WO2016157513A1 - Method and device for identifying amount of antigen-antibody interaction - Google Patents

Abstract

The present invention relates to a method for differentiating undesirable agglutination such as non-specific adsorption, and agglutination derived from molecules to be measured, such differentiation conducted by means of disagglutination, of only agglutination derived from molecules to be measured, which uses molecules comprising a portion (epitope) of the molecules to be measured captured by an antibody in a system for analyzing the agglutination (immunoagglutination) generated when a carrier (microparticles, beads, and the like) on which an antibody specific to the molecules to be measured has been fixed is exposed to a sample that comprises or possibly comprises the molecules to be measured.

Description

Method and apparatus for identifying antigen-antibody interaction amount

The present invention relates to an apparatus and method for measuring a molecule to be measured in a sample, specifically an apparatus for measuring a molecule to be measured in a sample by identifying an interaction between a specific antigen-antibody pair. And a method. The present invention also relates to a kit for measuring a molecule to be measured in a sample.

Immunoassay is widely used in the medical and pharmaceutical fields as an analytical method for detecting disease-derived proteins and pathogens using specific interactions between antigens and antibodies. Compared with general biochemical analysis, immunoassay has a low target concentration in a sample, and requires highly sensitive measurement. Improving the sensitivity of immunological tests is currently promoted, and it is expected to improve the quantitativeness of infectious disease tests, which account for 40% of immunological tests, and to discover new tumor markers.

Most of the current immunoassay devices bind an antibody labeled with an enzyme to a target substance, and measure the target substance from signals such as fluorescence and luminescence generated by the label as a catalyst (Enzyme-Linked Immunosorbent) (Assay, ELISA). Since these devices measure analog values from a plurality of molecules (about 10,000), signal fluctuation from the label becomes noise, and further enhancement of sensitivity is not easy. In recent years, a method has been proposed in which ELISA is performed simultaneously in a large number of microvolume spaces (more than tens of thousands, each volume is about 50 fl), and the presence or absence of target substances in each space is digitally measured and calculated. It has been demonstrated (digital ELISA method), and a detection sensitivity of 10 ⁻¹⁵ mol / L was obtained in the measurement of real samples (Non-patent Document 1). However, since the original principle is analog measurement, signal fluctuation cannot be suppressed yet, and it is difficult to stably perform high-sensitivity measurement.

In order to solve these problems associated with higher sensitivity, various methods have been developed that utilize antibodies attached to fine particles (beads). As a result, the association by the antigen-antibody reaction is converted into the association of beads, and the association phenomenon is expanded to facilitate measurement by scattered light and the like, thereby leading to higher sensitivity. However, this method involves a non-specific adsorption phenomenon of the beads themselves, so the background is high, and a technique for accurately extracting the aggregation phenomenon that occurs specifically in the antigen-antibody reaction is required. It has been developed (Patent Documents 1 to 3).

In Patent Document 1, a first step of adding a fine particle (bead) to which a specific binding substance (antibody) that specifically binds to a molecule to be measured is added to a specimen, agglutinating reaction, and measuring a change in absorbance; Next, the second step of measuring the change in absorbance measured by adding beads not added with antibodies and agglutination reaction, and the difference between the changes in absorbance obtained in the first step and the second step A method comprising three steps is described. Thereby, the background caused by non-specific adsorption of fine particles such as beads or the background caused by the inter-bead adsorption promoting substance (lipid etc.) contained in the molecule solution to be measured can be effectively removed.

The method of Patent Document 1 cannot remove a buffering action caused by a substance that causes a measurement interference action only when an antibody is present. Further, when the inter-bead adsorption promoting substance (lipid or the like) contained in the molecule solution to be measured is a small amount and is exhausted in the first step, it is impossible to calculate the degree of this effect in the second step. In this method, only the adsorption promoting substance between the fine particle bodies is an interference substance, and the influence of the interference substance can be eliminated only when it is contained in a sufficiently large amount in the solution.

In Patent Document 2, an antibody-luciferyl peptide complex in which an antibody capable of specifically binding to a molecule to be measured and a luciferyl peptide compound are used is used. The antibody-luciferyl peptide complex and the sample containing the molecule to be measured were allowed to coexist in the solution, the luciferyl peptide was dissociated by substitution of the molecule to be measured and the luciferyl peptide, and then cleaved from the dissociated luciferyl peptide A method for measuring luciferin using a luciferase reaction is described. This provides a detection system that can easily measure a molecule to be measured and has high specificity for the molecule to be measured.

