JP2020091285A - Subject substance detection sensitization method using colloidal particle - Google Patents

Abstract

To provide a signal sensitization method used for subject substance detection of a low concentration.SOLUTION: In the signal sensitization method, regarding colloidal particles binding to a subject substance directly or indirectly, the colloidal particles are bound to each other by a specific binding of a surface modification substance pair, or the operation is repeated, thereby increasing the number of colloidal particles and increasing the detection sensitivity. For detection, by direct optical detection of colloidal particle itself, or by optical, chemical, or electric detection of the subject using a reactant substance that is directly or indirectly bound to colloidal particle, the sensitivity can be increased.SELECTED DRAWING: None

Description

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

The dispersion stability of the hydrophobic colloidal particles is known as the Deryagin-Landau-Felway-Overbeek theory (DLVO theory) and is considered to be due to the electric double layer. On the other hand, the dispersion stability of hydrocolloid particles is considered to be due to steric hindrance due to hydration water.

Nano-sized colloidal particles are widely used for detection of test substances because of their optoelectronic properties, large surface area, and coloration found in metal colloids. Although there are many methods for detecting a test substance using colloidal particles (excluding molecular colloids), the colloidal particles can be bound directly or directly by binding them through an affinity substance for the test substance that modifies the surface of the colloidal particles. Most observe indirect signals. Therefore, when the concentration of the test substance is below a certain level, the sensitivity required for detection may not be obtained due to the shortage of the number of colloidal particles to be detected.

In various sensitization methods for detecting analytes (including those using molecular colloids) that do not use colloidal particles, specificity at each step poses a problem, so streptavidin-biotin binding, antigen-antibody reaction, and nucleic acid Hybridization and the like have been used. Signal sensitization is preferably simple and repeatable where possible. Hybridization of nucleic acids such as PCR and signal amplification for detection of a test nucleic acid by replication of a test substance satisfy this. However, in PCR and the like, a temperature change is essential for the amplification, and a dedicated device and a long reaction time are required. On the other hand, the signal amplification by the antigen-antibody reaction or streptavidin-biotin bond does not require special equipment or environment. These have been widely used when the test substance was a protein such as ELISA and Western blot, but most of them were only one sensitization step.

The detection system using particles has been used for observation with a microscope or an electron microscope, an agglutination reaction for an antigen, a lateral flow immunoassay method or a microchannel chip (see Patent Document 1) as an alternative thereto, blotting and the like. It was

As in Non-Patent Document 1, there is also a technique of amplifying a signal by combining streptavidin-biotin bonds and repeating the same. However, in the case where such a two-component system using molecular colloids is repeated, Many bind to the molecules used in the steps up to that point, reducing the amplification effect. This is considered to be a problem of signal amplification using molecular colloid.

Regarding Patent Document 2, a streptavidin-biotin bond is combined and sensitization is performed using qDOT particles. The size of qDOT is relatively small, and the types, sizes, and numbers of labeling molecules used for repeated sensitization are limited. Large molecules such as antibodies are not suitable. In the first step, a biotin-labeled antibody is used as the affinity substance for the test substance, but the subsequent increase is compared to the case where a large number of affinity substances are bound on the colloid particles larger than the affinity substance in this step. The size or number of substances/objects that can be used in the sensing step is limited. Furthermore, the test substance cannot be detected only by the first step, and even if the test substance has a relatively high concentration, the detection requires a plurality of steps.

JP 2017-78664 JP Japanese Patent No. 4654414

J Am Chem Soc. 2005 Jul 6;127(26):9328-9.

An object of the present invention is to provide a signal sensitization method used for detecting a low concentration of a test substance.

In the conventional method for detecting a test substance using colloidal particles, when the concentration of the test substance is low, sufficient sensitivity cannot be obtained. Therefore, the present inventors have conducted extensive studies on a method for increasing the sensitivity. ..

The present inventors have made it possible to bind colloid particles directly or indirectly to a test substance by specific binding of surface-modifying substance pairs to each other, or by repeating this process. Invented a method for increasing the detection sensitivity by increasing the number of colloidal particles. For the detection, the direct optical detection of the colloidal particles themselves or the reaction substance directly or indirectly bound to the colloidal particles is used to detect the test substance optically, chemically or electrically. It was found that the sensitivity can be increased.

In the conventional method of repeatedly binding molecular colloids, for example, assuming that a combination of reactants (A and B) are bound to each other, A and/or B must be present as a polymer in order to be bound alternately. , It was necessary to alternately add an excessive amount of A or B. Alternatively, when substances other than A and B are used one after another, these substances can be combined one after another (A to B, B to C, C to D, and so on). There have been cases where the combinations of substances are limited, and there are problems with the affinity or binding specificity between these substances. However, such problems can be avoided by binding such reactants on colloidal particles that are sufficiently larger than the reactants. For example, the colloidal particles bound with the reactant A and the colloidal particles bound with the reactant B can be considered as giant polymers of A or B, respectively. When these are applied alternately, unreacted A or B always remains on the particles in each step, so that bonding in the next step is possible. Alternatively, a combination of substances can be attached to the same colloidal particle. This makes it possible to create a combination of colloidal particles having high binding affinity and binding specificity. Furthermore, since the reactant is adsorbed or immobilized on the colloid particles at a much higher concentration than in the state of molecular colloid (pre-concentration), a high reaction rate is obtained on the surface of the colloid particles.

