeled particles is immobilized, and examples thereof include a line shape, a circular shape, and a belt shape. A line shape of 0.3 to 1.0 mm is more preferable. In addition, when the shape is a line, the amount of antibody immobilized is preferably 0.1 μg to 5 μg per unit length (cm).

4) Absorption pad 7
The absorption pad 7 is preferably provided on the immunochromatographic test strip of the present invention. The absorption pad 7 is a constituent member that absorbs the sample liquid and the labeled particles that have moved through the membrane by capillary action and always generates a constant flow.

5) Backing sheet 8
The backing sheet 8 is preferably provided on the immunochromatographic test strip of the present invention. The backing sheet 8 is a constituent member for stably fixing each member of the sample pad, the conjugate pad, the membrane, and the absorption pad. The backing sheet 8 is preferably a backing sheet with an adhesive.

There is no restriction | limiting in particular as a material of these each structural member, The normal member used for the test strip for immunochromatography can be used.
A glass fiber pad such as Glass Fiber Conjugate Pad (trade name, manufactured by MILLIPORE) is preferable as the sample pad and conjugate pad, and a nitrocellulose membrane such as Hi-Flow Plus 120 membrane (trade name, manufactured by MILLIPORE) is used as the membrane. As the absorbent pad, a cellulose membrane such as Cellulose Fiber Sample Pad (trade name, manufactured by MILLIPORE) is preferable.
Examples of the backing sheet with an adhesive include AR9020 (trade name, manufactured by Adhesives Research).

  There is no restriction | limiting in particular as a preparation method of the test strip for immunochromatography methods of this invention. When there are two types of detection objects included in the specimen, the sample pad, the first conjugate pad, the first membrane, the second conjugate pad, the second membrane, and the absorption pad are arranged between the members in the order of arrangement. In order to make the capillary phenomenon easy to occur, it can be produced by pasting both ends of each of these members with an adjacent member by about 1 to 2 mm (preferably on a backing sheet).

In the immunochromatographic test strip of the present invention, the transmittance of the labeled particles with respect to the membrane having the antibody-immobilized part for detecting the detection target is 3% or less, more preferably 1% or less, and still more preferably 0.5. % Or less is preferable. By setting the transmittance of the labeled particles in such a range, the labeled particles with respect to the first detection target that have entered the second membrane are adsorbed nonspecifically with the antibody of the determination unit of the second membrane. Can prevent false positives caused by. In addition, when particles containing a fluorescent substance are used as the labeled particles, the background generated when the labeled particles are adsorbed or bound to the membrane can be reduced, and the color of the antibody-immobilized portion can be more clearly recognized.
In this specification, “transmittance” refers to a lateral flow immunochromatography method in which a sample is dropped on a sample dropping portion among labeled particles held on a conjugate pad of a test strip for lateral flow type immunochromatography before use. It is the ratio of the labeled particles that have flowed through the membrane when using the test strip. Permeability is extracted from the material downstream of the membrane after using the lateral flow immunochromatographic test strip, relative to the fluorescence intensity of the labeled particles extracted from the conjugate pad of the lateral flow immunochromatographic test strip before use. It can be determined from the ratio of the fluorescence intensity of the labeled particles. As a method of setting the transmittance of the labeled particles to 3% or less, the first detection object is determined downstream of the first detection object determination unit and the first detection object control unit in the first membrane. There is a method of providing an antibody immobilization part for capturing the labeled particles and capturing the labeled particles for the first detection target.

  The width of the immunochromatographic test strip of the present invention is preferably 0.5 to 2 mm, more preferably 0.5 to 1 mm. In a test strip for immunochromatography, a certain amount of sample liquid needs to permeate through the test strip by capillary action. Therefore, the amount of sample solution required is proportional to the membrane area. By reducing the width of the strip, the area of the membrane is reduced, and the amount of specimen to be used can be reduced. For example, according to the present invention, the volume of the sample containing the detection target to be added can be set to 5 to 40 μL, preferably 5 to 20 μL, for example. Furthermore, by reducing the width of the immunochromatographic test strip, the manufacturing cost of the immunochromatographic test strip can be reduced.

  The labeled particles in the present invention are preferably silica nanoparticles containing a fluorescent substance (also referred to as “fluorescent silica nanoparticles” in the present specification). Rather than visually judging gold nanoparticles and colored latex particles collected in the antibody immobilization part for detecting the detection object, fluorescent silica nanoparticles accumulated in the antibody immobilization part for detecting the detection object This is because the method of detecting and determining the fluorescence has higher sensitivity, and even a narrow test strip can be determined with high accuracy.

  The fluorescent silica particles that can be used in the present invention are not particularly limited, and may be silica particles obtained by any arbitrary preparation method. For example, silica particles prepared by the sol-gel method described in Journal of Colloid and Interface Science, 159, 150-157 (1993), and fluorescent dye compound-containing colloidal silica particles prepared by the method described in WO 2007/074722. Etc.

  Specifically, the fluorescent silica nanoparticles are one or two or more silane compounds on a product obtained by reacting a fluorescent substance with a silane compound, and covalently bonding, ionic bonding, or other chemical bonding or adsorption. Can be prepared by condensation polymerization. Alternatively, it can be prepared by reacting a silane coupling agent to carry out condensation polymerization and allowing the condensation polymerization product to be covalently bonded, ionically bonded or otherwise chemically bonded or adsorbed on the surface of the fluorescent material. Further, it is also possible to prepare by mixing a fluorescent substance and a silane compound and sequentially subjecting the surface of the fluorescent substance to a covalent bond, an ionic bond or other chemically bonded or adsorbed silane compound to sequentially perform condensation polymerization of the silane compound. it can.

