JP7540903B2 - Method for detecting a test substance using a flow channel and colloidal particles - Google Patents

Description

The present invention relates to a method for detecting a test substance using a flow path.

1. Increase in detection sensitivity of test substance When detecting a test substance, a detection reagent that directly binds to the test substance and a sensitizing reagent that binds to the detection reagent are used, it is common to use a washing step after using the detection reagent, remove unbound detection reagent, and then use the sensitizing reagent. However, the procedure becomes complicated and an automated device is required. In order to increase sensitivity in a simple one-step detection method such as the immunochromatography method, it is necessary to complicate the structure including the flow path as in Patent Document 1, and it is necessary to adopt a costly technology such as automating multiple steps as in Patent Document 2.

The inventors previously discovered a phenomenon in which sensitivity increases when particles that cause surface plasmon resonance are used in combination with particles that do not. Such a particle combination has an advantageous effect on the detection sensitivity of the analyte.

2. Increase in S/N ratio There are methods to simultaneously detect two or more related test substances in one flow path. For example, in the case of immunochromatography related to upper respiratory tract infections, kits are commercially available that detect influenza types A and B and RSV (respiratory syncytial virus) individually (with each of the three test lines) in the same kit. In addition, even if there is only one test item, the test substance may consist of multiple types. For example, if the test substance is a biomarker protein of eukaryotes such as humans, there may be splicing variants, and if the test substance is a microorganism, it may be classified into multiple serotypes or genotypes. In these cases, it may not be possible to design the binding site of the affinity substance for the test substance to be a common region of the variants, so multiple affinity substances may be required for the same test item. To use multiple affinity substances for multiple test substances including variants, one particle can be bound to multiple types of affinity substances, or colloidal particles to which each type of affinity substance is bound can be used to detect the test substance. From the viewpoint of producing stable affinity substance-bound particles, etc., it is preferable to bind each affinity substance to each particle rather than to bind multiple affinity substances to one particle. In this way, when the test substance is composed of multiple types, only some of the types of the test substance may be included.

Or, even if there is only one type of test substance, the colloidal particles used for detection are often contained in excess relative to the test substance.

Under these conditions, particles that are not involved in the reaction increase the background signal (the signal from colloid particles remaining in the flow path other than the detection site), resulting in a decrease in the S/N ratio for the detection of the test substance.

3. A one-step, multifaceted detection method for target substances using multiple types of particles
Two or more different types of particles can be used to detect a test substance by different methods. For example, the combined use of colored particles and fluorescent particles can realize visual detection and high sensitivity. However, when binding to the same test substance, these particles may compete with each other. For this reason, in many cases, the sensitivity and S/N ratio are lower than when a single type of particle is used. As in Patent Document 3, it is possible to prepare composite particles of these particles, but there is a problem with the production cost. In addition, the S/N ratio of at least one of the constituent particles remains unchanged or is lower than when it is bound to the test substance alone.

International Publication No. 2020/040159 Patent No. 5610389 Patent No. 5288634

The aim is to provide a method for detecting low concentrations of test substances using flow channels and particles, and to solve the following problems:

1. Increased detection sensitivity of test substances Test substances can be detected with higher sensitivity than before, with the same amount of work and time as before.

2. Reduction of background signal and increase in S/N ratio When detecting multiple test substances using corresponding colloidal particles in the same detection kit, if the respective particles that bind to each other are used, not all of the test substances are necessarily contained in the sample. Only a portion of the colloidal particles can be involved in the detection of the test substances, and the S/N ratio during detection decreases due to the particles that are not involved in the reaction.

3. Multifaceted detection method for a test substance in one step using multiple types of particles When using multiple types of particles and performing a separate detection method for each type of particle, it can be difficult to perform this in a single step, or the S/N ratio during detection can decrease.

Conventional methods for detecting analytes using colloid particles and flow channels, such as immunochromatography (Figure 4), often use a substance that has affinity for the analyte as a surface modifier for the colloid particles. The present invention is characterized in that the surface of some or all of the colloid particles is modified with not only a substance that has affinity for the analyte as a surface modifier for the colloid particles, but also a substance that bonds colloid particles together. Such colloid particles are used to detect analytes using flow channels, such as immunochromatography.

Colloid particles bound to the analyte at the detection site are called analyte-bound particles, and colloid particles that are not directly bound to the analyte but are bound to the analyte via other colloid particles are called particle-bound particles. Conventional analyte detection methods using a flow path and colloid particles, such as immunochromatography, mainly detect signals from analyte-bound particles. The present invention achieves the following 1 to 3 by detecting signals from analyte-bound particles and particle-bound particles, either separately or together.

1. Increased detection sensitivity of analyte In the multi-step sensitization method (Patent Application No. 2019-213491) that uses the binding of colloidal particles that the inventors have already invented, a detection step and a washing step were required before the sensitization step. In contrast, this method combines the detection step and the sensitization step into one step without the washing step, reducing labor and time costs. Specifically, in an analyte detection method using a carrier flow path such as immunochromatography, analyte-bound particles and particle-bound particles flow in a mixed state through the flow path, and a complex of analyte: analyte-bound particles: particle-bound particles (: indicates binding) is detected at the detection site.

2. Increase in S/N ratio When detecting multiple types of test substances using colloid particles bound to affinity substances for each substance, or when there is only one type of test substance, the amount of the test substance may be relatively small compared to the number of colloid particles. In such cases, the S/N ratio can be increased by effectively using colloid particles that cannot bind to the test substance. Specifically, colloid particles that cannot bind to the test substance indirectly bind to the test substance as particle-binding particles, increasing the S/N ratio. By forming a complex with colloid particles that were not involved in the reaction via other particles, the test substance can be detected with a high S/N ratio.

3. Multifaceted one-step detection method for analyte using multiple types of particles By using particles compatible with two or more detection methods (e.g. colored particles and fluorescent particles), analyte can be detected in a single operation without reducing the S/N ratio.

That is, the present invention is as follows.
[1] A method for detecting a test substance using colloid particles at a detection site in a flow path on a carrier, the method comprising: detecting colloid particles that can bind to each other via a surface-modifying substance; flowing these colloid particles in a mixed state through part or all of the flow path on the carrier; and detecting a test substance in which some of the colloid particles indirectly bind to the test substance at the detection site via other colloid particles.
[2] The method according to [1], comprising colloidal particles having no substance with affinity for the test substance bound to the surface of the colloidal particles.
[3] The method according to [1] or [2], wherein at least one of the colloidal particles is a hydrophilic colloid.
[4] The method according to any one of [1] to [3], wherein at least one of the colloidal particles is a hydrophobic colloid.
[5] Any of the methods [1] to [4] that increase the detection sensitivity or S/N ratio of the test substance.
[6] Any of the methods [1] to [5] for detecting two or more different test substances.
[7] Any of the methods [1] to [6], in which directly-bound particles that are not bound to the test substance function in the same manner as indirectly-bound particles.
[8] The method according to any one of [1] to [7], wherein at least one of the colloidal particles is a quantum dot.
[9] Any of the methods [1] to [8] in which two or more streams join along the way.
[10] A method as in [9] in which the flow path branches upstream from the confluence.
[11] A method for detecting a test substance at a detection site in a flow path on a carrier using colloid particles bound to a substance having affinity for the test substance, the method using directly bound particles and one or more types of indirectly bound particles, the direct bound particles being colloidal particles having a modifier that binds to the test substance bound to their surface, the indirectly bound particles being colloidal particles having a modifier that binds to the modifier on the surface of the direct bound particles and/or a modifier that binds to the modifier on the surface of another indirectly bound particle bound to their surface, the colloidal particles flowing in a mixed state in some or all of the flow path on the carrier, and the sensitivity is increased by forming a complex of the test substance:directly bound particles:indirectly bound particles (":" indicates bond), thereby detecting the test substance.
[12] The method according to [11], wherein a carrier or a porous carrier having an average flow path diameter of 1000 μm or less is used for part or all of the flow paths.
[13] The method according to [11] or [12], wherein the directly-bound particles, the indirectly-bound particles and the test substance are mixed in advance and applied to the carrier.
[14] Any of the methods [11] to [13], using a carrier at which a carrier having a flow path for direct bonded particles and a carrier having a flow path for indirect bonded particles join, the direct bonded particles are applied to the upstream part of the carrier having the flow path for direct bonded particles, and the indirect bonded particles are applied to the upstream part of the carrier having the flow path for indirect bonded particles, and the particles are mixed at the carrier junction and flow downstream of the carrier junction in a mixed state.
[15] The method according to any one of [11] to [14], wherein the flow path after mixing of the directly bound particles and the indirectly bound particles is the same carrier up to the analyte detection site.
[16] Any of the methods [1] to [15], in which the carrier is a test strip for immunochromatography and the test substance is detected by the principle of immunochromatography.
[17] The method according to any one of [1] to [16], wherein at least one of the colloidal particles used is a latex particle.
[18] The method according to any one of [1] to [17], wherein at least one of the colloidal particles used is a gold colloidal particle.
[19] Any of the methods [1] to [18], wherein the test substance is a substance of biological origin.
[20] The method according to any one of [1] to [19], wherein the test substance is derived from an infectious microorganism including a virus.
[21] A kit for use in any one of the methods [11] to [20], comprising at least directly bonded particles and indirectly bonded particles, wherein these particles form a complex.
[22] A kit for use in any one of the methods [1] to [10] or the kit of [21], which is an immunochromatography kit.
[23] A method for increasing the sensitivity of analyte detection in a method for detecting an analyte at a detection site in a flow path on a carrier using colloidal particles bound to a substance having affinity for the analyte, the method using directly bound particles and one or more types of indirectly bound particles, the direct bound particles being colloidal particles having bound to their surface a modifier that binds to the analyte, and the indirectly bound particles being colloidal particles having bound to their surface a modifier that binds to the modifier on the surface of the direct bound particles and/or a modifier that binds to the modifier on the surface of other indirectly bound particles, the colloidal particles flowing in a mixed state in some or all of the flow path on the carrier, and forming a complex of analyte:directly bound particles:indirectly bound particles (":" indicates bond) at the detection site, thereby increasing the sensitivity of analyte detection.