That is, the present invention is as follows.
[1] A method for detecting a test substance using colloidal particles to which a substance having an affinity for the test substance is bound, comprising the steps of (i) A method of increasing the sensitivity of detection of a test substance, which comprises the step iii) and the detection sensitivity of the test substance is increased by the formation of a complex of colloidal particles added in each step:
(i) BS (Binding Step) step of directly or indirectly binding the test substance and the first colloidal particles;
(ii) SS (Sensitizing Step) step of adding n times the sensitized modified colloidal particles (MC) to which the surface modification substance for binding the colloidal particles is added to the complex of the test substance and the first colloidal particle [ n is a natural number], and the modified colloid particles for sensitization (Modified colloid particles) MC(m) added in the mth SS(m) step are colloidal particles C(m) and surface modification substances M for binding colloidal particles. (m) are bound to each other, and the colloidal particles MC(m+1) added in the SS(m+1) step are colloidal particles C(m+1) with a surface modifier M(m+1) for binding colloidal particles. ) Are bound together, M(m) and M(m+1) have affinity, and MC(m) and MC(m+1) are bound to M(m) and M(m+1). Step of forming a complex through [m is a natural number and m+1≦n. ]; and
(iii) A DS (Detecting Step) step of detecting a test substance by detecting a signal generated directly or indirectly from the formed colloidal particles.
[2] A method for detecting a test substance using colloidal particles to which a substance having an affinity for the test substance is bound, which is a method for increasing the sensitivity of detection of the test substance. A method of increasing the sensitivity of detection of a test substance, which comprises the step iii) and the detection sensitivity of the test substance is increased by the formation of a complex of colloidal particles added in each step:
(i) Test substance and first colloidal particle [The surface-modified colloidal particle is referred to as MC(0), and the surface modifier M(0) is bound to the colloidal particle C(0). BS (Binding Step) step of directly or indirectly binding
(ii) SS (Sensitizing Step) step of adding n times the sensitized modified colloidal particles (MC) to which the surface modification substance for binding the colloidal particles is added to the complex of the test substance and the first colloidal particle [ n is a natural number], and the modified colloid particles for sensitization (Modified colloid particles) MC(m) added in the mth SS(m) step are colloidal particles C(m) and surface modification substances M for binding colloidal particles. (m) are bound together, colloidal particles MC(m-1) added in the previous step [SS(m-1) step if m>1 and BS step if m=1] Is a surface modification substance M(m-1) for binding colloidal particles to colloidal particles C(m-1).M(m-1) and M(m) have an affinity and MC(m-1) -1) and MC(m) form a complex through the bond of M(m-1) and M(m) [m is a natural number and m≤n. ]; and
(iii) A DS (Detecting Step) step of detecting a test substance by detecting a signal generated directly or indirectly from the formed colloidal particles.
[3] The method of [1] or [2], which detects or measures a direct signal of colloidal particles in at least one of the BS step and/or the SS step.
[4] The method according to any one of [1] to [3], wherein the colloidal particles have a diameter of 10 nm or more.
[5] The method according to any one of [1] to [4], wherein n is 1 to 10.
[6] The method according to any one of [1] to [5], wherein in the BS step, the test substance is bound to the insoluble carrier with or without the affinity substance for the test substance.
[7] The method according to any one of [1] to [6], wherein in the BS step, the test substance and the first colloidal particles to which a substance having an affinity for the test substance is bound are bound.
[8] The method according to any one of [1] to [7], in which the colloidal particles used in the SS step are composed of various types.
[9] The modified colloidal particles MC(n) for sensitization to which the surface modifier for bonding colloidal particles used in the SS step is added are the modified colloidal particles MC(m) and m for sensitization added in the m-th SS step. The method according to any one of [1] to [8], which comprises two types of modified colloidal particles MC(m+1) for sensitization added in the +1st SS step.
[10] The colloidal particles used in the SS step are composed of two types, colloidal particles C(m) used in the mth SS step and colloidal particles C(m+1) used in the m+1th SS step. The method according to any one of [1] to [9].
[11] The method according to any one of [1] to [7], wherein the colloidal particles used in the SS step are composed of one type.
[12] First colloidal particles to which a substance having an affinity for the test substance used in the BS process is bound, and SS(m) [m is an even number] modified sensitized colloidal particles MC(m) used in the process Are the same, any one of [1] to [7].
[13] The modified sensitizing colloidal particles MC(1) used in the SS(1) step and the sensitizing modified colloidal particles MC(m) used in the SS(m) [m is an odd number] step are the same. The method according to any one of 1] to [7] and [12].
[14] The method according to any one of [1] to [13], wherein the combination having the affinity of the surface modifier M(m) for binding colloidal particles and M(m+1) is a combination of an antigen and an antibody.
[15] The method according to any one of [1] to [14], wherein the test substance is a biological substance.
[16] The method according to any one of [1] to [14], wherein the test substance is a substance derived from an infectious microorganism including a virus.
[17] The method according to any one of [1] to [16], wherein an enzymatic reaction is used in the DS step which is the detection step.
[18] The method according to any one of [1] to [17], wherein the test substance detection method is an immunochromatography method.
[19] Colloidal particles to which at least a substance that binds to a test substance is bound and colloidal particle binding to be used in n SS steps [n is a natural number] for use in any of the methods of [1] to [18] It is a kit containing modified colloidal particles for sensitization (MC) combined with a surface modification substance for modification, and modified colloidal particles for modified sensitization (MC(m)) added in the mth SS(m) step are The surface modifier M(m) for binding the colloidal particles is bound to the colloidal particles C(m).The colloidal particles MC(m+1) added in the SS(m+1) step are the colloidal particles C(m+ The surface modifier M(m+1) for binding colloidal particles is bound to 1), and M(m) and M(m+1) have affinity, and MC(m) and MC(m+1) ) Is a kit that forms a complex through the binding of M(m) and M(m+1).
[20] The kit of [19], which is a kit for immunochromatography.

The colloidal particles are bound to each other through the binding between the colloidal particle surface modifier pairs. By repeating the binding between the colloidal particles, the detection sensitivity of the test substance increases exponentially.

It is a figure which shows the outline of the method of this invention, and is a figure which shows the outline of the method of using two types of colloidal particles. It is a figure showing the outline of the method of the present invention, and is a figure showing the outline of the method of using many kinds of colloid particles. It is a figure which shows that the gold colloid particle has couple|bonded with the latex particle through the antigen-antibody reaction of surface modification substances. It is a figure which shows that the latex particle has couple|bonded with the gold colloid particle through the bond between biotin and streptavidin. It is a figure which shows that the influenza detection result by the immunochromatography kit for influenza virus detection using latex particles is sensitized by the gold colloid particles. It is a figure which shows the sensitization result of BS process, SS(5) process, and SS(10) process. It is a figure which shows the sensitization result at the time of making a hydrophilic colloid particle couple|bond together via the surface modifier for colloid particle bonding. It is a figure which shows the result of having sensitized using two types of colloidal particles in SS(1) process. It is a figure which shows the result which used the luminescence reaction by ECL(enhanced chemiluminescence) for the detection process (DS process).

The present invention provides a method for detecting a test substance in a specimen using colloidal particles bound to a test substance, which is a sensitizing method for increasing the detection sensitivity of a test substance using colloidal particles for sensitization. is there.

In the method of the present invention, the analyte and the colloidal particles are first bound, and further the sensitizing colloidal particles are bound to form a complex of the colloidal particles. The colloidal particle is a colloidal particle which can directly or indirectly bind a colloidal particle and/or a detection reaction substrate capable of directly detecting a signal for detecting a test substance, or is a bound colloidal particle. The binding increases the number of colloidal particles in the complex formed by the colloidal particles, and the signal intensity increases according to the number of colloidal particles.

The method of the present invention includes n times of sensitization step SS(n) step of adding colloidal particles, the mth sensitization step SS(m) step colloidal particles and the m+1th sensitization step SS( The colloidal particles in the step (m+1) are bound to each other through the modifier on the surface of the colloidal particles to form a complex. The colloidal particles used in the sensitizing step SS(m) step and SS(m+1) step are represented by C(m) and C(m+1), respectively.

By repeatedly causing the binding between the colloidal particles, the number of colloidal particles contained in the complex increases, and as a result, the detection of the test substance can be directly or indirectly sensitized.