  Preferred embodiments of the method for preparing the fluorescent silica nanoparticles include N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, and epoxy group. Obtained by reacting a fluorescent substance having an active group such as a cyano group with a silane coupling agent having a reactive substituent (for example, an amino group, a hydroxyl group, or a thiol group) that reacts with the active group and covalently bonding it. The product obtained can be prepared by polymerizing one or more silane compounds.

The fluorescent silica nanoparticles can be prepared, for example, by performing the following steps (a) and (b).
(A) A fluorescent substance-silane coupling agent composite compound obtained by reacting a fluorescent substance (1) having an active ester group such as N-hydroxysuccinimide (NHS) ester with a silane coupling agent (2) having an amino group Step (b) for producing (3) The fluorescent substance-silane coupling agent composite compound (3) obtained in (a) is polymerized with a silica compound (4) under basic conditions to produce fluorescent silica nanoparticles ( 5) Forming Step The fluorescent substance (1) having an NHS ester group used in the step (a) can be prepared by activating a carboxyl group with carbodiimide and subsequent esterification with NHS. However, commercially available products can also be obtained.

The reaction between the fluorescent substance (1) and the silane coupling agent (2) is performed by dissolving in a solvent such as DMSO (dimethyl sulfoxide) or DMF (N, N-dimethylformamide) and then at room temperature (for example, 25 ° C.) It can carry out by reacting under stirring under conditions.
The ratio of the fluorescent material (1) and the silane coupling agent (2) used in the reaction is not particularly limited, but the fluorescent material (1): the silane coupling agent (2) = 1: 0.5-2 ( (Molar ratio) is preferable, and a ratio of 1: 0.8 to 1.2 (molar ratio) is more preferable.

  Although there is no restriction | limiting in particular in the said fluorescent substance, From a viewpoint of the detection by a general purpose detector (for example, AE-6931FXCF print graph (brand name, ATTO company make)) and the compatibility of data, blue fluorescence (440-490 nm), A fluorescent substance having yellow fluorescence (540 to 590 nm), orange fluorescence (590 to 620 nm) or red fluorescence (620 to 740 nm) is preferable. Specific examples of the fluorescent substance having an active group include DY550-NHS ester, DY555-NHS ester, DY630-NHS ester, DY635-NHS ester (all trade names, manufactured by Dynamics GmbH), 5-TAMRA-NHS ester, Alexa Fluor 546-NHS ester, Alexa Fluor 555-NHS ester, Alexa Fluor 633-NHS ester, Alexa Fluor 647-NHS ester (both trade names, manufactured by Invitrogen), Oyster 643-NHS ester, Oyster 656-NHS ester (both trade names, Denv Biolabels), 5- (and -6) -carboxytetramethylrhodamine succinimidyl ester (trade) Name can include a fluorescent substance having an NHS ester group of emp Biotech GmbH, Ltd.) and the like.

  Specific examples of the silane coupling agent having a reactive substituent that reacts with the active group of the fluorescent substance include γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino]. Propyl-triethoxysilane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxysilane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, 3-aminopropyltrimethoxy Examples include silane coupling agents having an amino group such as silane. Of these, APS is preferable.

  Thus, an active group such as a carbonyl group of the fluorescent substance (1) and a reactive substituent that reacts with the active group such as an amino group of the silane coupling agent (2) are combined with an amide bond (—NHCO—). Etc. are formed to obtain the fluorescent substance-silane coupling agent composite compound (3). That is, in the fluorescent substance-silane coupling agent composite compound (3), the fluorescent substance and the silica component are bonded through an amide bond or the like.

  Next, in the step (b), the fluorescent substance-silane coupling agent composite compound (3) is reacted with the silica compound (4). The silane compound (4) 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-tri Mention may be made of ethoxysilane. Among these, TEOS, MPS, or APS is preferable.

  The ratio of the fluorescent material-silane coupling agent composite compound (3) and the silica compound (4) is not particularly limited, but the mole of the silica compound (4) with respect to 1 mole of the fluorescent material-silane coupling agent composite compound (3). The ratio is preferably 50 to 40000, more preferably 100 to 2000, and further preferably 150 to 1000.

  This reaction is preferably carried out in the presence of alcohol, water and ammonia. Here, examples of the alcohol include lower alcohols having 1 to 3 carbon atoms such as methanol, ethanol, and propanol.

  The ratio of water and alcohol in the reaction system is not particularly limited, but preferably 0.5 to 20 parts by volume of alcohol, more preferably 2 to 16 parts by volume, and still more preferably 4 to 10 parts by volume with respect to 1 part by volume of water. It is the range of the capacity section. The amount of ammonia is not particularly limited, but the ammonia concentration is preferably 30 to 1000 mM, more preferably 60 to 500 mM, and still more preferably 80 to 200 mM.

  This reaction can be performed at room temperature, and is preferably performed with stirring. Usually, the fluorescent dye compound-containing colloidal silica particles (5) of the present invention can be prepared by reaction for several tens of minutes to several tens of hours.

  In the step (b), the size (diameter) of the prepared fluorescent silica nanoparticles can be appropriately adjusted by adjusting the concentration of the silica compound (4) to be used or adjusting the reaction time. Smaller fluorescent silica nanoparticles can be prepared by reducing the concentration of the silica compound (4) used and / or by shortening the reaction time (eg, Blaaderenet al., “Synthesis and Characterization of Monodisperse Colloidal Organo- silica Spheres ”, J. Colloid and Interface Science, 156, 1-18, 1993). On the other hand, if the step (b) is repeated a plurality of times, larger fluorescent silica nanoparticles can be prepared. Thus, the particle diameter (diameter) of the obtained fluorescent silica nanoparticles can be freely adjusted to a desired size, for example, from the nm order to the μm order. Specifically, it is possible to prepare fluorescent silica nanoparticles having a minute size of 3 to 30 nm. Further, if necessary, it can be adjusted so as to have a desired particle size distribution by subsequent processing, and thus fluorescent silica nanoparticles in a desired particle size distribution range can be obtained.