At least one of the following 1, 2, and 3 can be achieved by a simple operation similar to that of a conventional detection method using a flow channel and particles.
1. Increase in detection sensitivity of analyte The detection sensitivity of the analyte is increased by detecting the signal of the analyte-bound particles and the signal of the particle-bound particles together.
2. Increase in S/N ratio The S/N ratio increases when direct-binding particles that were not involved in the detection of the analyte bind indirectly to the analyte as particle-binding particles.
3. A one-step, multifaceted method for detecting test substances using multiple types of particles Multiple test substance detection methods can be easily performed with a high S/N ratio.

FIG. 1 is a diagram showing an overview of a multi-stage sensitization method, and is a diagram showing an overview of a method using two types of colloidal particles. FIG. 1 is a diagram showing an overview of a one-step sensitization method, and is a diagram showing an overview of a method using two types of colloidal particles. FIG. 1 is a diagram showing an example of the immunochromatographic test piece of the present invention. FIG. 3 is a diagram showing an outline of an assay method using the immunochromatographic test strip shown in FIG. 2. FIG. 1 shows an example of a conventional immunochromatographic test piece. This is a diagram showing an example of an immunochromatographic test piece in which a conjugate pad is used for each of two flow paths and a sample pad is installed. By simply adding a sample dropwise to the sample pad, the same detection sensitivity for the analyte as in the method of FIG. 3 can be obtained. This is a diagram showing an example of an immunochromatographic test piece in which a conjugate pad and a membrane are used for each of two flow paths, and a sample pad is installed. By simply adding a drop of a sample to the sample pad, the same detection sensitivity of a test substance as in the method of FIG. 3 can be obtained. FIG. 13 is a graph showing an increase in detection sensitivity when a biotinylated antibody is used as a test substance, streptavidin-modified particles are used as test substance-binding particles, and biotin-modified particles are used as particle-binding particles. This figure shows the effect of the composition of the surface modifier for particle binding on colloidal particles on sensitivity and background signal. The formation of a detection-inhibiting complex is observed at the site indicated by the arrow. Streptavidin and biotin are used to bind corresponding particles together, and the sensitivity (shade of the dots) and the degree of formation of the detection-inhibiting complex (site indicated by the arrow) differ depending on the biotin concentration. FIG. 13 is a diagram showing the effect of combined use of first and second indirectly bonded particles on sensitivity. FIG. 1 shows the results of enhancement when this enhancement method was applied to a sandwich lateral flow immunochromatography method using two antibodies that bind to respiratory syncytial virus (RSV) (part 1). FIG. 2 shows the results of enhancement when this enhancement method was applied to a sandwich lateral flow immunochromatography method using two antibodies that bind to respiratory syncytial virus (RSV) (part 2).

Colloid particles with a modifier that binds to the analyte bound to their surface are called direct-binding particles. Colloid particles with a modifier that binds to the analyte bound to their surface can also be called analyte-binding particles, since they bind to the analyte. Colloid particles with no modifier that binds to the analyte bound to their surface, but with a modifier that binds to the modifier on the direct-binding particle surface and/or a modifier that binds to the modifier on the surface of other indirect-binding particles bound to their surface, are called indirect-binding particles. Colloid particles with a modifier that binds to the modifier on the direct-binding particle surface and/or a modifier that binds to the modifier on the surface of other indirect-binding particles bound to their surface, can also be called particle-bound particles, since they indirectly bind to other particles. A particle that can bind to a specific particle is called the "corresponding particle" for that particle. Conversely, a particle that cannot bind to a specific particle is called the "non-corresponding particle" for that particle. Corresponding particles are bound to each other by their respective "surface modifiers for binding colloid particles to each other". These surface modifiers are called the "surface modifiers for binding colloid particles" for each particle. A single colloid particle may have one or more types of surface modifiers for binding colloid particles.

In order to clearly demonstrate the effect of the particle-bound particles, the examples of the present invention mainly use indirectly bound particles as the particle-bound particles, but the present invention can also be implemented with only a combination of directly bound particles. In other words, an affinity substance for the test substance may be bound to a portion or the entire surface of some or all of the colloidal particles. The indirectly bound particles that are the counterpart particles of the directly bound particles are called first indirectly bound particles, and the indirectly bound particles that are the counterpart particles of the first indirectly bound particles are called second indirectly bound particles.

Detection methods that detect signals from analyte-bound particles at a detection site in a flow channel include immunochromatography. Figure 4 shows a typical structure of a test piece (device) that has been used in the past for immunochromatography. To increase detection sensitivity, an excess amount of colloid particles is included in the kit, but to the extent that it does not affect the background signal. When the concentration of the analyte is relatively low, only a portion of the colloid particles can bind to the analyte, and the analyte is detected by the signal emitted by the analyte-bound particles that have bound to the analyte. Conversely, the majority of the colloid particles cannot be involved in the detection of the analyte, and this reduces the S/N ratio due to an increase in the background signal, etc.

In the present invention, a single-step operation allows corresponding particles to flow in a partially or entirely mixed state through the flow path. At the detection site, not only the bound analyte-binding particles but also the particle-bound particles contribute to the detection of the analyte. As a result, the detection sensitivity and S/N ratio are increased, and various detection methods according to the type of particle become possible.

Colloid particles to which one of the colloid particle-binding surface modifiers is bonded are bonded to the colloid particle-binding surface modifier on the corresponding particle via bonding between the colloid particle-binding surface modifiers.

The particle-bound particle bound to the analyte-bound particle via the surface modifier is called the first particle-bound particle. The particle-bound particle bound to the first particle-bound particle is called the second particle-bound particle, and similarly, the particle-bound particle bound to the nth particle-bound particle is called the n+1th particle-bound particle (n is a natural number). In this case, the analyte-bound particle and the nth particle-bound particle may each be composed of one type or two or more types of particles.