1. Colloid Particles Examples of colloid particles used in the present invention include hydrophilic colloid particles such as latex particles, metal colloid particles, and hydrophobic colloid particles such as silica colloid particles. Hydrophobic colloid particles also include particles to which protective colloid is bound. Here, the protective colloid is a hydrocolloid and refers to a colloid that surrounds the hydrophobic colloid to protect the hydrophobic colloid. By the protection, it is possible to prevent the coagulation of the colloidal particles due to the change of the solution due to the heating or the addition of salts. Further, the hydrophobic colloid particles may be particles that cause surface plasmon resonance. Light near a specific wavelength is absorbed by surface plasmon resonance. The latex particles include colored latex particles and latex particles containing a fluorescent dye. Latex particles refer to particles that form an emulsion that is colloidally dispersed in water. Although the material of the particles is not limited, it is possible to use a material that is used as a material for a solid phase carrier that binds proteins such as antibodies, antigens, ligands and receptors in the technical field of test agents. Examples thereof include polystyrene, styrene copolymers such as styrene-acrylic acid copolymers, polycarbonate, polymethylene methacrylate (PMMA), resins such as polyvinyltoluene, silica, and cellulose. Of these, particles based on styrene are preferred. The styrene-based particles refer to particles made of a copolymer of polystyrene, styrene, or a derivative of styrene and a polymerizable unsaturated carboxylic acid, a polymerizable unsaturated sulfonic acid, or the like. Examples of the styrene derivative include chloromethylstyrene and divinylbenzene, the polymerizable unsaturated carboxylic acid includes acrylic acid and methacrylic acid, and the polymerizable unsaturated sulfonic acid includes styrene sulfonic acid soda. Is mentioned. In the present invention, latex particles based on styrene are referred to as polystyrene latex particles. The diameter of the latex particles is several tens nm to several hundreds nm, preferably 50 nm to 800 nm, more preferably 200 to 600 nm. The metal colloidal particles may be of any type and shape such as metal, alloy, metal oxide, metal compound and the like, and examples thereof include gold colloidal particles. Further, it also includes a core-shell type in which one metal colloid particle is coated with one layer of silica or the like. Furthermore, composite particles in which particles are bonded in various forms can also be used. The diameter of the metal colloid particles is 10 to 200 nm, preferably 10 to 100 nm, more preferably 10 to 50 nm. Whatever the colloidal particles, the size of the colloidal particle surface modifier is similar to that of the molecule, or it is necessary to use a sufficiently large particle in order to leave unreacted colloidal particle surface modifier due to steric hindrance. preferable. For example, when an antibody is used as a molecule of the colloidal particle surface modifier, the diameter of the colloidal particle is preferably 10 mm or more.

Colloid particles are used in the BS and SS steps, but it is not always necessary to directly detect the signal. Detecting the signal of the colloidal particles directly includes detecting the colored substance or the metal colloidal particles themselves, including optical observation (coloring, fluorescence, phosphorescence, luminescence, absorption) of the particles themselves. The indirect signal detection means that the detection of the colloid particles themselves is not used for the detection of the test substance. For indirect signal detection, a chemical reaction using a substrate can be used. For example, by directly binding peroxidase to the surface of the colloidal particles, or indirectly binding an enzyme such as streptavidin-conjugated horseradish peroxidase via biotin bound to the surface of the colloidal particles, the final coloration or chemical The test substance can be detected by luminescence. Further, even if colloidal particles capable of directly detecting a signal are used, it is possible to further indirectly detect a signal when a low-concentration test substance is not detected (see Example 7). Regardless of whether the final detection method of the test substance is direct or indirect, in each step including the BS step, the presence or absence of a direct signal from the colloid particles is confirmed before proceeding to the next step. Preferably. Accordingly, when the test substance has a relatively high concentration, it may be possible to detect or measure the test substance in any step up to the DS step. Alternatively, it may be possible to reduce the test cost by judging the necessity of the subsequent steps and, if unnecessary, if unnecessary.

In the method of the present invention, colloidal particles to which a surface modifier for binding the colloidal particles is bound are used. In the present invention, the "surface modifier for binding colloidal particles" is referred to as the "surface modifier for binding colloidal particles". The colloidal particles to which one of these surface modifying substances for binding colloidal particles is bound can be bound to the colloidal particles to which the other one is bound via the surface modifying substance for binding colloidal particles. Sensitization step The colloidal particle binding surface modifier bound to the colloidal particles used in the SS(m) step and SS(m+1) step is represented by M(m) and M(m+1), respectively.

In addition, colloidal particles to which a surface modifier for binding colloidal particles is bound are called modified colloid particles for sensitization, and are used in the sensitization process SS(m) and SS(m+1) processes. The modified colloidal particles for use are represented by MC(m) and MC(m+1), respectively.

The colloidal particles in which the surface modifier M(m) for binding colloidal particles is bound to the colloidal particles C(m) are the modified sensitizing colloidal particles MC(m).

One kind of surface modifying substance for bonding colloidal particles may be bonded to one colloidal particle, or plural kinds of surface modifying substances for bonding colloidal particles may be bonded. The combination of surface modifiers for binding colloidal particles, which are involved in the binding between the surface modifiers for binding colloidal particles, is the nth SS(n) step and the n-1th SS(n- In step 1), they may be the same or different.

In the sensitization step SS(m), the number of colloidal particles C(m) may be one type or multiple types. The multiple types refer to two or more types, for example, three types, four types, five types, six types, seven types, eight types, nine types, or ten types. For example, sensitization step SS(m) for both latex particles and gold colloid particles modified with M(m), modified with M(m) capable of binding to surface modifier M(m-1) for binding colloidal particles. Can be used in. At this time, the structure of M(m) for each particle is not necessarily the same as long as it can bind to M(m-1). When n is a natural number and the sensitization step is performed n times, particles having different sizes can be used in combination in any step from SS(1) to SS(n). At this time, the combined use does not necessarily have to be a mixture, and for example, infiltrating large particles first and then infiltrating small particles can be one step.

Examples of the combination of two kinds of surface modifiers for binding colloidal particles include a combination of an antigen and an antibody. Not only a combination of an antibody and an antigen, but also a combination of a ligand and a receptor or a combination of a receptor and a ligand, for example. Good. Examples of such an affinity substance include a polypeptide capable of binding to a test substance and other compounds. Specific examples include a combination of avidin and biotin, a combination of streptavidin and biotin, a combination of tamavidin and biotin. Biotin, avidin, streptavidin, tamavidin, etc. may be directly bound to the colloid particles, or a conjugate with a substance that binds to the test substance may be prepared and the conjugate may be bound to the colloid particles. The biotin may be maleimide biotin having a maleimide group or the like. It is preferable that a non-specific reaction is suppressed on the surface of the colloid particles by binding a blocking agent in addition to the surface modifier for binding the colloid particles. In some cases, the blocking agent can also serve as the surface modifier for binding colloidal particles. For example, biotinylated BSA can be used as a blocking agent and a surface modifier for binding colloidal particles, and streptavidin can be bonded as a surface modifier for binding colloidal particles in the next step. Alternatively, a substance that contributes to the DS process can be used as a surface modifier other than the purpose of binding to colloidal particles. For example, when M(m) (m is a natural number) is streptavidin, biotinylated horseradish peroxidase (HRP)-bonded colloidal particles are used as MC(m+1), and biotin is used as a surface modifier for binding the colloidal particles. , And HRP can be made to function when a color reaction using TMB or a luminescence reaction using ECL is used in the DS step.