  The average particle diameter of the fluorescent silica nanoparticles is preferably 20 to 600 nm, and more preferably 100 to 300 nm. If the particle size is too small, the fluorescence intensity of the fluorescent silica particles accumulated in the antibody-immobilized portion decreases, and sufficient sensitivity cannot be obtained. On the other hand, if the particle size is too large, the membrane is clogged, the membrane permeability of the fluorescent silica nanoparticles is remarkably lowered, and sufficient sensitivity cannot be obtained.

In the present invention, the average particle size is determined from the total projected area of 100 silica nanoparticles randomly selected from an image of a transmission electron microscope (TEM), a scanning electron microscope (SEM), etc. 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 of selected silica nanoparticles (100) is obtained.
The fluorescent silica nanoparticles that can be used in the present invention are preferably monodispersed, and the variation coefficient of particle size distribution, so-called CV value, is preferably 15% or less, more preferably 10% or less, and even more preferably 8% or less.

  The labeled particles used in the present invention are preferably spherical or nearly spherical fluorescent silica particles. The nearly spherical fluorescent silica particles specifically refer to particles having 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. You may refine | purify using a conventional method, such as an ultrafiltration membrane, except a coexisting ion and a coexisting unnecessary substance.

In the present invention, the fluorescent substance is in an immobilized state in the fluorescent silica nanoparticles. There is no restriction | limiting in particular regarding a fluorescent substance, It is an organic molecule, an inorganic compound, a semiconductor particle, etc.
When the fluorescent substance is fixed and included in the fluorescent silica nanoparticles, the fluorescent substance is dispersed and present in the silica nanoparticles, so that the sensitivity can be increased as compared with the free fluorescent dye compound. That is, the luminance can be increased as compared with a free fluorescent substance.
In addition, the fluorescent silica nanoparticles of the present invention can include and incorporate many fluorescent substances in the silica nanoparticles without causing self-quenching. For this reason, the highly sensitive label | marker which can be used also in a micro area | region is possible.

It is preferable that the density | concentration of the fluorescent substance with respect to the said fluorescent silica nanoparticle is 20 mmol / L or more, and it is more preferable that it is 40-80 mmol / L. Here, the “concentration of the fluorescent substance with respect to the fluorescent silica nanoparticles” is obtained by dividing the number of moles of the fluorescent substance by the volume of the fluorescent silica nanoparticles. The number of moles of the fluorescent substance is determined from the absorbance or fluorescence intensity of the fluorescent silica nanoparticles, and the volume of the fluorescent silica nanoparticles is determined by centrifuging or ultrafiltration of the fluorescent silica nanoparticles from the fluorescent silica nanoparticle dispersion. And dried, and the mass was determined. The density of the silica particles was determined as 2.3 g / cm ³ .

Next, surface modification of fluorescent silica nanoparticles will be described.
Silica is generally known to be chemically inert and easy to modify. The fluorescent silica nanoparticles in the present invention can also easily bind desired molecules to the surface, and the surface can be made mesoporous or smooth.

Regarding the surface modification of the fluorescent silica nanoparticles, specifically, depending on the type of the silica compound (4) used in the step (b), the fluorescent silica nanoparticles having an acceptor group capable of binding with a desired molecule on the surface are used. be able to. Examples of the acceptor group include an amino group, a hydroxyl group, a thiol group, a carboxyl group, a maleimide group, and a succinimidyl ester group.
Table 1 shows the relationship between the silica compound (4) used in the reaction and the acceptor group formed on the surface of the fluorescent silica nanoparticles obtained thereby.

  In addition, about the fluorescent silica nanoparticle (5), when it is desired to introduce an acceptor group different from the acceptor group introduced onto the surface by the silica compound (4) used in the reaction, the fluorescent silica nanoparticle (5) is further processed. This can be achieved by treating with a silica compound different from the silica compound (4) used in (b). This treatment can be performed by performing the same operation as in the above step (b) using a silica compound different from the silica compound (4) used in the step (b).

  In order to use the fluorescent silica nanoparticles in an immunochromatography method, the surface is preferably modified with a substance that recognizes a detection target contained in a specimen (for example, a biomolecule such as an antibody, an antigen, DNA, or RNA). When the fluorescent silica nanoparticles whose surface is modified with a substance that recognizes an analyte are used as the labeled particles, highly sensitive detection or quantification is possible by detecting the fluorescence emitted by the fluorescent silica nanoparticles.

Although there is no restriction | limiting in particular as a method of modifying the surface of the said fluorescent silica nanoparticle group with a desired biomolecule etc., The example of following (i)-(iii) is mentioned.
(I) Fluorescent silica nanoparticles having a thiol group prepared on the surface using MPS or the like may be modified with a biomolecule or the like on the surface via a disulfide bond, a thioester bond, or a bond via a thiol substitution reaction it can.
(Ii) In particular, when the biomolecule or the like has an amino group, the thiol group of the fluorescent silica nanoparticle and the amino group of the biomolecule or the like are succinimidyl-trans-4- (N-maleimidylmethyl). It can also couple | bond using crosslinking agents, such as a cyclohexane- 1-carboxylate (SMCC) and N- (6-maleimido caproyloxy) succinimide (EMCS).
(Iii) Fluorescent silica nanoparticles having an amino group prepared on the surface using APS or the like are bonded to this amino group and a thiol group possessed by a biomolecule using a crosslinking agent such as SMCC or EMCS, as described above. can do. Moreover, this amino group and the amino group which a biomolecule etc. have can also be couple | bonded with crosslinking agents, such as glutaraldehyde. Furthermore, the surface can be modified with a biomolecule or the like via an amide bond or a thiourea bond.