In the flow path or at the detection site, the analyte binds to a first colloid particle to become an analyte-bound particle. A second colloid particle binds to the analyte-bound particle to become a first-particle-bound particle, and a third colloid particle binds to the first-particle-bound particle to become a second-particle-bound particle. Since corresponding particles can bind to each other, the n+2-particle-bound particle (n is a natural number) and the n-th particle-bound particle may be the same colloid particle. Other colloid particles may also bind if they are corresponding particles. Particles directly or indirectly bound to the analyte are analyte-bound particles or particle-bound particles, respectively. The signals emitted by these analyte-bound particles and particle-bound particles can be detected for each type of particle or together.

Compared to conventional methods that detect only the signal from analyte-bound particles, the present invention can also detect the signal from particle-bound particles. When the signal from the analyte-bound particles and the signal from the particle-bound particles are the same or contain the same type of signals (for example, gold colloid particles with a diameter of about 40 nm that appear red and red-colored latex particles), these signals can be detected together. As a result, the detection sensitivity and S/N ratio of the analyte can be increased depending on the number of particles compared to conventional methods. Alternatively, when multiple different types of signals are emitted, they can be detected using the corresponding detection method for each.

It is also possible to use colloidal particles that emit different types of signals for some of the particles (e.g. fluorescent particles and colored particles), in which case two or more signals can be detected in one detection, making it possible to perform detection according to costs and the required sensitivity.

The present invention can be applied to a detection method using a device in which the above-mentioned complex can be formed at any location. Examples of such devices include devices for chromatography such as immunochromatography, microchips and capillaries having microchannels made of glass or resin. Here, a microchannel refers to a channel with an inner diameter on the order of micrometers, and a device having such a channel through which colloid particles can move and detect a specimen is called a microchip or microcapillary. In these devices, when a liquid containing colloid particles and a test substance is added to the device, the colloid particles and the test substance move by flowing through the channel on the carrier constituting the device, and form a complex at any location. For example, in the case of an immunochromatography device that utilizes the principle of immunochromatography, an immunochromatography test piece made of a porous carrier serves as the channel. The colloid particles and the test substance move through the channel, and a complex is formed or captured at a detection site where a substance capable of binding to the test substance (for example, when the test substance is an antigen, an antibody that binds to the antigen) is immobilized. This complex is composed of a test substance and a test substance-bound particle, or a test substance, a test substance-bound particle, and a particle-bound particle.

Among colloid particles, some of the corresponding particles may form a complex after mixing before reaching the detection site. In many cases where this affects the detection of the analyte, such complexes do not reach the detection site at the time of detection and may inhibit detection. Complexes that do not reach the detection site at the time of detection are called detection inhibition complexes. Detection inhibition complexes often have an effect due to their large size. The effect of detection inhibition complexes may be reduced by shortening the flow path length, enlarging the flow path diameter, increasing the number of types of indirectly bound particles (corresponding to the maximum value of n for the nth indirectly bound particle), optimizing the number of directly bound particles and indirectly bound particles, or reducing the amount of surface modifier for binding colloid particles.

In the present invention, the corresponding particles are applied to the flow path at approximately the same time, but it is preferable that the particles are mixed and flow continuously (slowly). It is also preferable to continuously add these colloidal particles to the extent that it does not affect the increase in the background signal.

1. Colloid particles Examples of colloid particles used in the present invention include hydrophilic colloid particles such as latex particles, metal colloid particles, hydrophobic colloid particles such as silica colloid particles, and particles having a core-shell structure including quantum dots. Hydrophobic colloid particles also include those to which protective colloids are bonded. Here, the term "protective colloid" refers to a hydrophilic colloid that protects the hydrophobic colloid by surrounding it. By protecting it, it is possible to prevent the aggregation of colloid particles caused by changes in the solution due to heating, addition of salts, etc. Hydrophobic colloids often maintain dispersion by electrical repulsion. For this reason, it is preferable that at least one of the colloid particles used in the present invention is a hydrophilic colloid in terms of ease of bonding between colloid particles. The hydrophobic colloid particles may be particles that cause surface plasmon resonance. By causing surface plasmon resonance, light in the vicinity of a specific wavelength is absorbed. The latex particles include colored latex particles and latex particles containing fluorescent dyes. Latex particles refer to particles that form an emulsion dispersed in water in a colloidal state. The material of the particles is not limited, but those used as materials for solid-phase carriers that bind proteins such as antibodies, antigens, ligands, and receptors in technical fields such as diagnostic drugs can be used. For example, styrene copolymers such as polystyrene, styrene-acrylic acid copolymers, resins such as polycarbonate, polymethyl methacrylate (PMMA), and polyvinyl toluene, silica, cellulose, and the like can be mentioned. Among these, styrene-based particles are preferred. Styrene-based particles refer to particles made of copolymers of polystyrene, styrene, or styrene derivatives and polymerizable unsaturated carboxylic acids or polymerizable unsaturated sulfonic acids. Examples of styrene derivatives include chloromethylstyrene and divinylbenzene, examples of polymerizable unsaturated carboxylic acids include acrylic acid and methacrylic acid, and examples of polymerizable unsaturated sulfonic acids include sodium styrene sulfonate. In the present invention, styrene-based latex particles are called polystyrene latex particles. The diameter of the latex particles is several tens to several hundreds of nm, preferably 50 to 800 nm, and more preferably 200 to 600 nm. The metal colloid particles may be of any type and shape, such as metal, alloy, metal oxide, or metal compound, and may be, for example, gold colloid particles. Also included are core-shell type metal colloid particles in which one metal colloid particle is coated with a layer of material such as silica. Furthermore, composite particles in which particles are bonded together in various ways may also be used. The diameter of the metal colloid particles is 10 to 200 nm, preferably 10 to 100 nm, and more preferably 10 to 50 nm. Regardless of the type of colloid particles, it is preferable to use particles that are approximately the same size as or sufficiently larger than the combined size of a general surface modifier molecule for binding colloid particles and a linker molecule that bonds the molecule to the colloid particles (for general molecules such as antibodies, the particle diameter is 10 nm or more) in order to leave unreacted surface modifier for binding colloid particles due to steric hindrance.

It is not necessary for all colloid particles to be directly involved in the detection method. For example, when detecting signals from colored colloid particles to detect a test substance, if colored colloid particles are used for all particles, the background signal may become high due to the formation of detection-inhibiting complexes. For this reason, the S/N ratio can be increased by using, for example, uncolored colloid particles for some of the particles (Example 4).

One type of surface modifier for binding colloid particles may be bonded to one colloid particle, or multiple types of surface modifiers for binding colloid particles may be bonded to one colloid particle.

As a combination of two types of surface modifiers for binding colloid particles that bind to each other, a combination of an antigen and an antibody can be mentioned, but it is not limited to a combination of an antibody and an antigen, and may be, for example, a combination of a ligand and a receptor or a receptor and a ligand. Examples of such affinity substances include polypeptides and other compounds that can bind to the test substance. Specifically, a combination of avidin and biotin can be mentioned. In addition to the surface modifier for binding colloid particles, a blocking agent can be bound to the colloid particle surface to suppress non-specific reactions. The blocking agent can also serve as the surface modifier for binding colloid particles. For example, biotinylated BSA can be used as a blocking agent and a surface modifier for binding colloid particles to the direct binding particles, and streptavidin can be bound as a surface modifier for binding colloid particles to the first particle binding particles.

At least one type of surface modifier for binding colloidal particles is used, which is a combination of substances that have affinity for each other, in order to bond corresponding particles together. One or more types of surface modifier for binding colloidal particles may be used, but it is preferable to use one type.

Methods for immobilizing surface modifiers onto colloidal particles include physical bonding, electrical bonding, and chemical bonding.

Colloidal particles are used by dispersing them in a liquid. This liquid is called a colloidal particle dispersion.