As for the surface modifying substances M(m) and M(m+1) for binding colloidal particles in the two subsequent steps, at least one combination of substances having affinity for each other is used. The surface modifier M(m) for binding colloidal particles in each step may be one kind or many kinds, but is preferably one kind. Total sensitization process from SS(1) process to SS(n) process Surface modification substance (group) (M(1) to M(n)) for binding colloidal particles used in SS(1) to SS(n) processes The composition may be one kind or multiple kinds, but two kinds are alternately used in each step (in any integer m of 0 or more, M(m) and M(m+2) are the same substance or composition. Preferably).

As a method for immobilizing the surface modifier on the colloidal particles, physical bonding, electrical bonding, chemical bonding or the like can be used.

2. Test Substance and Specimen The test substance may be any substance such as nucleic acid, protein, sugar, or other compound, and may be a substance of biological origin. Further, it may be a complex thereof, for example, a pathogenic microorganism such as a bacterium or a virus, or a substance in the living body or the environment.

As the sample, an aqueous phase sample or a diluted solution of the sample with a buffer solution can be mainly used. Examples of the sample include serum, plasma, blood, urine, saliva, interstitial fluid, spinal fluid, pharyngeal or nasal swab, pharyngeal or nasal lavage fluid, body fluids such as nasal aspirate, feces, fecal suspension, and culture fluid. Be done.

3. Method for Increasing Detection Sensitivity The method of the present invention comprises a step of binding colloidal particles and a test substance, a step of binding colloidal particles to each other to sensitize the reaction, and detecting a signal from the colloidal particles. The step of detecting a substance is included.

In the present invention, the step of binding the colloidal particles and the test substance is called the BS (biding step) step, the step of binding the colloidal particles to each other to sensitize the reaction is called the SS (sensitizing step) step, and The step of detecting a test substance by detecting a signal is called a DS (detecting step) step. The SS step is expressed as SS(n) [n represents a natural number], and the step of adding the nth colloidal particle to form a complex is the nth sensitization step. ) Call it a process. The SS step of binding the colloidal particles to each other to sensitize the reaction may be performed once, but is preferably repeated twice or more. The upper limit is not limited, but is preferably 10 times. That is, n is 1 to 10 in the SS(n) step.

In the BS process, the first colloidal particles and the test substance are mixed and bonded. Any colloidal particles in the BS process may be used as long as they can be directly or indirectly bound to the test substance. Here, directly binding the test substance to the colloid particles means binding a substance having an affinity for the test substance to the surface of the colloid particles and binding the substance to the test substance. Indirectly binding the substance to the colloidal particles means binding the test substance to the substance bound to the surface of the colloidal particles via another substance. For example, another substance A is bound to the test substance by pretreatment of the sample, and the test substance is bound to the colloidal particles to which the substance B having an affinity for the substance A is bound. Preferably, a substance having an affinity for the test substance is bound to the surface and directly modified using the modified colloidal particles. That is, the first colloidal particles are bound with a colloidal particle binding surface modifying substance for binding the colloidal particles together with a substance that directly or indirectly binds to the test substance. The surface modification substance for binding colloidal particles is the same as the substance that binds to the test substance, and the surface modification substance for binding colloidal particles may also serve as an affinity substance that binds to the test substance. The substance having an affinity for the test substance and the surface modifier for binding to the colloidal particles, which is bound to the colloidal particles, may be referred to as a surface modifier. The affinity substance for the test substance is not limited as long as it is a substance that binds to the test substance, for example, when the test substance is an antigen, it is an antibody against the test substance, when the test substance is an antibody, An antigen to which an antibody binds.

Then, the second colloidal particles are added as sensitized modified colloidal particles to which a surface modifying substance for bonding colloidal particles is bound. To the second colloidal particles, another colloidal particle-bonding surface modifying substance that is bonded to the colloidal particle-bonding surface modifying substance of the first colloidal particle is bonded. As a result, the first colloidal particles and the second colloidal particles are bound to each other by the binding of the surface modifiers for binding the colloidal particles to form a complex. This process is called the SS(1) process.

In the method of the present invention, the third colloid particle, the fourth colloid particle, the mth colloid particle, the (m+1)th colloid are further processed in n sensitization steps. The particles are added as sensitizing colloidal particles, and the mth colloidal particle and the (m+1)th colloidal particle are bonded by bonding the surface modifiers for bonding the colloidal particles to each other to form a composite of colloidal particles. Increase the size. Here, m represents a sensitizing process in an arbitrary order among n sensitizing processes, m is a natural number, and m+1≦n. The second and subsequent colloidal particles, that is, the colloidal particles used in the SS(n) step, are referred to as sensitized modified colloidal particles because they are used for sensitization because they have surface-modifying substances for binding colloidal particles. be able to. Further, the sensitizing method of the present invention can be carried out in one stage or in multiple stages, but when performing sensitization in multiple stages, it can be called a multi-stage sensitizing method. The modified colloidal particles for sensitization used in the SS(m) step, which is the m-th sensitization step, are represented by MC(m). The surface modifier for binding the colloidal particles bound to the sensitized modified colloidal particles is represented by M(m). Two kinds of substances may be used as the surface modifying substance for binding the colloidal particles, which is bound to the colloidal particles used in each step, or different substances may be used in each step.

In the method of the present invention, the number of kinds of colloidal particles is not limited, and one kind or two or more kinds of colloidal particles can be used. The colloidal particles used in each step may be the same or different. When two kinds of colloidal particles are used, the colloidal particle C(n) (n is an integer of 0 or more) and the colloidal particle C(n+2) used in the next and subsequent sensitization step may be the same. In this case, hydrophilic colloid particles such as latex particles and hydrophobic colloid such as metal colloid may be alternately used to bond the hydrophilic colloid particles and the hydrophobic colloid particles together. Further, hydrophilic colloids such as latex particles may be bound to each other to form a complex. Also, the same colloidal particles as the first colloidal particles used in the BS step may be used in the SS step.