  In the present invention, the fluorescent silica nanoparticles may be directly modified with a target biomolecule, but a desired molecule obtained by modifying the fluorescent silica nanoparticles (eg, antibody, DNA, RNA, sugar chain, receptor, peptide, etc.) However, it itself becomes an acceptor molecule and can be used as a target biomolecule by utilizing a specific molecular recognition reaction such as an antigen-antibody reaction, a biotin-avidin reaction, or hybridization utilizing base sequence complementarity. It is preferable to bind specifically. Here, the molecular recognition reaction includes (1) hybridization between DNA molecules or DNA-RNA molecules, (2) antigen-antibody reaction, (3) reaction between enzyme (receptor) and substrate (ligand), etc. A reaction derived from a specific interaction between biomolecules.

In the present invention, the fluorescent silica nanoparticles are preferably subjected to a blocking treatment after surface modification with a desired biomolecule or the like.
On the surface of the fluorescent silica nanoparticle surface-modified with a desired biomolecule, there exists a portion that easily adsorbs nonspecifically to the biomolecule or the membrane. This is considered to be caused by the fact that when the surface is modified with a desired biomolecule or the like, the portion that is likely to bind to the biomolecule on the surface of the fluorescent silica nanoparticle is not completely covered with the desired biomolecule or the like. It is done. If such a non-specific adsorption-prone portion is present on the surface of the fluorescent silica nanoparticle, even if it is a negative sample, the fluorescent silica nanoparticle is nonspecifically bound to the membrane or immobilized antibody in the antibody immobilization part (determination part). When the fluorescent silica nanoparticles move through the membrane due to capillary action, the fluorescent silica nanoparticles adsorb non-specifically to the membrane and the background increases and the sensitivity decreases. In order to prevent such a phenomenon, it is preferable to block the surface of the fluorescent silica nanoparticle by adding an appropriate polymer after modifying the fluorescent silica nanoparticle with a desired biomolecule or the like.
The polymer that can be used for the blocking treatment is not particularly limited, but hydrophilic polymers such as PEG (polyethylene glycol) and polyvinyl alcohol, proteins such as BSA (bovine serum albumin) and casein, alginic acid, dextran, Examples include polysaccharides such as hyaluronic acid. In the present invention, when the blocking treatment is performed on the fluorescent silica nanoparticles, the polymer may be used alone, or two or more kinds may be used in combination.

  The test strip for lateral flow type immunochromatography of the present invention is easy to operate even for general consumers who are not skilled in the technique, and from the point of view of POCT, the plastic material of the observation window for visually observing the detection line of the test strip The housing (casing) is preferably used. For example, the housing etc. which are described in Unexamined-Japanese-Patent No. 2000-356638 etc. are mentioned.

Next, the detection method or quantification method of the detection target of the present invention will be described.
According to the detection method or quantification method of the detection object of the present invention, a sample is added to the sample addition member of the test strip for lateral flow immunochromatography of the present invention, and two or more types of detection objects contained in the sample are once added. To detect or quantify.
The specimen that can be added to the sample addition member of the lateral flow immunochromatographic test strip of the present invention is not particularly limited, but nasal aspirate, nasal wipe, pharyngeal wipe, saliva, whole blood, serum, Examples include plasma, ground food, urine, and feces.

  Examples of detection objects that can be detected or quantified by the detection method or quantification method of the detection object of the present invention include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, peptides, and bioactive organic compounds. Etc. More specifically, influenza A virus nucleoprotein, influenza A virus hemagglutinin, influenza A virus neuramidase, influenza A virus membrane protein, influenza B virus nucleoprotein, influenza B hemagglutinin , Influenza B virus neuramidase, influenza B virus membrane protein, immunoglobulin E, food allergen, bacterial toxin and the like.

Substance that recognizes the detection target contained in the specimen by electrostatic attraction, van der Waals force, hydrophobic interaction, etc., in order to bind the label particles and the substance that recognizes the detection target contained in the specimen May be adsorbed on the labeling particles, or may be bonded by a chemical bond with a crosslinking agent or a condensing agent. Further, a thiol of a substance that recognizes a detection target contained in the sample by introducing a thiol group using a silane coupling agent having a thiol group such as MPS (γ-mercaptopropyltriethoxysilane) on the surface of the labeled particle. It may be bonded to the group by an S—S bond. For example, when detecting influenza A virus by the detection method or quantification method of the detection object of the present invention, the surface of the labeled particles is mouse anti-influenza A virus nucleoprotein monoclonal antibody or mouse anti-influenza A virus hemagglutinin monoclonal. It is preferable to modify with an antibody or a mouse anti-influenza A virus neuramidase monoclonal antibody or a mouse anti-influenza A virus membrane protein monoclonal antibody. influenza virus nucleoprotein monoclonal antibody or mouse anti-influenza B virus hemagglutinin monoclonal antibody, or mouse anti-influenza B virus neuraminidase, It is preferable to be modified with monoclonal antibodies or mouse anti-influenza B virus membrane protein monoclonal antibodies.

  In addition, when the labeled particles aggregate when binding a substance that recognizes the detection target contained in the specimen, such as biomolecules (for example, antibodies, antigens, DNA, RNA) on the surface of the labeled particles, Surface treatment may be performed on the surface of the labeled particles by an alternate adsorption method. 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.

Next, the fluorescence detection system for immunochromatography of the present invention will be described.
The fluorescence detection system for immunochromatography of the present invention has the test strip for lateral flow immunochromatography of the present invention. The test strip used in the fluorescence detection system for immunochromatography of the present invention is preferably lined with a backing sheet. Furthermore, it is preferable to have an excitation light source and an optical filter that does not transmit excitation light but transmits fluorescence.