2. Flow Channel In the method of the present invention, colloidal particles move by flowing through a flow channel on the carrier.
The inner diameter of the pores of the channel of the porous carrier through which the colloid particles or the specimen move (carrier channel diameter) must be sufficiently larger than the colloid particle diameter. When using the method of the present invention, it is preferable that the detection inhibition complex does not affect the measurement results or background signal. The carrier channel diameter, depending on the colloid particle diameter used, is on average 1000 μm or less, preferably 500 μm or less, more preferably 200 μm or less, and even more preferably 100 μm or less, and is preferably about 100 nm to 100 μm. In this respect, a porous carrier is preferable as the carrier forming the channel, and among them, a nitrocellulose membrane is preferable. Furthermore, a microchannel, in which the channel diameter is on the order of micrometers, which is the same as that of the nitrocellulose membrane, is preferable. A channel having a channel diameter on the order of micrometers is called a microchannel.

Any carrier with a flow path can be used as long as colloidal particles can flow through it. For example, a carrier with a micro flow path consisting of individual flow paths can be used. Another example is a porous carrier that does not have a specific flow path and is composed of many flow paths. Any material can be used for the carrier as long as it functions as a flow path and does not cause problems with detection sensitivity and specificity. For example, in the case of a carrier with a micro flow path, glass and resin can be used, and in the case of a carrier used in immunochromatography, natural or synthetic polymers such as nitrocellulose, cellulose acetate, nylon, polyethersulfone, polyvinyl alcohol, polyester, glass fiber, polyolefin, cellulose, polystyrene, or a material made of a mixture of these can be used.

The corresponding particles can be mixed before being applied to the flow path. If they are mixed before being applied to the flow path, it is preferable that they are applied to the flow path immediately after mixing.

Alternatively, the corresponding particles can be mixed on the flow path by devising the structure of the flow path. For example, consider the case where the direct bonded particles, the first indirect bonded particles, and the second indirect bonded particles are mixed on the flow path (see Example 3). The direct bonded particles and the first indirect bonded particles flow through different flow paths until they are mixed. These corresponding particles are mixed at the part where the flow paths merge. When they are mixed on the flow path, the formation of the detection inhibition complex can be suppressed. Of the multiple flow paths leading to the merger, at least the flow path through which the direct bonded particles flow is called the direct bonded particle flow path. The flow paths other than the direct bonded particle flow path are called the indirect bonded particle flow path. The part where the flow paths merge and the particles are mixed is called the flow path mixing section. The flow path after mixing is called the mixing flow path. It is preferable that the corresponding particles flow through different flow paths upstream of the flow path mixing section. Conversely, the non-corresponding particles do not contribute much to the formation of the detection inhibition complex whether they flow through different flow paths or the same flow path upstream of the flow path mixing section. For example, the second indirect bonded particles do not contribute much to the formation of the detection inhibition complex whether they flow through the direct bonded flow path or through a flow path where only the second indirect bonded particles flow. In this way, by having non-compatible particles flow through the same channel upstream of the channel mixing section, the strip structure can be simplified (see Example 3 and Figure 2, which shows the strip structure used for this).

In laminar flows with low Reynolds numbers, it is difficult for particles to bind together in the flow channel by diffusion to form a complex. Therefore, binding between particles is likely to occur mainly at sites where colloid particles can be immobilized, such as the detection site. Conversely, if there is a change in the material of the flow channel or the direction of the flow channel after corresponding particles are mixed, it is expected that the Reynolds number will increase. The formation of detection-inhibiting complexes, which is thought to be due to such turbulent flow, may be promoted. For example, when the present invention is implemented with a conventional lateral flow immunochromatographic strip as shown in Figure 4, the formation of detection-inhibiting complexes may be observed when colloid particles move from the conjugate pad (CP) to the membrane (see Example 2). Due to the formation of this detection-inhibiting complex, many of the particles that would normally move to the membrane stagnate on the flow channel. In order to suppress the formation of detection-inhibiting complexes, the mixing flow channel can be reduced in size or a flow channel with a relatively small Reynolds number can be used. In addition, in order to increase the critical Reynolds number, a carrier having the same material or the same characteristics (pore size, pore shape, wettability, etc.) can be used from the mixing flow channel to the detection site. It is also preferable to use carriers or materials that do not have a large change in flow rate or direction throughout the entire flow channel. For example, the particles can be applied directly to the membrane (see Example 1), or a structure as shown in FIG. 5 can be used (see Example 4). In this way, in order to prevent the formation of detection-inhibiting complexes and to avoid excessive cost of testing, it is preferable to mix the particles on the flow channel. For example, the particles on the flow channel can be mixed using an external force such as a stirrer. Alternatively, it can be possible to use structural innovations such as the merging of multiple porous carriers or multiple flow channels. Compared to using a stirrer, structural innovations of the flow channel are passive and less likely to cause turbulence, so they are preferable as a particle mixing method. It is thought that the colloid particle mixing efficiency in laminar flow due to diffusion is inversely proportional to the square of the flow channel diameter of the flow channel mixing section. In order to increase the colloid particle mixing efficiency, a flow channel with a small thickness (the combined thickness of the direct binding particle flow channel and the indirect binding particle flow channel overlap) relative to the flow channel diameter of the flow channel mixing section or the width of the flow channel mixing section (the width of the surface where the two flow channels contact) can be used.

A flow path mixing section is formed by using multiple porous carriers, each of which is in contact with, connected to, or overlapped with the other, and corresponding particles can flow through different porous carriers upstream of the flow path mixing section. The corresponding particles mixed in the flow path mixing section form a complex downstream of the flow path mixing section (including the detection site). In the case of a lateral flow immunochromatography kit, the flow path mixing section can be set on a conjugate pad (Figure 5) or on a membrane (Figure 6). In particular, when the flow path mixing section is a flow path that is thin relative to its width, such as two nitrocellulose membranes, the flow path mixing section can be designed in such a way that the wide surfaces of the flow paths are in contact with each other. As a method for mixing in this manner, a method similar to that described in Patent Publication No. 4865664 can be used. Specifically, two separate carriers are overlapped to form a flow path mixing section, and the detection site is located downstream of the confluent carrier. The first carrier and the second carrier are overlapped to form a bifurcation. The second carrier may be overlapped in a shape that branches out in different directions from the first carrier. Alternatively, the first and second carriers may branch in the same direction or may be in contact with each other, as long as they are sandwiched between a film or the like to prevent them from mixing (see Figures 5 and 6). The test piece shown in Figures 5 and 6 is an immunochromatographic test piece in which a conjugate pad is used for each of the two flow paths and a sample pad is installed. Combinations of corresponding particles can be contained in advance in two or more separate conjugation pads.

Mixing by the merging of multiple microchannels is extremely difficult because the flows are close to laminar. However, a method has been reported that allows mixing by diffusion in such laminar flows without significantly increasing the Reynolds number (Science, 295 (2002) 647). Even when this mixing method is used, the particles mixed in the channel mixing section form a complex downstream of the channel mixing section (including the detection site). Particles are bound together by binding between corresponding particles in this way. As a result, not only the analyte and the analyte-bound particles can be detected at the analyte capture site, but also a complex formed by further binding of particle-bound particles.

3. Test substances and specimens Test substances may be any of nucleic acids, proteins, sugars, and other compounds, and may be substances of biological origin. They may also be complexes of these, such as pathogenic microorganisms such as bacteria and viruses, or substances found in living organisms or in the environment.

As the specimen, a specimen in the aqueous phase or a diluted specimen with a buffer solution can be used. Examples of specimens include body fluids such as serum, plasma, blood, urine, saliva, tissue fluid, cerebrospinal fluid, pharyngeal or nasal swabs, pharyngeal or nasal washes, and nasal aspirates, as well as feces, fecal suspensions, and culture fluids.

In the method of the present invention, colloid particles or colloid particle complexes are successively bound to the test substance, forming larger complexes, thereby sensitizing the reaction. The test substance is detected by detecting signals from these complexes.