In the BS step of binding the first colloidal particles and the test substance, the test substance is adsorbed/fixed/bound to the insoluble carrier, which binds to the test substance or binds to the test substance. Alternatively, the colloidal particles and the test substance may be combined in a liquid system without using such an insoluble carrier. The test substance may be directly or indirectly bound to the insoluble carrier. Examples of the insoluble carrier include synthetic resins such as ELISA plates, other colloidal particles, and immunochromatographic test pieces for immunochromatography. Further, a gel containing a band of a specific substance obtained by electrophoresis or Western blotting, a membrane to which a substance is transferred, or the like may be used. It is also possible to utilize hybridization between the immobilized nucleic acid and the nucleic acids bound to the colloidal particles. When an insoluble carrier is used, a complex of colloidal particles is formed on the insoluble carrier, and the size of the complex increases as the number of steps represented by n in the SS(n) step progresses. When an insoluble carrier is not used, a complex of colloidal particles is formed in the liquid system, and the size of the complex increases as the number of steps represented by n in the SS(n) step progresses. In either case, in the DS step, the signal intensity generated from the colloidal particles increases according to the number of colloidal particles forming the complex, and the detection sensitivity of the test substance increases depending on the number of sensitization steps.

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

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 fluorescence intensity with a fluorescence measuring device.

When the colloidal particles are particles to which an enzyme such as alkaline phosphatase or horseradish peroxidase is bound, the signal generated from the colloidal particles is the color development at the specific wavelength of the specific substance produced by the enzymatic reaction, and the intensity of this color development. Can be measured with a spectrophotometer or the like to measure the signal intensity.

When two kinds of colloidal particles are used, and each colloidal particle is a combination of an energy donor and an energy acceptor for fluorescence resonance energy transfer, the colloidal particles form a complex and the colloidal particles are densely packed. As a result, a change in fluorescence resonance energy transfer (FRET) occurs, and the intensity of the signal can be measured by measuring this change as a signal with a fluorescence measuring device.

Hereinafter, an example of the method of the present invention will be described with reference to the drawings. The method shown in FIG. 1 is a case where two types of colloidal particles are used and an insoluble carrier 1 to which a substance that binds to the test substance 2 is bound is used. That is, this is the case where the test substance 2 is bound to the insoluble carrier 1. When a sample containing the test substance is added, the substance that binds to the test substance 2 on the insoluble carrier 1 binds to the test substance 2 and is captured. In the method shown in FIG. 1, the surface modifying substance for binding colloidal particles is the same as the substance that binds to the test substance, and the surface modifying substance for binding the colloidal particles also serves as the substance that binds to the test substance.

In FIG. 1, as the colloidal particles, the first colloidal particles represented by a black circle and the second colloidal particles represented by a white circle are used. Here, the nth colloidal particles mean the nth colloidal particles added to the reaction system. In the case shown in FIG. 1, the first colloidal particles are colloidal particles having an affinity substance for the test substance bound thereto. It is also used as a sensitizing colloidal particle. To the first colloidal particles, only one type of colloidal particle-binding surface modifier that binds to the test substance is bound. The surface modifier for binding the colloidal particles also serves as an affinity substance for the test substance. Only the substance that binds to the substance that binds to the test substance that has bound to the first colloid particle is bound to the second colloid particle. That is, there is only one type of colloidal particle binding surface modifier that binds to one type of colloidal particle. The substance that binds to the substance that binds to the test substance may be the test substance itself or a substance whose structure is partially or wholly similar to the test substance. As the substance having a structure similar to that of the test substance, for example, when the test substance is a polypeptide or a protein, a substance having an amino acid sequence highly homologous to a partial amino acid sequence of the test substance can be mentioned.

First, in the BS step, the first colloidal particles 5 bind to the test substance 2 captured by the substance on the carrier 1. At this time, a complex represented by the test substance 2: the first colloidal particles 5 (“:” indicates binding) is formed.

Next, in the SS(1) step, the second colloidal particles 8 (MC(1)) which are the modified colloidal particles for sensitization are added. As a result, the second colloidal particles 8 (MC(1)) are bonded to the first colloidal particles 5. The binding between the colloidal particles is performed by the surface modification substance 4 for binding the colloidal particles of the first colloidal particle 4 and the surface modification substance 7 for binding the colloidal particles of the second colloidal particle 8 (MC(1)) (M(1 )). In the SS(1) step, a complex in which the first colloidal particles 5 and the second colloidal particles 8 (MC(1)) are bound is formed. At this time, a complex represented by test substance 2: first colloidal particle 5: second colloidal particle 8 (MC(1)) (“:” indicates binding) is formed.

Next, in the SS(2) step, the same colloidal particles as the first colloidal particles 5 are added as the third colloidal particles 11 (MC(2)) which are the modified sensitizing colloidal particles. Further, in the SS(3) step, the same colloidal particles as the second colloidal particles 8 (MC(1)) are added as the fourth colloidal particles 14 (MC(3)) which are the modified colloidal particles for sensitization. To do. As a result, as shown in SS(3) of FIG. 1, test substance 2: first colloid particle 5: second colloid particle 8 (MC(1)): third colloid particle 11( MC(2)): A large complex containing two kinds of colloidal particles is formed, which is represented by the fourth colloidal particle 14 (MC(3)) (":" indicates a bond). A signal is generated according to the number of colloid particles, and the test substance can be detected by measuring the signal intensity. As the number of SS(1) step, SS(2) step,... SS(n) step and the number of sensitization steps for a certain amount of the test substance increases, the complex becomes larger and is included in the complex. Since the number of colloidal particles generated increases and the resulting signal also increases, the measurement sensitivity of the test substance is enhanced.

Although FIG. 1 shows the SS(3) step, the number of times the SS step is repeated is not limited. In the method shown in FIG. 1, the first colloidal particles used in the BS step and the sensitized modified colloidal particles MC(m) used in the SS(m) [m is an even number] step are the same, and the SS(1) step The sensitized modified colloidal particles MC(1) and the sensitized modified colloidal particles MC(m) used in the SS(m) [m is an odd number] step are the same.

FIG. 2 shows a method using a large number of three or more kinds of colloidal particles, in which one kind of colloidal particle is combined with one kind of surface-modifying substance for bonding colloidal particles.

First, in the BS step, the first colloidal particles 5 bind to the test substance 2 captured by the substance on the carrier 1.

Next, in the SS(1) step, the second colloidal particles 8 (MC(1)) which are the modified colloidal particles for sensitization are added. As a result, the second colloid particles 8 (MC(1)) are bonded to the first colloid particles. The bonding between the colloidal particles is performed by the surface modification substance 4 for bonding the colloidal particles of the first colloidal particle 4 and the surface modification substance 7 for bonding the colloidal particles of the second colloidal particle 8 (CM(1)) (M(1 )). In the SS(1) step, a complex in which the first colloidal particles 5 and the second colloidal particles 8 (MC(1)) are bound is formed.

Next, in the SS(2) step, the third colloidal particles 17 (MC(2)), which are modified colloidal particles for sensitization, are added. The third colloidal particles 17 (MC(2)) added here are different from the first colloidal particles 5 and the second colloidal particles 8 (MC(1)) in surface modification for binding to colloidal particles. Material 15 (M(2)) is bound. The third colloidal particle 17 (MC(2)) binds to the second colloidal particle 8 (MC(1)). The third colloidal particle 17 (MC(2)) and the second colloidal particle 8 (MC(1) are bound to each other by modifying the surface of the third colloidal particle 17 (MC(2)) for binding to the colloidal particle. It occurs through the binding of the substance 15 and the surface modification substance 7 for binding the colloidal particles of the second colloidal particles 8 (MC(1)).