In the fluorescence detection system of the present invention, the wavelength of the excitation light source is not particularly limited, and it is preferable to emit excitation light having a wavelength of 200 nm to 700 nm. Examples of the excitation light source include a mercury lamp, a halogen lamp or a xenon lamp, a blue LED, and a green LED.
In the fluorescence detection system of the present invention, when the light source is a lamp or a light emitting diode, it is more preferable that a filter for transmitting only light of a specific wavelength from the excitation light source is provided. Furthermore, the fluorescence detection system of the present invention is particularly preferably provided with a charge coupled device, a photomultiplier tube or a photodiode that receives the fluorescence, thereby detecting fluorescence of intensity or wavelength that cannot be visually confirmed, Since the fluorescence intensity can be measured, the sample can be quantified, and highly sensitive detection and quantification are possible.

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

(1) Preparation of Labeled Silica Nanoparticles 3.0 mg of 5- (and -6) -carboxyrhodamine 6G succinimidyl ester (trade name, manufactured by HiLyte Biosciences) was dissolved in 1 ml of dimethylformamide (DMF). 1.3 μl of APS was added thereto and reacted at room temperature (23 ° C.) for 1 hour.
Ethanol 128 ml, TEOS (tetraethoxysilane) 600 μL, distilled water 28.8 ml, 28 mass% ammonia water 400 μL were added to the obtained reaction liquid 400 μL, and reacted at room temperature for 24 hours to polymerize and add TEOS.
The reaction solution was centrifuged for 30 minutes at a gravitational acceleration of 18000 × g, and the supernatant was removed. 4 ml of distilled water was added to the precipitated silica particles to disperse the particles, and the mixture was again centrifuged for 30 minutes at a gravitational acceleration of 18000 × g. This washing operation was further repeated twice to remove unreacted TEOS, ammonia and the like contained in the labeled silica nanoparticle dispersion, thereby obtaining 149.2 mg of silica nanoparticles having an average particle diameter of 172 nm. Yield about 92%.

  FIG. 2 is a view showing an SEM photograph image of the obtained labeled silica nanoparticles. In the figure, spherical-shaped substances that appear white are the obtained labeled silica nanoparticles.

(2) Preparation of conjugate pad for detecting influenza A virus In a centrifuge tube, 700 μL of 50 mM KH ₂ PO ₄ (pH 6.5) and silica nanoparticle dispersion liquid containing 5- (and -6) -carboxyrhodamine 6G (10 mg / mL) ) 200 μL was added and stirred gently. Add 100 μL (10 μg / mL) of mouse anti-influenza A virus nucleoprotein monoclonal antibody (trade name: Influenza A NP, manufactured by santa cruz biotechnology) to the centrifuge tube and mix for 1 hour at room temperature. A monoclonal antibody was adsorbed on the silica nanoparticles. To this, 100 μL of 1% PEG (polyethylene glycol, molecular weight 20000, manufactured by Wako Pure Chemical Industries) was added and stirred gently, and further 100 μL of 10% BSA was added and stirred gently.
The mixture was centrifuged at 12000 × g for 15 minutes, the supernatant was removed, and the precipitate was phosphate buffer (10 mM KH ₂ PO ₄ (pH 7.2), 0.05% PEG20,000, 1% BSA, 0.1 1 mL of% NaN ₃ ), centrifugation, removal of the supernatant, and further precipitation with phosphate buffer (10 mM KH ₂ PO ₄ (pH 7.2), 0.05% PEG 20,000, 1% BSA, 0.1 1 mL of% NaN ₃ ) was added, the mixture was centrifuged, and the supernatant was removed. 10.67 mL of phosphate buffer (10 mM KH ₂ PO ₄ (pH 7.2), 0.05% PEG 20,000, 1% BSA, 0.1% NaN ₃ ) was added to the resulting precipitate to disperse the precipitate. A dispersion of silica nanoparticles (187.5 μg / mL) obtained by adsorbing a mouse anti-influenza A virus nucleoprotein monoclonal antibody was obtained.
Glass Fiber Conjugate Pad (trade name: GFCP, manufactured by MILLIPORE) (8 × 150 mm) was uniformly coated with 0.8 mL of a dispersion of silica nanoparticles obtained by adsorbing the mouse anti-influenza A virus nucleoprotein monoclonal antibody. . A conjugate pad 143 containing silica nanoparticles adsorbed with mouse anti-influenza A virus nucleoprotein monoclonal antibody was prepared by drying under reduced pressure overnight at room temperature in a desiccator.

(3) Preparation of conjugate pad for detection of influenza B virus Conjugate pad 145 for detection of influenza B virus is a mouse anti-influenza B virus nucleoprotein monoclonal antibody instead of a mouse anti-influenza A virus nucleoprotein monoclonal antibody. (Product name: Influenza BNA, manufactured by santa cruz biotechnology) was used in the same manner as the conjugate pad for detecting influenza A virus.

(4) Production of antibody-immobilized membrane for detecting influenza A virus A test line (antibody-immobilized) at a position of about 8 mm from the end of the membrane (length: 25 mm, trade name: Hi-Flow Plus120 membrane, manufactured by MILLIPORE) Part)) a solution containing 0.5 mg / mL of mouse anti-influenza A virus nucleoprotein monoclonal antibody ((50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) at a coating amount of 0.75 μL / cm, An antibody immobilization section 148 for influenza A virus was provided.
Subsequently, a solution containing 0.5 mg / mL of anti-IgG antibody (Goat Anti Mouse IgG, manufactured by Dako) as a control line having a width of about 1 mm at a position of about 12 mm from the end of the membrane (50 mM KH ₂ PO ₄ , pH 7. 0) Sugar Free) was applied at a coating amount of 0.75 μL / cm, and a control line 149 was provided.
Subsequently, a solution ((50 mM KH ₂ PO ₄ ) containing 10.0 mg / mL of anti-IgG antibody (Goat Anti Mouse IgG, manufactured by Dako) as a labeled particle capturing unit having a width of about 2 mm at a position of about 16 mm from the end of the membrane. , PH 7.0) Sugar Free) was applied at a coating amount of 1.0 μL / cm and dried at 50 ° C. for 30 minutes to provide a labeled particle capturing unit 150.