The first colloid particle and the test substance are bound. The colloid particles used here may be any as long as they can be bound to the test substance directly or indirectly. Here, the direct binding of the test substance and the colloid particle means that a substance having affinity for the test substance is bound to the surface of the colloid particle, and the substance is bound to the test substance, and the indirect binding of the test substance and the colloid particle means that the test substance is bound to the substance bound to the surface of the colloid particle via another substance. For example, another substance A is bound to the test substance by pretreatment of the specimen, and the test substance is bound to the colloid particle to which substance B having affinity for the substance A is bound. Preferably, the direct binding is performed using colloid particles modified by binding a substance with affinity for the test substance to the surface. That is, the first colloid particle is bound to a surface modifier for binding colloid particles to each other and a substance that directly or indirectly binds to the test substance. The surface modifier for binding colloid particles may be the same as the substance that binds to the test substance, and the surface modifier for binding colloid particles may also serve as an affinity substance that binds to the test substance. Both the substance that has affinity for the test substance and is bound to the colloid particles and the surface modifier for binding colloid particles are called surface modifiers. For example, if the test substance is an antigen, the affinity substance for the test substance is an antibody for the test substance, and if the test substance is an antibody, the affinity substance for the test substance is an antigen to which the antibody binds. However, the affinity substance for the test substance is not limited as long as it is a substance that binds to the test substance.

The second colloid particle is bound to another surface modifier for binding colloid particles, which binds to the surface modifier for binding colloid particles of the first colloid particle. The first colloid particle and the second colloid particle are bound to each other by the bond between the surface modifiers for binding colloid particles, forming a complex.

The third and subsequent colloid particles bind to other colloid particles in the same way as the second colloid particle. In other words, the n+1th (n is a natural number) colloid particle is bound to another colloid particle binding surface modifier that binds to the nth colloid particle's colloid particle binding surface modifier. The nth colloid particle and the n+1th colloid particle bind to each other through the binding of the colloid particle binding surface modifier to form a complex. These n+1 sensitization steps increase the size of the colloid particle complex. This sensitization method can be performed in one step or multiple steps. When performed in one step, these reactions occur in parallel. In other words, binding reactions occur individually and in random order in parallel. A reaction occurs in which the first colloid particle binds to the test substance, or an unspecified nth colloid particle binds to the n+1th colloid particle, forming a complex. This complex further binds to other colloid particles or to each other. Regarding the binding between a particle and a complex or between complexes, the binding between an unspecified n-th colloid particle and an n+1-th colloid particle causes these bonds. When sensitization is performed in one step, it is called a one-step binding mode. When sensitization is performed in multiple steps, the first colloid particle binds to the analyte, followed by the second and third colloid particles in turn. When sensitization is performed in multiple steps, it is called a multi-step binding mode. In both the one-step binding mode and the multi-step binding mode, in order for the signal emitted by the analyte and the complex to be detected at the detection site, the analyte must be captured or fixed at the detection site and bound to a colloid particle complex, and the complex must contain at least the first colloid particle, which is the analyte-binding particle. The type of colloid particle binding surface modifier bound to the colloid particle is not limited. One or more types of colloid particle binding surface modifiers can be used for the unspecified n-th colloid particle. A combination of surface modifiers for binding colloid particles, which may be the same or different in composition, can be used between the nth colloid particle and the n+1th colloid particle.

Similarly, one or more types of colloid particles can be used. One or more types of colloid particles can be used for an unspecified nth colloid particle. Also, the same or different configurations can be used between the unspecified nth colloid particle and the n+1th colloid particle.

Figure 1-1 shows the principle of sensitization by the multi-stage binding mode. Two types of colloid particles, each bound to one type of modifier, are used. In addition, in order to detect the analyte 2, an insoluble carrier 1 bound to a substance that binds to it is used. That is, Figure 1-1 shows an example of capturing and detecting the analyte 2 on the insoluble carrier 1. When a sample containing the analyte 2 is added, the substance that binds to the analyte 2 on the insoluble carrier 1 binds to the analyte 2 and captures it. In Figure 1-1, the colloid particles used are the first colloid particle represented by a black circle and the second colloid particle represented by a white circle. Here, the nth colloid particle means the colloid particle added nth to the reaction system. In the case shown in Figure 1-1, the first colloid particle is a direct binding particle bound to a substance that has affinity for the analyte, and is also used as an indirect binding particle. The first colloid particle is bound to one type of modifier that binds to the analyte (surface modifier 4 in Figure 1-1). The surface modifier 4 in FIG. 1-1 serves both as an affinity substance for the test substance and as a surface modifier for binding colloid particles to bind to the second colloid particle 8 (through the surface modifier for binding colloid particles 7) in step B in FIG. 1-1. A substance (surface modifier 7 in FIG. 1-1) that binds to the substance that binds to the surface modifier 4 in FIG. 1-1 is bound to the second colloid particle. The surface modifier 7 in FIG. 1-1 may be the test substance itself, or may be a substance that is similar in structure to the test substance. Examples of substances that are similar in structure to the test substance include substances that have a partial structure in common with the test substance. For example, if the test substance is a polypeptide or protein, examples of such substances include substances that have an amino acid sequence that is highly homologous to the partial amino acid sequence of the test substance.

First, in step A, the first colloid particle 5 binds to the test substance 2 captured by the substance on the carrier 1. At this time, a complex represented by the test substance 2:first colloid particle 5 (":" indicates binding) is formed.

Next, in step B, second colloid particles 8, which are sensitizing modified colloid particles, are added. As a result, the second colloid particles 8 are bonded to the first colloid particles 5. This bonding between the colloid particles occurs via the bond between the colloid particle bonding surface modifier 4 of the first colloid particle and the colloid particle bonding surface modifier 7 (M(1)) of the second colloid particle 8. In step B, a complex is formed in which the first colloid particle 5 and the second colloid particle 8 are bonded. At this time, a complex represented by the test substance 2: first colloid particle 5: second colloid particle 8 (":" indicates a bond) is formed.

Next, in step C, the same colloid particles as the first colloid particles 5 are added as the third colloid particles 11, which are sensitizing modified colloid particles. Furthermore, in step D, the same colloid particles as the second colloid particles 8 are added as the fourth colloid particles 14, which are sensitizing modified colloid particles. As a result, as shown in step D of FIG. 1-1, a large complex containing two types of colloid particles is formed, which is represented as the test substance 2: the first colloid particles 5: the second colloid particles 8: the third colloid particles 11: the fourth colloid particles 14 (":" indicates a bond). A signal corresponding to the number of colloid particles is generated, and the test substance can be detected by measuring the signal intensity. For a given amount of test substance, as the number of sensitization steps is increased, the complex becomes larger, the number of colloid particles contained in the complex increases, and the signal generated as a result also increases, so that the measurement sensitivity of the test substance is enhanced.

Figure 1-1 shows up to step D, but the number of times the steps are repeated is not limited. In the method shown in Figure 1-1, the first colloid particles used in step A are the same as the colloid particles used in step C, and the colloid particles used in step B are the same as the colloid particles MC used in step D.

Furthermore, Figure 1-2 shows the principle of sensitization by the one-step binding method. Figure 1-2 is an example using the same materials as Figure 1-1. The more the mixing is by diffusion alone (the flow is laminar), the more the particles bind to each other at the detection site where the interparticle velocity difference is large (the captured particles can be considered to be stationary). For this reason, the one-step binding method shows a reaction similar to that of the multi-step binding method in Figure 1-1 over time. However, some of the particles form complexes before reaching the detection site, and after reactions such as those in Figure 1-2B and Figure 1-2C, they form complexes such as D in the multi-step binding method in Figure 1-1. Figure 1-2B shows the possibility of various complexes binding during the complex formation process. Also, Figure 1-2C shows that, although it is a one-step method like the multi-step sensitization method, various complexes and particles bind, and the number of particles in the complex continues to increase.

In the method of the present invention, the number of types of colloidal particles is not limited, and one or more types of colloidal particles can be used. The inventors have previously demonstrated that sensitivity can be further increased by using resonant particles (particles that cause surface plasmon resonance; metal colloid particles, etc.) and retained particles (particles that are larger than the resonant particles and do not cause surface plasmon resonance; latex colloid particles, etc.) in combination as colloidal particles used for detection. This increase in sensitivity can be applied to some or all of the directly bound particles and/or indirectly bound particles. The resonant particles bind to the surface of the retained particles. The resonant particles absorb light near a specific wavelength by causing surface plasmon resonance. At the same time, the retained particles also emit a signal, so that the total signal including the signal due to surface plasmon resonance and the signal from the retained particles can be detected, enabling highly sensitive detection.