Further, in the SS(3) step, the fourth colloidal particles 20 (MC(3)) which are the modified colloidal particles for sensitization are added. The fourth colloidal particles 20 (MC(3)) added here include the first colloidal particles 5, the second colloidal particles 8 (MC(1)) and the third colloidal particles 17 (MC(3)). A surface modifier 19 (M(3)) for binding colloidal particles different from MC(2)) is bound. The fourth colloidal particle 20 (MC(3)) binds to the third colloidal particle 17 (MC(2)). The bonding between the fourth colloidal particle 20 (MC(3)) and the third colloidal particle 17 (MC(2)) is the surface for bonding the fourth colloidal particle 20 (MC(3)). It occurs through the binding of the modifier 19 (M(3)) and the surface modifier 15 (M(2)) for binding the colloidal particles of the third colloidal particles 17 (MC(2)). As a result, as shown in SS(3) of FIG. 2, a large complex containing the first colloid particle 5 to the fourth colloid particle 20 (MC(3)) is formed. A signal is generated according to the number of colloid particles, and the test substance can be detected by measuring the signal intensity. The colloid contained in the complex becomes larger as the number of S(1) process, S(2) process,... Since the number of particles increases and the resulting signal also increases, the measurement sensitivity of the test substance is enhanced.

The reaction solution used in the method of the present invention does not inhibit the binding generated in the BS step, does not affect the reaction result of the BS step, and may be anything as long as the reaction between the colloidal particle surface modifiers specifically occurs, It is preferable to use a buffer solution. You may add salt, surfactant, alcohol etc. to a buffer solution.

In each BS step and SS(n) step, it is preferable to perform washing for the purpose of removing colloidal particles that could not be bound between the steps.

Although it is possible for a human (examiner) to perform each step of sensitization, it is preferable to perform multi-step sensitization. Examples include an automatic analyzer, a syringe pump, an immunochromatography method in which a channel is devised, and a mode using a capillary phenomenon such as a microchannel chip. The micro-channel chip refers to a micro-fluidic device having a micro-channel manufactured by using a microfabrication technology inside a member, and includes a two-liquid transport system using a pump or the like and an interaction system by piston transport. Even when these are used, a two-component reaction using particles used for SS(n) and colloidal particles used for SS(n+1), or a three-component reaction in which a cleaning liquid used for cleaning is added Can be In the method of the present invention, it is preferable to reduce the number of types of colloidal particles and the number of reagents used. This makes it possible to downsize the device and simplify the operating procedure. It is also possible to set an amplification factor that indicates how much sensitization can be performed for each repeating unit represented by SS(n).In this case, it is also possible to provide by using the sensitization factor. is there.

The present invention also includes reagents and kits for increasing the detection sensitivity of an object to be detected to detect the object to be detected. The kit is a reagent or kit for use in the above sensitization method.

The kit includes at least colloidal particles to which a substance that binds to a test substance is bound, and colloidal particles for sensitization. The colloidal particles for sensitization include colloidal particles MC(m) for sensitization added in the SS(m) step, which is the m-th sensitization step, and SS(m+1 The colloidal particles MC(m+1) added in the step (2) may be the same colloidal particles or different colloidal particles. The surface modifiers M(m) and M(m+1) for binding colloidal particles are bound to the sensitizing colloidal particles MC(m) and MC(m+1), respectively.

The kit is, for example, an immunochromatography kit, and the substance that binds to the test substance bound to the colloidal particles is an antibody or an antigen that binds to the test substance by an antigen-antibody reaction. The kit further includes a test piece for immunochromatography, a blusher, a buffer solution and the like. The colloidal particles to which a substance that binds to the test substance is bound may be contained as a reagent different from the immunochromatographic test strip, or may be contained in the labeling site of the immunochromatographic test strip. ..

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

<Example 1>
Test method An anti-mouse antibody was used as a test substance. This example is an example in the case where a test substance is bound to an insoluble carrier.

Immunochromatography was carried out using a membrane in which 1 microliter of 10 mg/ml anti-mouse antibody was impregnated in a dot shape and dried.

In the first liquid, latex colloidal particles (blue) (BS process developing solution) to which a mouse-derived anti-norovirus antibody was bound (immobilized) as a surface-modifying substance for particle binding were added to the membrane. This process is a BS process. Subsequently, gold colloidal particles (red) (SS(1) step developing solution) to which a partial antigen peptide against the mouse-derived anti-Norovirus antibody was bound as a surface-modifying substance for particle binding were added in the second liquid. This process is the SS(1) process. As a negative control, one excluding each colloidal particle was used.

Test Results As shown in FIG. 3, a complex represented by anti-mouse antibody: latex particle bound with mouse-derived anti-norovirus antibody: gold colloid particle bound with partial antigen peptide (“:” indicates binding). Purple dots were detected indicating the formation of

Discussion From the results of this example, it was shown that the antigen-antibody reaction between the particle-bonding surface-modifying substances of the colloidal particles can be used for binding the colloidal particles together.

It was also shown that the partial antigen peptide for the mouse-derived anti-norovirus antibody used in the BS step can be applied to the colloidal particles used in the SS(1) step.

The purple dots showed that the gold colloid particles were bonded to the latex particles, but the gold colloid particles did not aggregate (with a change in absorption spectrum due to an irreversible aggregation reaction).

<Example 2>
Test Method A substance obtained by binding biotin to bovine serum albumin (BSA) through a maleimide group (hereinafter, biotinylated BSA) was used as a test substance. This example is an example in the case where a test substance is bound to an insoluble carrier.

Immunochromatography was carried out using a membrane in which 1 microliter of 10 mg/ml biotinylated BSA was impregnated in a dot shape and dried.

Gold colloidal particles (red) (Streptavidin-bonded surface-modifying substance for particle binding) (BS process developing solution) were added to the membrane in the first liquid, and then maleimidobiotin was used as a surface-modifying substance for particle bonding in the second liquid. The latex colloidal particles (blue) (SS(1) step developing solution) bound as above were added. As a negative control, one excluding each colloidal particle was used.

Test results As shown in FIG. 4, formation of a complex (“:” indicates binding) represented by colloidal gold particles bound with biotinylated BSA:streptavidin antibody:latex colloidal particles bound with maleimidobiotin was confirmed. The purple dots shown were detected.

Consideration From the results of this example, it was shown that the binding between the colloidal particles can be achieved by using the avidin-biotin binding between the particle-binding surface modifiers bound to the colloidal particles.