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 or more. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature to prepare an antibody-immobilized membrane 144 for detecting influenza A virus.

(5) Production of antibody-immobilized membrane for detecting influenza B virus The antibody-immobilized membrane 146 for detecting influenza B virus is an influenza A virus using mouse anti-influenza B virus nucleoprotein monoclonal antibody and anti-IgG antibody. It was produced by applying only the antibody immobilization part 151 for influenza B virus and the control line 152 in the same manner as the detection antibody-immobilized membrane.

(6) Preparation of test strip The obtained two types of membranes 144 and 146, the obtained two types of conjugate pads 143 and 145, a sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE) 142, Absorbent pads (Cellulose Fiber Sample Pad (CFSP) (manufactured by MILLIPORE) 147 are connected in series on a backing sheet (trade name AR9020, manufactured by Adhesives Research), and are cut into strips having a width of 1.5 mm and a length of 90 mm. A test strip 141 having the configuration shown in FIG. 4 was obtained.
In addition, as shown in FIG.1 (b), each component was affixed and affixed about 2 mm with the adjacent member on each both ends, respectively.

(7) Detection of influenza A virus and influenza B virus Two test strips are prepared, and 30 μL of a solution containing 5 × 10 ² FFU / mL of influenza A virus is dropped on one test strip sample pad. Allowed to stand for 10 minutes. To the sample pad of the other test strip, 30 μL of a solution containing 5 × 10 ² FFU / mL of influenza B virus was dropped and allowed to stand for 10 minutes.
The fluorescence image of the test strip was acquired using AE-6935GS VISRAYS-GS (product name, manufactured by ATTO Corporation). The obtained image was analyzed with image analysis software CS Analyzer 3 (product name, manufactured by Ato Corporation).
As a result, in the test strip in which the liquid containing influenza A virus was dropped, the fluorescence of the control line of the antibody-immobilized membrane for detecting influenza A virus and the control line of the antibody-immobilized membrane for detecting influenza B virus was detected. In the test strip where the liquid containing influenza B virus was dropped, the control line for the antibody-immobilized membrane for detecting influenza A virus and the test line and control line for the antibody-immobilized membrane for detecting influenza B virus The color development was confirmed.

(8) Evaluation of labeled particle transmittance The antibody-immobilized membrane for detecting influenza A virus having the labeled particle capturing part prepared in (4) above, and the conjugate for detecting influenza A virus prepared in (2) above A pad, a sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE), and an absorbent pad are connected in series on a backing sheet (trade name AR9020, manufactured by Adhesives Research), in a strip shape of 1.5 mm width and 60 mm length Cut to obtain a test strip. In addition, instead of Cellulose Fiber Sample Pad (CFSP, manufactured by MILLIPORE), Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE) was used as the absorbent pad.

30 μL of a phosphate buffer solution (50 mM KH ₂ PO ₄ , pH 7.0) was dropped on the test strip prepared in this manner and allowed to stand.
After 1 hour, only the absorbent pad was separated from the test strip, immersed in 3 ml of phosphate buffer (50 mM KH ₂ PO ₄ , pH 7.0) and shaken for 1 hour. After 1 hour, the liquid was recovered (extract liquid 1), and the fluorescence intensity at 555 nm was measured when an excitation wavelength of 530 nm was irradiated with a fluorescence spectrophotometer FP-6500 (trade name, manufactured by JASCO Corporation).

Subsequently, only the conjugate pad was separated from the test strip before dropping the phosphate buffer, immersed in 3 ml of phosphate buffer (50 mM KH ₂ PO ₄ , pH 7.0), and shaken for 1 hour. One hour later, the liquid was recovered (extract liquid 2), and the fluorescence intensity at 555 nm was measured with a fluorescence spectrophotometer FP-6500 (trade name, manufactured by JASCO Corporation) at an excitation wavelength of 530 nm.
Fluorescence intensity of the absorbent pad extract of the test strip of the antibody-immobilized membrane 144 for detecting influenza A virus having a labeled particle capturing portion, and the fluorescence intensity of the conjugate pad extract of the test strip before the phosphate buffer is dropped. From the ratio, the transmittance of particles was obtained. The results are shown in Table 2.

In Table 2, the fluorescence intensity of the extract 1 indicates the fluorescence intensity of the fluorescent silica particles that have passed through the antibody-immobilized membrane 144 for detecting influenza A virus . The fluorescence intensity of the extract 2 indicates the fluorescence intensity of the total amount of particles that have entered the antibody-immobilized membrane 144 for detecting influenza A virus . Therefore, it can be seen that the value obtained by dividing the fluorescence intensity of the extract 1 by the fluorescence intensity of the extract 2 is the transmittance of the membrane of this test strip, and the transmittance is 0.6%.