Particle-bound particles can be used up to the nth particle-bound particle (n is any natural number). This n can be optimized for the reaction rate between particles in the flow channel and the background signal. For example, when the detection-inhibiting complex becomes large before reaching the detection site, such as when the flow channel is relatively long, the reaction rate can be reduced by increasing n without changing the total number of particles. Conversely, when the size of the particle is small compared to the carrier pore size, n can be set to a small number. For example, when using indirectly bound particles (explicitly particle-bound particles), it may be advantageous to use up to the second indirectly bound particles in terms of detection sensitivity. The direct-bound particles and the first indirectly bound particles, and the first indirectly bound particles and the second indirectly bound particles are corresponding particles. In this case, the direct-bound particles and the second indirectly bound particles may be bound to the first indirectly bound particles by a similar mechanism. In many cases, a larger amount of surface modifier for particle binding can be bound to the second indirectly bound particles than to the direct-bound particles, and the detection sensitivity is advantageous to use up to the second indirectly bound particles (see Example 2).

Test substance detection kits that simultaneously detect multiple test substances are commercially available that use colloid particles and flow channels (for example, a kit that distinguishes between influenza types A and B in the same kit). In detection using such kits, the test sample does not necessarily contain all types of test substances to be detected. For example, if the test sample contains test substance A but does not contain test substance B other than A, the direct binding particles that detect B do not contribute to the reaction at all, and the S/N ratio for the detection of A decreases. In such a case, if each direct binding particle binds to a common first particle binding particle (for example, a common substance is used for the surface modifier for colloid particle binding of A and B that binds to the indirect binding particle), the direct binding particle of B functions as the second particle binding particle. Conversely, it also functions in the same way when the test sample does not contain A but does contain B. In either case, the sensitivity and S/N ratio may be improved.

The test substance may be directly or indirectly bound to an insoluble carrier. Examples of insoluble carriers include other colloidal particles, resins, and immunochromatographic test strips used in immunochromatography.

When the colloidal particles are colored colloidal particles, the signal generated from the colloidal particles is reflected light containing a specific wavelength, and the intensity of the signal can be measured by visually observing the light or by measuring the intensity with a densitometer or spectrophotometer, etc.

When the colloidal particles are fluorescent particles, the signal generated from the colloidal particles is fluorescence of a specific wavelength, and the intensity of the signal can be measured by measuring the intensity of the fluorescence using a fluorescence measuring device.

When two types of colloidal particles are used, each of which is a colloidal particle that is bound to an energy donor and an energy acceptor for fluorescence resonance energy transfer, the colloidal particles form a complex and become densely packed, causing a change in fluorescence resonance energy transfer (FRET). This change is used as a signal and can be measured with a fluorescence measuring device to measure the signal intensity.

The reaction solution used in the method of the present invention may be any solution as long as it does not inhibit the binding of the direct binding particles to the test substance and the reaction between the colloid particle surface modifiers and occurs specifically, and it is preferable to use a buffer solution. Salts, surfactants, alcohol, etc. may be added to the buffer solution.

The present invention also includes reagents and kits. The kits are not only reagents and kits using directly bound particles, but also indirectly bound particles. The kits are, for example, immunochromatographic kits that utilize the principle of immunochromatography, and the substance that binds to the test substance bound to the colloidal particles is an antibody or antigen that binds to the test substance through an antigen-antibody reaction. The kits further include a test strip for immunochromatography, a blotter, a buffer solution, and the like. The colloidal particles bound to the substance that binds to the test substance may be included as a reagent separate from the immunochromatographic test strip, or may be included in the labeling site of the immunochromatographic test strip.

An example of the immunochromatography method of the present invention will be described below. In the following case, two separate carriers are overlapped to form a flow path mixing section, and a carrier in which a first carrier and a second carrier are overlapped to form a bifurcation is used as a carrier with a detection site downstream of the merged carrier. In addition, a substance that binds to the test substance is immobilized at the detection site on the carrier.

(1) Immunochromatographic test strip An immunochromatographic test strip comprises a support having a detection site where a substance such as an antibody that captures a test substance (such as an antigen) is immobilized, a site for applying a specimen, an absorption band that absorbs the developed specimen liquid, and a backing sheet for bonding these components together.

The support is a material that has the ability to immobilize antibodies to capture the test substance (antigen), and does not impede the horizontal passage of liquid. It is made of cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, etc. The support is sometimes simply called a membrane.

The detection site refers to a part of the support where an antibody that captures the test substance (antigen) is immobilized. The detection site is provided with at least one site where an antibody for capturing the antigen is immobilized. The detection site may be included in the support, for example, an antibody may be immobilized on the support.

A sample pad, which is a porous material for applying the sample, may be provided at the site for applying the sample. The sample pad is the most upstream site of the immunochromatographic test piece. Commonly used materials such as filter paper, glass fiber, and nonwoven fabric can be used for this purpose.

The absorption band is a member that is supplied to the support and absorbs the components that are not involved in the reaction at the detection site. The material can be a highly water-retentive filter paper or sponge made of a general natural or synthetic polymer compound.

When designing a flow path mixing section for an immunochromatographic test piece used in the method of the present invention, at least two carriers are present, a carrier for a flow path for direct-bound particles and a carrier for a flow path for indirect-bound particles, and the two carriers overlap and merge upstream of the detection section, forming a flow path mixing section at the carrier merging section. For example, two carriers having two respective flow paths may be overlapped with a second carrier that does not have a detection site upstream of the detection site of the first carrier. In this case, the first carrier becomes a flow path for direct-bound particles, and the second carrier functions as a flow path for indirect-bound particles.

The direct-binding particles and indirect-binding particles may be applied upstream of the carrier, or may be held on the carrier in advance. Such holding may be performed directly on the flow path carrier, or may have a holding site made of a different material at the upstream part of each flow path, such as the conjugate pad in a lateral flow immunochromatographic strip. In the case of the immunochromatographic test strip having the bifurcated carrier described above, the direct-binding particles may be immobilized by impregnating and drying the conjugate pad upstream of the first carrier, and the indirect-binding particles may be immobilized by impregnating and drying the conjugate pad upstream of the second carrier. Using this method, a test strip with a structure such as that shown in Figure 5 or Figure 6 can be used, and the test substance can be detected by applying the sample liquid to only one place (such as a sample pad), which may simplify the operation.

Figures 2 and 4 to 6 show examples of immunochromatographic test strips used in the present invention.

(2) Assay Method Using the immunochromatographic test strip of (1), an assay can be carried out as follows.
A mixed suspension of a specimen containing a test substance and direct-binding particles is applied to the upstream part of the first carrier, and a suspension containing indirect-binding particles is applied to the upstream part of the second carrier. The application can be performed by dropping the suspension onto the sample pad, or by putting the suspension to be applied into a container and immersing the sample pad of the immunochromatographic test strip in it. The mixed solution of the test substance and the direct-binding particles and the indirect-binding particles join at the overlapping part of the first carrier and the second carrier to form a flow channel mixing part. The test substance, the direct-binding particles, and the indirect-binding particles move in a mixed state to the detection area of the carrier. In the detection area of the carrier, a complex of the substance bound to the test substance: the test substance: the direct-binding particles: the indirect-binding particles (":" indicates binding) is formed, and the test substance can be measured by detecting signals from the direct-binding particles and the indirect-binding particles.

Even if different particles that are incompatible flow through individual flow paths, no detection-inhibiting complexes are formed. In addition, a sample containing a test substance may be mixed with indirectly bound particles, or may be mixed with both directly bound and indirectly bound particles.

Figure 3 shows an overview of an example of an assay method for the immunochromatographic test strip whose structure is shown in Figure 2. In the method shown in Figure 3, the sample is added by placing the suspension to be applied in a container and immersing the sample pad of the immunochromatographic test strip in it. A suspension 21 containing directly bound particles and a test substance, or directly bound particles, a test substance and a second indirectly bound particle, is applied to the first carrier 16, and a suspension containing a suspension 22 containing indirectly bound particles is applied to the second carrier 17. The suspension applied to the first carrier and the suspension applied to the second carrier are mixed in the flow channel mixing section, and the complex captured at the detection site included in the support 19 can be detected. In Figure 3, the arrow F indicates the direction in which the liquid flows.