<Example 3>
Test method
Using a membrane in which 1 μl of 10 mg/ml anti-influenza virus antibody was bound in a dot shape (immobilized), latex particles (blue) and streptavidin bound to another biotinylated anti-influenza virus antibody were bound to this membrane The inactivated influenza virus was detected and sensitized using the sensitized colloidal particles (red). After the inactivated influenza virus was mixed with the latex particle suspension, the strip containing the membrane was immediately impregnated. This detection step corresponds to the BS step. Next, sensitization up to the SS(1) step was performed using the sensitizing colloidal particles. Specifically, colloidal gold particles (red) (SS(1) step developing solution) to which streptavidin was bound as a surface-modifying substance for particle binding in the SS(1) step were added subsequent to the BS step. Color development obtained after the SS(1) step of the sample solution that does not contain the influenza virus VLP that is the test substance in the BS step (negative control), and the color development and SS after the BS step when using the sample solution containing VLP (1) The color development after the process was compared.

Test results As shown in FIG. 5, only in the presence of the test substance, influenza virus antibody: influenza virus VLP: biotinylated influenza virus antibody-conjugated latex colloid particles: complex represented by gold colloid particles conjugated with streptavidin Purple dots were detected indicating the formation of bodies (":" indicates binding).

Discussion It was shown that the present invention is useful for sensitization of the immunochromatographic method for detecting influenza virus as a test substance. Further, in this example, colloids of different colors were used in the BS step and the SS(1) step in order to prove the binding between the colloid particles, but by using colloids of similar colors and fluorescent particles, the same kind of A stronger signal is expected to result in a higher sensitizing effect.

<Example 4>
Test method Streptavidin was used as a test substance. This example is an example in the case where a test substance is bound to an insoluble carrier.

Multistep sensitization was performed by immunochromatography using a membrane in which 1 microliter of a solution containing 1000, 100, 10 or 0 nanograms of streptavidin was impregnated in a dot shape with streptavidin as a test substance and dried.

Biotin-bonded latex colloidal particles (blue) were added in the first BS step, and streptavidin-bonded gold colloidal particles (red) were added in the sensitization step SS(1). In the steps after the SS(2) step, latex colloid particles bound with biotin (blue) and gold colloid particles bound with streptavidin (red) were alternately added. The BS, SS(5) and SS(10) steps were compared). As the buffer, Tris buffer containing 1% Tx-100 was used. Each SS step was performed within 1 minute from the previous step.

Test Results As shown in FIG. 6, after the BS step, blue dots were observed only when 1000 nanograms of streptavidin was used as a test subject. On the other hand, after the SS(10) step, purple dots were observed even when 100 nanograms of streptavidin was used. The sensitized purple coloration was dependent on the concentration of the test substance.

Discussion It was suggested that the purple dots were the result of the two types of particles, the latex colloid particles and the gold colloid particles used, alternately binding and forming a large complex. This indicates that the detection sensitivity was increased by repeating the sensitization step (SS(n)) in which colloid particles were newly added. It has been suggested that the sensitization using the present invention increases the sensitivity depending on the concentration of the test substance and the number of sensitization steps. Further, the sensitizing step is performed for a very short time (while the particles pass through the detection site), and it is considered that the sensitizing effect per time is very high.

<Example 5>
Test method As a test substance, 1 μl of 0.5 mg/ml streptavidin was impregnated into the membrane, dried, and a BS step was performed using maleimide-biotin-bonded latex (blue), and then SS (red) was used for streptavidin-bonded latex (red). 1) Process was performed. As a negative control, a buffer excluding latex particles in the BS step or SS(1) step was used. As the buffer, Tris buffer containing 1% Tx-100 was used.

Test Results As shown in FIG. 7, purple dots indicating the formation of a complex (“:” indicates binding) represented by streptavidin:maleimidobiotin-bonded latex particles (blue):streptavidin-bonded latex particles (red). Was observed.

Discussion It was suggested that the hydrocolloid particles can be bound to each other through the surface modifier for binding the colloid particles.

<Example 6>
Overview
The lateral flow immunoassay by sandwich using two kinds of antibodies against RS virus (respiratory syncytial virus) is used as BS step, and the sensitization step of SS(1) only is performed using a mixture of latex and gold colloid as C(1). It was

Test method
After the membrane was impregnated with 1 μl of anti-RS virus antibody with OD280 = 3.0, dried, and mixed with inactivated RS virus antigen serially diluted 4 times and biotinylated anti-RS virus antibody-bound latex (red, 350 nm in diameter). Immediately, the strip containing the membrane was impregnated into the BS process. Next, the SS(1) step was carried out using streptavidin-bonded latex (red, diameter 350 nm) and streptavidin-bonded gold colloid (diameter 40 nm) to compare the detection sensitivities with and without sensitization. As a negative control, one containing no antigen was used. As the buffer, Tris buffer containing 1% Tx-100 was used.

Test results As shown in FIG. 8, the minimum detection concentration was four times or more than that in the SS(1) step.

Discussion Two or more kinds of particles with different sizes modified by the same surface modifier for binding to colloidal particles were used in the same sensitization process, and the sensitivity was significantly increased. It is considered to be useful for multi-step sensitization and detection by chemiluminescence using biotinylated HRP or the like as the DS step if it is after the SS(1) step.

<Example 7>
Overview
Lateral flow immunoassay by sandwich using two kinds of antibodies against RS virus (respiratory syncytial virus) is used as BS process, sensitization is performed up to SS(2), and detection is performed using ECL (enhanced chemiluminescence) in DS process. It was Not only latex particles whose surface was modified with biotinylated HRP in SS(2), but also biotinylated HRP itself was made to act.

Test method
1 μl of anti-RS virus antibody with OD280 = 3.0 was impregnated into the membrane, dried and mixed with inactivated RS virus antigen and biotinylated anti-RS virus antibody-bound latex (red, 350 nm in diameter), and then immediately impregnated with the strip containing the membrane. The BS process was performed. Next, the SS(1) step was performed using streptavidin-bonded latex (red, diameter 350 nm). Subsequently, biotinylated HRP-bonded latex (red, 350 nm in diameter) was impregnated, and further biotinylated HRP was impregnated. The process up to this point was performed as the SS(2) process. Here, the strip was cut, and only the membrane portion was used for the subsequent examination. Luminata forte (Merck Millipore) was used in the DS process, and the detection sensitivities were compared with and without sensitization. As a negative control, one containing no antigen was used. As the buffer, Tris buffer containing 1% Tx-100 was used.

Test results See FIG. RSV was detected by chemiluminescence by ECL in the DS process using HRP of SS(2) as a substrate. The test substance was detected by using biotinylated HRP as a surface modifier for colloidal particles. The sensitivity was further increased by using biotinylated HRP, which is a molecular colloid, after the SS (2) step and before the detection step.

Discussion The combined use of colloidal particles and molecular colloids may improve the signal/noise ratio. Further, streptavidin, which is an unreacted colloidal particle surface modifier due to steric hindrance and a difference in the number of M(1) and M(2) (in this example, the number of streptavidin molecules of M(1) per unit number of particles is Is more than twice as many as the number of biotin molecules in the BS process.) may be effectively used. On the contrary, if biotin is large in the unreacted colloidal particle surface modifier, biotinylated particles can be used in the final sensitization step, and streptavidin HRP molecules and streptavidin HRP-bonded colloidal particles can be used as reaction substrates.