(9) Preparation of a test strip using a membrane having two test lines on the same membrane 700 μL of 50 mM KH ₂ PO ₄ (pH 6.5) and the 5- (and -6) -carboxyrhodamine 6G-containing silica nanoparticles in a centrifuge tube Dispersion (10 mg / mL) 200 μL was added and stirred gently. Add 100 μL (10 μg / mL) of mouse anti-influenza A virus nucleoprotein monoclonal antibody (trade name: Influenza A NP, manufactured by santa cruz biotechnology) to the centrifuge tube and mix for 1 hour at room temperature. A monoclonal antibody was adsorbed on the silica nanoparticles. To this, 100 μL of 1% PEG (polyethylene glycol, molecular weight 20000, manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred gently, and further 100 μL of 10% BSA was added and stirred gently.
The mixture was centrifuged at 12000 × g for 15 minutes, the supernatant was removed, and the phosphate buffer (10 mM KH ₂ PO ₄ (pH 7.2), 0.05% PEG20,000, 1% BSA, 0.1) was added to the precipitate. 1 mL of NaNa ₃ ), centrifuged, and the supernatant was removed. The precipitate was further added to phosphate buffer (10 mM KH ₂ PO ₄ (pH 7.2), 0.05% PEG 20,000, 1% BSA, 0. 1 mL of 1% NaN ₃ ) was added, centrifuged, and the supernatant was removed. To the obtained precipitate, 5.33 mL of phosphate buffer (10 mM KH ₂ PO ₄ (pH 7.2), 0.05% PEG20,000, 1% BSA, 0.1% NaN ₃ ) was added to disperse the precipitate, and the mouse A dispersion of silica nanoparticles (375 μg / mL) obtained by adsorbing an anti-influenza A virus nucleoprotein monoclonal antibody was obtained.

  Subsequently, a mouse anti-influenza B virus nucleoprotein monoclonal antibody (trade name: Influenza BNA, manufactured by santa cruz biotechnology) was used instead of the mouse anti-influenza A virus nucleoprotein monoclonal antibody. A dispersion of silica nanoparticles (375 μg / mL) obtained by adsorbing a mouse anti-influenza B virus nucleoprotein monoclonal antibody was obtained.

  Subsequently, 1 mL of a silica nanoparticle dispersion liquid (375 μg / mL) on which the mouse anti-influenza A virus nucleoprotein monoclonal antibody is adsorbed, and silica nanoparticle on which the mouse anti-influenza B virus nucleoprotein monoclonal antibody is adsorbed 1 mL of a particle dispersion (375 μg / mL) is mixed, and silica nanoparticles formed by adsorbing mouse anti-influenza A virus nucleoprotein monoclonal antibody and silica nanoparticles formed by adsorbing mouse anti-type B influenza virus nucleoprotein monoclonal antibody A mixed dispersion was obtained.

  0.8 mL of the mixed dispersion was uniformly applied to Glass Fiber Conjugate Pad (GFCP, manufactured by MILLIPORE) (8 × 150 mm). It contains silica nanoparticles adsorbed with mouse anti-influenza A virus nucleoprotein monoclonal antibody and silica nanoparticles adsorbed with mouse anti-influenza B virus nucleoprotein monoclonal antibody, and dried under reduced pressure overnight at room temperature in a desiccator. A conjugate pad 133 was produced.

A mouse anti-influenza A virus nucleoprotein monoclonal antibody is 0.5 mg / ml as a test line for influenza A virus at a position about 8 mm from the end of the membrane 134 (length 25 mm, trade name Hi-Flow Plus120 membrane, manufactured by MILLIPORE). A solution containing (mL (50 mM KH ₂ PO ₄ , pH 7.0) + 5% sucrose) in mL was applied at a coating amount of 0.75 μL / cm, and an antibody immobilization part 136 for influenza A virus was provided.
Subsequently, a solution containing 0.5 mg / mL mouse anti-influenza B virus nucleoprotein monoclonal antibody as a test line for influenza B virus at a position of about 12 mm from the end of the membrane 134 ((50 mM KH ₂ PO ₄ , pH 7 0.0) + 5% sucrose) at a coating amount of 0.75 μL / cm, and an antibody immobilization part 137 for influenza B virus was provided.
Subsequently, a solution containing 0.5 mg / mL of an anti-IgG antibody (Goat Anti Mouse IgG, manufactured by Dako) as a control line having a width of about 1 mm at a position of about 16 mm from the end of the membrane 134 ((50 mM KH ₂ PO ₄ , pH 7.0) Sugar Free) was applied at a coating amount of 0.75 μL / cm and dried at 50 ° C. for 30 minutes.

Next, the entire membrane 134 was immersed in a blocking buffer at room temperature for 30 minutes as a blocking treatment.
The membrane was transferred to a membrane washing / stable buffer and allowed to stand at room temperature for 30 minutes or more. The membrane was pulled up, placed on a paper towel and dried overnight at room temperature to prepare an antibody-immobilized membrane having two test lines on the same membrane.

  Adsorbing antibody-immobilized membrane 134 having two test lines on the same membrane, silica nanoparticles formed by adsorbing mouse anti-influenza A virus nucleoprotein monoclonal antibody and mouse anti-influenza B virus nucleoprotein monoclonal antibody Backing conjugate pad 133 containing silica nanoparticles, sample pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE) 132, absorption pad (Glass Fiber Conjugate Pad (GFCP), manufactured by MILLIPORE) 135 Assembled on a sheet (trade name AR9020, manufactured by Adhesives Research) into a 1.5mm wide, 60mm long strip Cross to obtain a test strip 131.

(10) Background evaluation The fluorescence intensity of the portion a shown in FIG. 3 of the test strip using the membrane having two test lines on the same membrane prepared in the above (9) is measured with a fluorescence plate reader (Spectra Max). , Manufactured by Molecular Devices Co., Ltd.), with an excitation wavelength of 530 nm and a fluorescence wavelength of 555 nm. As a result, the fluorescence intensity was 657.
Subsequently, 30 μL of a solution containing 5 × 10 ² FFU / mL of influenza B virus was dropped onto a sample pad of a test strip using a membrane having two test lines on the same membrane, and allowed to stand for 1 hour.
Subsequently, the background fluorescence intensity of the part a shown in FIG. 3 was measured using a fluorescence plate reader (Spectra Max, manufactured by Molecular Devices) at an excitation wavelength of 530 nm and a fluorescence wavelength of 555 nm. As a result, the fluorescence intensity was 1840.