It is preferable to apply an external force perpendicular to the first carrier and the second carrier. This external force makes it easier for the particles that have flowed through the first carrier and the particles that have flowed through the second carrier to mix. The external force can be applied, for example, by pressing down on the carrier from above.

The test strip shown in Figures 5-6 is an immunochromatographic test strip that uses a conjugate pad in each of the two flow paths and has a sample pad installed. When using the test strip shown in Figures 5-6, a sample containing a test substance is applied to the sample pad, and the liquid (containing the test substance) that spreads on the test strip flows separately to conjugation pad 1 (24) and conjugation pad 2 (25). Conjugation pad 1 (24) contains direct-bonded particles, or direct-bonded particles and second indirect-bonded particles, and conjugation pad 2 (25) contains indirect-bonded particles (or vice versa). The liquids that spread are merged and mixed on the test strip to become one liquid. The complex captured at the detection site 19 can be detected. In Figures 4-6, arrow F indicates the direction in which the liquid flows.

The present invention will be specifically described with reference to the following examples, but the present invention is not limited to these examples.

Example 1
Test Method: A biotinylated mouse antibody modified with an amino group biotin labeling reagent (Thermo) so that the molar ratio of biotin/antibody was 5 was used as the test substance.

1μl of each of 40, 10, 5, and 2.5μg/ml biotinylated mouse antibodies was impregnated in a dot pattern on a nitrocellulose membrane, dried, and immobilized. Immunochromatography was performed using this strip.

A mixture of streptavidin-modified latex colloid particles, which are direct-binding particles, and biotin-modified latex colloid particles, which are indirect-binding particles, was applied to the membrane.

Test results: The direct-binding particles and indirect-binding particles migrated on the membrane and accumulated on the detection area where the biotinylated mouse antibody was immobilized. As shown in FIG. 7, the addition of indirect-binding particles (biotin-modified particles) enhanced the detection signal of the test substance at each test substance concentration. The coloration by the direct-binding particles (streptavidin-modified particles) and indirect-binding particles was observed depending on the test substance concentration. Even when the test substance concentration was 40 μg/ml, no signal from the indirect-binding particles was detected when the direct-binding particles were not applied. No line formation due to the aggregation of colloids in the flow channel or an increase in background signal was observed.

Discussion: In the absence of direct-binding particles, no signal from the analyte was detected with the indirect-binding particles, and a sensitizing effect was observed with the indirect-binding particles. This suggests that the indirect-binding particles indirectly bound to the analyte via the direct-binding particles. This suggests the formation of a complex of analyte: analyte-bound particles: particle-bound particles (: indicates binding). This suggests that when particles are applied directly to the membrane, little detection-inhibiting complex is formed.

Example 2
Test method: A test was performed using a strip of the shape shown in FIG. 4, which is a conventional immunochromatographic test piece in which the sample pad 26, the conjugation pad 24, and the support 19 are in contact with each other in series and the flow path is not branched. 1 μl of 1 mg/ml of the test substance capture substance (anti-influenza virus antibody) was blotted onto a nitrocellulose membrane and then dried. A mixture of the test substance (inactivated influenza virus antigen), directly bound particles (anti-influenza virus antibody-modified latex with various numbers of biotins added with a biotin/antibody molar ratio of 1 to 16), and first particle-bound particles (streptavidin-modified latex) was applied onto the sample pad. The same latex particles (red, diameter about 400 nm) were used before binding to the surface modifier.

Test results The results are shown in Figure 8. Sensitivity increased from S1 to S5. For S1 to S4, latex stagnation was observed at the site where the sample transferred from the conjugation pad to the membrane (site indicated by the arrow), but this decreased in S5 compared to S1 to S4.

Discussion Based on the inventor's previous studies, it was considered that there was a trade-off between the binding reaction of avidin and biotin due to an increase in the number of biotins and the inhibition of the antigen-antibody reaction due to an increase in the number of biotins. It is considered that in S1 to S4, the inhibition of the antigen-antibody reaction due to biotin modification was suppressed by reducing the number of biotins, and the sensitivity was increased. Since the formation of the detection inhibition complex was suppressed in S5, the number of biotins was less than the optimal number when only the reaction between avidin and biotin was considered, and the number of particles bound to the particles may have been suppressed. However, it is considered that the detection inhibition complex was less likely to be formed and many latex particles were able to participate in the reaction, resulting in a further increase in sensitivity compared to S4. In this way, the S/N ratio may be improved by optimizing the amount of the surface modification substance for binding colloidal particles.

Example 3
Test Method
1 μl of 1 mg/ml of the test substance capture substance (anti-RSV (Respiratory syncytial virus) antibody) was blotted onto a nitrocellulose membrane and then dried. The test substance (RSV inactivated antigen), direct binding particles (anti-RSV antibody modified latex with biotin added so that the molar ratio of biotin/antibody was 1), first indirect binding particles (streptavidin modified latex), and second indirect binding particles (8 times the amount of biotin molecules bound to the second indirect binding particles compared to the direct binding particles) were applied, and the effect of each indirect binding particle was examined. The same latex particles (red, diameter about 400 nm) were used before binding to the surface modifier. The strip structure shown in Figure 2 was used, and each suspension was impregnated from the end of each of the two branched membranes as shown in Figure 3. Specifically, the mixture of the test substance, direct binding particles, and second indirect binding particles was applied from the same flow path, and the first indirect binding particles were applied from a different flow path. In the flow channel mixing section, a nitrocellulose membrane for the first indirectly bound particle flow channel was pressed against the nitrocellulose membrane for the direct-bound particle flow channel upstream of the detection site by clipping it with a clip, using the same method as that described in Japanese Patent No. 4865664. The negative control was a sample without the RSV antigen (Group-4).

As shown in Figure 9, when only the directly bound particles were used, very faint dots were observed (Group-1), but when the first indirectly bound particles were used, the dots became darker and the signal was enhanced to a detectable level (Group-2). When the second indirectly bound particles were also used, the dots became darker (Group-3), but when the antigen was not included, no dots were observed (Group-4).

Discussion In conventional detection methods using only direct-binding particles, when the analyte is weak, only the complex of analyte:direct-binding particles (":" indicates binding) is detected at the analyte capture site, resulting in a low detection signal (Group-1). In contrast, when indirect-binding particles are used in combination, not only the complex of analyte:direct-binding particles but also the complex of analyte:analyte-binding particles:particle-bound particles is thought to be formed. These complexes are captured or further accumulated at the analyte capture site, and the detection sensitivity is thought to have increased. Compared to direct-binding particles, the second indirect-binding particles have more biotin, which is a surface modifier for particle binding, bound to them. In this way, when the affinity substance for the analyte on the surface of the direct-binding particles and the surface modifier for particle binding are different, the use of second particle-bound particles can sometimes increase the detection sensitivity and improve the S/N ratio (Comparison between Group-2 and Group-3). It was also suggested that by using appropriate materials and flow path designs in this method, it is possible to merge the flow paths for directly bound particles and indirectly bound particles using a carrier, and that even when using second or more indirectly bound particles, the number of flow paths can be limited to two.