According to the present invention, it is possible to universally increase the sensitivity for various test substance detection methods.

1 carrier 2 test substance 3 first colloidal particle 4 surface modifying substance for binding to colloidal particle and affinity for test substance 5 surface modifying substance binding colloidal particle 6 colloidal particle C(1)
7 Surface modifier M(1) for binding colloidal particles
8 Modified colloidal particles for sensitization MC(1)
9 Colloidal particles C(2)
10 Surface modifier M(2) for binding colloidal particles
11 Modified colloidal particles for sensitization MC(2)
12 Colloidal particles C(3)
13 Surface modifier M(3) for binding colloidal particles
14 Modified colloidal particles for sensitization MC(3)
15 Surface modifier M(2) for binding colloidal particles
16 Colloidal particles C(2)
17 Modified colloidal particles for sensitization MC(2)
18 Colloidal particles C(3)
19 Surface modifier M(3) for binding colloidal particles
20 Modified colloidal particles for sensitization MC(3)

Claims (20)

In the method for detecting a test substance using colloidal particles having a substance having an affinity for the test substance, a method for increasing the sensitivity of detection of a test substance, comprising the following (i) to (iii) Including the steps, the detection sensitivity of the test substance is increased by forming a complex of colloidal particles added in each step, and a method of increasing the detection sensitivity of the test substance:
(i) BS (Binding Step) step of directly or indirectly binding the test substance and the first colloidal particles;
(ii) SS (Sensitizing Step) step of adding n times the sensitized modified colloidal particles (MC) to which the surface modification substance for binding the colloidal particles is added to the complex of the test substance and the first colloidal particle [ n is a natural number], and the modified colloid particles for sensitization (Modified colloid particles) MC(m) added in the mth SS(m) step are colloidal particles C(m) and surface modification substances M for binding colloidal particles. (m) are bound to each other, and the colloidal particles MC(m+1) added in the SS(m+1) step are colloidal particles C(m+1) with a surface modifier M(m+1) for binding colloidal particles. ) Are bound together, M(m) and M(m+1) have affinity, and MC(m) and MC(m+1) are bound to M(m) and M(m+1). Step of forming a complex through [m is a natural number and m+1≦n. ]; and
(iii) A DS (Detecting Step) step of detecting a test substance by detecting a signal generated directly or indirectly from the formed colloidal particles. In the method for detecting a test substance using colloidal particles having a substance having an affinity for the test substance, a method for increasing the sensitivity of detection of a test substance, comprising the following (i) to (iii) Including the steps, the detection sensitivity of the test substance is increased by forming a complex of colloidal particles added in each step, and a method of increasing the detection sensitivity of the test substance:
(i) Test substance and first colloidal particle [The surface-modified colloidal particle is referred to as MC(0), and the surface modifier M(0) is bound to the colloidal particle C(0). BS (Binding Step) step of directly or indirectly binding
(ii) SS (Sensitizing Step) step of adding n times the sensitized modified colloidal particles (MC) to which the surface modification substance for binding the colloidal particles is added to the complex of the test substance and the first colloidal particle [ n is a natural number], and the modified colloid particles for sensitization (Modified colloid particles) MC(m) added in the mth SS(m) step are colloidal particles C(m) and surface modification substances M for binding colloidal particles. (m) are bound together, colloidal particles MC(m-1) added in the previous step [SS(m-1) step if m>1 and BS step if m=1] Is a surface modification substance M(m-1) for binding colloidal particles to colloidal particles C(m-1).M(m-1) and M(m) have an affinity and MC(m-1) -1) and MC(m) form a complex through the bond of M(m-1) and M(m) [m is a natural number and m≤n. ]; and
(iii) A DS (Detecting Step) step of detecting a test substance by detecting a signal generated directly or indirectly from the formed colloidal particles. The method according to claim 1 or 2, wherein a direct signal of the colloidal particles is detected or measured in at least one of the BS step and/or the SS step. The method according to claim 1, wherein the colloidal particles have a diameter of 10 nm or more. The method according to any one of claims 1 to 4, wherein n is 1 to 10. The method according to claim 1, wherein in the BS step, the test substance is bound to the insoluble carrier with or without a substance having an affinity for the test substance. The method according to any one of claims 1 to 6, wherein in the BS step, the test substance and the first colloidal particles to which a substance having an affinity for the test substance is bound are bound. The method according to claim 1, wherein the colloidal particles used in the SS step are composed of many types. The modified colloidal particles for sensitization MC(n), which is combined with the surface modifier for binding the colloidal particles used in the SS step, are the modified colloidal particles for sensitization MC(m) and the m+1st added in the mth SS step. 9. The method according to any one of claims 1 to 8, which comprises two types of modified sensitizing modified colloidal particles MC(m+1) added in the SS step. The colloidal particles used in the SS step are composed of two types, colloidal particles C(m) used in the mth SS step and colloidal particles C(m+1) used in the m+1th SS step. 10. The method according to any one of items 9 to 9. The method according to any one of claims 1 to 7, wherein the colloidal particles used in the SS step are composed of one type. The first colloidal particles that have a substance that has an affinity for the test substance used in the BS step are the same as the modified sensitizing colloidal particles MC(m) used in the SS(m) [m is an even number] step. The method according to any one of claims 1 to 7, wherein: The modified colloidal particles MC(1) for sensitization used in the SS(1) step and the modified colloidal particles MC(m) for sensitization used in the SS(m) [m is an odd number] step are the same. 13. The method according to any one of 7 and 12. 14. The method according to any one of claims 1 to 13, wherein the combination having an affinity for the surface modifier M(m) for binding colloidal particles and M(m+1) is a combination of an antigen and an antibody. The method according to any one of claims 1 to 14, wherein the test substance is a biological substance. The method according to any one of claims 1 to 14, wherein the test substance is a substance derived from an infectious microorganism including a virus. The method according to any one of claims 1 to 16, wherein an enzymatic reaction is used in the DS step which is a detection step. The method according to any one of claims 1 to 17, wherein the test substance detection method is an immunochromatography method. For use in the method according to any one of claims 1 to 18, for binding colloidal particles to which at least a substance that binds to a test substance is bound, and for colloidal particle binding used in n SS steps [n is a natural number]. A kit containing modified colloid particles for sensitization (MC) to which a surface modifier is bound. Modified colloid particles for sensitization (Modified colloid particles) MC(m) added in the mth SS(m) step are colloids. The surface modifier M(m) for binding colloidal particles is bound to the particles C(m), and the colloidal particles MC(m+1) added in the SS(m+1) step are colloidal particles C(m+1 ) Is bound with a surface modifier M(m+1) for binding colloidal particles, M(m) and M(m+1) have an affinity, and MC(m) and MC(m+1) Is a kit that forms a complex through the binding of M(m) and M(m+1). The kit according to claim 19, which is a kit for immunochromatography.