Next, using the fluorescence plate reader (Spectra Max, manufactured by Molecular Devices), the fluorescence intensity of the portions a and b shown in FIG. Measurements were taken at 530 nm and a fluorescence wavelength of 555 nm. As a result, the fluorescence intensity of the part a was 651, and the fluorescence intensity of the part b was 648.
Subsequently, 30 μL of a solution containing 5 × 10 ² FFU / mL of influenza B virus was dropped on the sample pad of the test strip prepared in the above (6), and allowed to stand for 1 hour.
Subsequently, the fluorescence intensity at the excitation wavelength of 530 nm and the wavelength of 555 nm is measured using the fluorescence plate reader (Spectra Max, manufactured by Molecular Devices) for the background fluorescence intensity of the portions a and b of the test strip shown in FIG. Measured with As a result, the fluorescence intensity of the part a was 1189, and the fluorescence intensity of the part b was 1204.

  The above evaluation results are summarized in Table 3. From this result, compared to a test strip using a membrane having two test lines on the same membrane, the series type test strip produced by the method of Example 1 has about half the fluorescence intensity of the adsorbed particles, It can be said that it is suitable for line judgment.

DESCRIPTION OF SYMBOLS 1 Test strip 2 Sample pad 3 Conjugate pad 4 Membrane 5 Conjugate pad 6 Membrane 7 Absorption pad 8 Backing sheet 41 First detection target judgment part 42 First detection target control part 43 Labeled particle capture part 61 Second detection target determination unit 62 Second detection target control unit 131 Test strip 132 Sample pad 133 Conjugate pad 134 Membrane 135 Absorption pad 136 Antibody immobilization unit for influenza A virus 137 For influenza B virus Antibody immobilization part 141 Test strip 142 Sample pad 143 Conjugate pad 144 Antibody immobilization membrane for detecting influenza A virus 145 Conjugate pad 146 Type B Influenza virus antibody immobilized membrane 147 absorbent pad 148 A influenza virus antibody immobilization part 149 control lines 150 labeled particle capture unit 151 B influenza virus antibody immobilization part 152 control line

Claims (8)

A test strip for a lateral flow type immunochromatography method for detecting or quantifying at least two kinds of detection objects contained in a sample at a time,
Sample addition member,
A conjugate pad for immunochromatography in which labeled particles for the first detection target are immobilized;
An antibody immobilization unit for detecting the first detection object and an antibody immobilization for capturing the label particles with respect to the first detection object with respect to the direction in which the label particles flow with respect to the first detection object. A first membrane, sequentially provided with parts,
A conjugate pad for immunochromatography in which labeled particles for the second detection target are immobilized,
A second membrane having an antibody immobilization part for detecting a second detection object, and the absorbent pad possess in series connected in this order,
The immunochromatography conjugate pad on which the labeled particles for the first detection target are immobilized and the immunochromatography conjugate pad on which the labeled particles for the second detection target are immobilized are separated, and the first Second detection is performed on the downstream side of the immunochromatography conjugate pad on which the labeled particles for the detection target are immobilized and on the upstream side of the immunochromatography conjugate pad on which the labeled particles for the second detection target are immobilized. A test strip for lateral flow type immunochromatography , in which an antibody immobilization part for capturing labeled particles for a first detection target is provided immediately before a conjugate pad for immunochromatography on which a label for target is fixed . Ri Ah in silica particles of said first label particles and label particles relative to the second object to be detected with respect to the detection object, respectively it flat Hitoshitsubu径20 to 600 nm, to detect the first detection target The transmittance of the labeled particles with respect to the first detection object is 3% or less with respect to the first membrane provided with the antibody immobilization part and the antibody immobilization part for capturing the labeled particles with respect to the first detection object. characterized in der Rukoto, lateral flow type immunochromatography test strip of claim 1. 3. The lateral flow immunochromatography method according to claim 1, wherein the labeled particles for the first detection target and the labeled particles for the second detection target are fluorescent silica nanoparticles, respectively . Test strip.   The width | variety of the said test strip is 0.5 mm-2 mm, The volume of the sample containing the detection target object added to the said member for sample addition is 5-40 microliters, The any one of Claims 1-3 characterized by the above-mentioned. 2. A test strip for lateral flow immunochromatography according to item 1. A specimen is added to the sample addition member of the lateral flow type immunochromatographic test strip according to any one of claims 1 to 4, and two types of detection objects contained in the specimen are detected in one test. Alternatively, a method for detecting or quantifying a detection target using a test strip for lateral flow immunochromatography, characterized in that quantification is performed. The method for detecting or quantifying a detection target using the lateral-flow immunochromatographic test strip according to claim 5, wherein the detection target is an influenza A virus or an influenza B virus. In the lateral flow immunochromatographic test strip, the labeled particles for the first detection target and the labeled particles for the second detection target are silica nanoparticles having an average particle diameter of 20 to 600 nm each containing a fluorescent substance, The silica nanoparticle accumulated in the antibody immobilization unit for detecting the detection target and the antibody immobilization unit for detecting the second detection target is irradiated with an excitation light source, and fluorescence emitted from the silica nanoparticle is emitted. The lateral flow immunochromatography method according to claim 5 or 6, wherein a detection target contained in a specimen is detected or quantified by receiving light with a charge coupled device or a photomultiplier tube or visually confirming fluorescence. For detecting or quantifying a detection object using a test strip . A sample is added to the sample addition member of the lateral flow type immunochromatographic test strip according to any one of claims 1 to 4, and two types of detection objects contained in the sample are detected in one test or A method for detecting or quantifying a detection object using a lateral flow type immunochromatographic test strip for quantification, wherein the labeled particles for the first detection object and the labeled particles for the second detection object are fluorescent. A test strip for lateral flow immunochromatography, which is a silica nanoparticle, and further reduces the fluorescence background intensity at the time of fluorescence measurement in an antibody immobilization section for detecting the first detection object and the second detection object A detection method or a quantification method of a detection object using the method.