Example 4
Test method Two test lines and one control line were formed on a strip in the shape of Figure 5. 0.5 μl of 1 mg/ml of a test substance capture substance (anti-RSV (Respiratory syncytial virus) antibody or an antibody that does not cross-react with RSV) was blotted onto a nitrocellulose membrane for the two test lines. Anti-mouse antibody was blotted onto the control line. After these blots, the membrane was dried. Mouse antibodies were used as affinity substances for the test substances of two types of direct binding particles (direct binding particles A and direct binding particles B; both types are latex colloid particles with a diameter of about 400 nm colored red). Direct binding particles A were modified with anti-RSV antibodies, and biotin was added so that the molar ratio of biotin/antibody was 1. Direct binding particles B were modified with mouse antibodies that do not react with RSV, and biotin was added so that the molar ratio of biotin/antibody was 1. Directly bound particles A and directly bound particles B were applied to the conjugate pad 1 shown in FIG. 5, and first particle-bound particles (uncolored streptavidin-modified latex colloid particles) were applied to the conjugate pad 2, respectively, and then dried (Group-4). The particles were applied thinly to the center of each conjugate pad, avoiding the overlapping tip where the flow channels join. In order to examine the effect of the present invention, strips using non-biotinylated directly bound particles (Group-1 and Group-2), strips with twice the concentration of non-biotinylated directly bound particles (Group-2), and strips without indirectly bound particles (Group-3) were also prepared. The test substance (RSV inactivated antigen) was applied to these strips. The effect of each indirectly bound particle was examined.

Test results The results are shown in Figure 10. The position of the line blotted with RSV antibody among the two test lines is shown in the figure. When the total number of directly bound particles was doubled, the test line signal and background signal increased (compare Group-1 and Group-2). When indirectly bound particles were added, the background signal increased (compare Group-4 and Group-3), but the test line signal was higher than when the number of directly bound particles was doubled (compare Group-4 and Group-1). There was no difference in sensitivity with or without biotin modification (compare Group-2 and Group-3). No stagnation of colloidal particles at the transition to the membrane was observed as seen in Example 3.

Observations
When only two types of direct-binding particles are used and no indirect-binding particles are used, modification with a surface modifier for particle binding (biotin) is not necessary. The surface modifier for particle binding may inhibit the binding of the direct-binding particles to the test substance, but this inhibition was not observed when the amount of the surface modifier for particle binding was adjusted (compare Group-2 and Group-3). The number of directly-binding particles for a specific test substance correlates with the sensitivity and the intensity of the background signal (compare Group-1 and Group-2). Directly-binding particles A bind to and detect the test substance, inactivated RSV, but directly-binding particles B do not contribute to this detection. In order to maintain detection sensitivity, the number of directly-binding particles contained in a kit that simultaneously detects two or more test substances, as in this embodiment, is often greater than that of a kit that detects a single test substance. For this reason, the background signal is likely to increase (compare Group-1 and Group-2). The use of indirectly-binding particles can increase the detection sensitivity without changing the number of directly-binding particles (compare Group-3 and Group-4). Since uncolored latex is used for the indirectly-binding particles, the increase in signal is thought to be due to the directly-binding particles. It is believed that a portion of the directly bound particles B and directly bound particles A, which could not contribute to the reaction without the indirectly bound particles, contribute to the increase in the signal as particle-bound particles. From the above, it is believed that the present invention is useful for increasing the detection sensitivity and S/N ratio in a test substance detection method for simultaneously detecting two or more types of test substances.

Example 5
Test method Two test lines and one control line were formed on a strip in the shape of Figure 5. 0.5 μl of 1 mg/ml of the test substance capture substance (anti-influenza virus antibody or antibody that does not cross-react with influenza virus) was blotted onto the nitrocellulose membrane on the two test lines. Anti-mouse antibody was blotted onto the control line. After these blots, the membrane was dried. Direct binding particles (40 nm diameter gold colloid manufactured by BBI Co., Ltd. bound to biotinylated anti-influenza virus antibody), indirect binding particles (blue streptavidin-modified latex colloid particles; diameter approximately 400 nm), and the test substance (inactivated influenza virus) were applied from the sample pad (Group-2). This (Group-2) was also compared with those not applied with indirect binding particles (Group-1) or direct binding particles (Group-3).

Test Results The results are shown in Figure 11. Purple test and control lines were formed in Group-2. Red test and control lines were formed in Group-1, and neither test nor control lines were formed in Group-3.

Discussion The purple lines indicate that corresponding particles are bonded to each other. It has been suggested that the present invention can be implemented using hydrophobic colloids such as gold colloids.

1. Carrier 2. Test substance 3. First colloid particle 4. Surface modifier for binding colloid particles and substance having affinity for test substance 5. Colloid particle bound to surface modifier 6. Colloid particle C(1)
7 Surface modification substance for colloidal particle binding M(1)
8. Sensitizing modified colloidal particles MC(1)
9. Colloidal particles C(2)
10 Surface modification substance for colloidal particle binding M(2)
11. Sensitizing modified colloidal particles MC(2)
12 Colloidal particles C(3)
13 Surface modification substance for colloidal particle binding M(3)
14. Sensitizing modified colloidal particles MC(3)
15 Immunochromatographic test piece 16 First carrier 17 Second carrier 18 Flow path mixing section 19 Support (membrane) including detection site
20 Absorbent pad (filter paper)
21 Suspension containing directly bound particles and a test substance, or directly bound particles, a test substance, and a second indirectly bound particle 22 Suspension containing indirectly bound particles 23 Impermeable membrane such as a film 24 Conjugation pad 1
25 Conjugation Pad 2
26 Sample pad 27 Backing sheet

The present invention makes it possible to generally and simply increase the sensitivity of various analyte detection methods.

Claims (14)

A method for detecting a test substance at a detection site on a flow path on a carrier by the principle of immunochromatography using colloid particles bound to a substance having affinity for the test substance, the method using direct-bound particles and one or more types of indirect-bound particles, the direct-bound particles being colloid particles having a modifier substance bound to their surface that binds to the test substance, the indirect-bound particles being colloid particles having a modifier substance bound to their surface that binds to the modifier substance on the surface of the direct-bound particles and/or a modifier substance bound to the modifier substance on the surface of another indirect-bound particle, A method for detecting a test substance using a carrier in which carriers having flow paths for directly bound particles merge, and in which a carrier having a flow path for directly bound particles and a carrier having a flow path for indirectly bound particles branch off upstream of the junction, in which directly bound particles are applied to the upstream part of the carrier having a flow path for directly bound particles and indirectly bound particles are applied to the upstream part of the carrier having a flow path for indirectly bound particles, and the particles are mixed at the carrier junction and flow downstream of the carrier junction in a mixed state , forming a complex of the test substance:directly bound particles:indirectly bound particles (":" indicates bond), thereby increasing sensitivity and detecting the test substance. The method according to claim 1, comprising colloidal particles on whose surface there is no substance with affinity for the test substance bound. The method according to claim 1 or 2, wherein at least one of the colloid particles is a hydrophilic colloid. The method according to any one of claims 1 to 3, wherein at least one of the colloidal particles is a hydrophobic colloid. The method according to any one of claims 1 to 4 , wherein two or more different test substances are detected. The method according to any one of claims 1 to 5 , wherein at least one of the colloidal particles is a quantum dot. 7. The method according to claim 1, wherein a carrier or a porous carrier having an average flow path diameter of 1000 μm or less is used for a part or all of the flow paths. The method according to any one of claims 1 to 7 , wherein the flow path after mixing of the directly bound particles and the indirectly bound particles is the same carrier up to the analyte detection site. 9. The method according to claim 1 , wherein at least one of the colloidal particles used is a latex particle. The method according to any one of claims 1 to 9 , wherein at least one of the colloidal particles used is a gold colloidal particle. The method according to any one of claims 1 to 10 , wherein the test substance is a substance of biological origin. The method according to any one of claims 1 to 11 , wherein the test substance is derived from an infectious microorganism. The method of claim 12, wherein the infectious microorganism is a virus. A kit comprising an immunochromatographic test piece, and direct-bound particles and indirect-bound particles for use in the method according to any one of claims 1 to 13 , wherein the immunochromatographic test piece is a carrier at which a carrier having a flow path for direct-bound particles and a carrier having a flow path for indirect-bound particles join, the carrier having the flow path for direct-bound particles and the carrier having the flow path for indirect-bound particles branch off upstream of the joining point, the direct-bound particles are colloidal particles having a modifier that binds to the test substance bound to their surface, the indirect-bound particles are colloidal particles having a modifier that binds to the modifier on the surface of the direct-bound particles and/or a modifier that binds to the modifier on the surface of other indirect-bound particles bound to their surface , and the kit wherein the direct-bound particles and indirect-bound particles form a complex on the carrier .