In the method of Patent Document 2, it is necessary to synthesize and use a dedicated luciferyl peptide complex for each molecule to be measured, which increases the cost of the reagent system. In addition, since an optical detection system is required, the housing becomes large and expensive. Furthermore, after the substitution reaction between the molecule to be measured and the luciferyl peptide complex, a peptide degradation reaction process is required enzymatically, which is further costly, and this reaction efficiency affects the detection efficiency.

In Patent Document 3, a buffer containing the same antibody or antigen as that carried on an insoluble carrier (such as microparticles) is used as a buffer solution for measurement in the reagent configuration for a sample that has undergone a reaction during the first sample measurement. Antigen antibody to determine whether it is a specific reaction or a non-specific reaction by measuring the second time after replacing the solution and measuring the degree of decrease in the reaction with the buffer relative to the first reaction A method for determining the reaction is described. As a result, it is possible to confirm whether or not aggregation of the insoluble carrier occurs specifically in the antigen-antibody reaction.

In the method of Patent Document 3, it is necessary to divide the molecule solution to be measured, perform the antigen-antibody reaction twice or more, and measure the reaction intensity such as absorbance each time. In addition, usually a large amount of expensive antibody solution is consumed.

JP 2009-042057 A Special table 2011-037791 JP 2003-004741 A International Publication WO2007 / 032266 JP 2010-019553 A (International Publication WO 2008/053822)

An object of the present invention is to provide a method for accurately calculating the influence of a non-specific aggregation element contained in a specimen in a measurement method using the immunoagglutination method as a measurement principle.

The present inventors dissociate the aggregated beads by recognizing the molecule to be measured in a system that captures and detects the glycoprotein or protein that is the molecule to be measured with an antibody and detect the disaggregation phenomenon, for example, a blocking current. By observing with a measuring device, it was found that the aggregation by specific binding and the aggregation by non-specific binding can be distinguished, and the effective detection sensitivity can be increased.

That is, the present invention is an apparatus for detecting a molecule to be measured in a sample,
A reaction unit for performing a reaction in which a first carrier immobilizing a first antibody that binds to one site of a molecule to be measured and a second carrier immobilizing a second antibody that binds to another site of the molecule to be measured are brought into contact with a sample; ,
A sample introduction part for introducing the sample into the reaction part;
A reagent supply unit for supplying the first carrier and the second carrier to the reaction unit;
A reagent supply unit for supplying a capture site molecule comprising or including a site to which the first antibody of the molecule to be measured binds and / or a site to which the second antibody binds to the reaction unit;
A measurement unit for measuring aggregation or adhesion between the first carrier and the second carrier;
The present invention relates to an apparatus comprising: a calculation unit that analyzes a measurement result obtained by the measurement unit.

The present invention also provides a method for detecting a molecule to be measured in a sample,
A first carrier immobilizing a first antibody that binds to one site of the molecule to be measured and a second carrier immobilizing a second antibody that binds to another site of the molecule to be measured are brought into contact with the sample, whereby the first carrier A step of agglomerating or adhering to the second carrier,
Measuring aggregation or adhesion between the first carrier and the second carrier;
A capture site molecule consisting of or containing the site to which the first antibody of the molecule to be measured binds and / or the site to which the second antibody binds is added to the reaction solution, whereby the molecule to be measured and the first antibody Specific binding and / or specific binding between the molecule to be measured and the second antibody is inhibited or dissociated,
Measuring aggregation or adhesion between the first carrier and the second carrier;
The present invention relates to a method including a step of calculating specific binding between a molecule to be measured and a first antibody and a second antibody based on a difference in aggregation degree or adhesion degree of a carrier before and after addition of the capture site molecule.

Furthermore, the present invention is a kit for detecting a molecule to be measured in a sample,
A first carrier immobilizing a first antibody that binds to one site of a molecule to be measured;
A second carrier immobilizing a second antibody that binds to another site of the molecule to be measured;
The present invention relates to a kit comprising a site to which a first antibody binds and / or a capture site molecule comprising or including a site to which a second antibody binds.

According to the present invention, the non-specificity between the carriers that is inevitably generated when the antigen-antibody interaction phenomenon is efficiently measured by using a carrier in which the antibody is immobilized on a carrier containing fine particles such as beads or other base materials. The amount of antigen-antibody interaction can be calculated regardless of the number of interactions.

According to the present invention, the following solution is given to the contents pointed out as a problem remaining in the method of Patent Document 1.

The first step of Patent Document 1 is used as it is, using a capture site molecule (a molecule consisting of or including only a peptide molecule or sugar chain molecule as an epitope) in which the molecule to be measured is captured by an antibody in the second step. By deaggregating, the difference in the degree of aggregation before and after deaggregation is measured. As a result, the degree of aggregation caused only by the molecule to be measured, regardless of the amount of adsorption promoting substances (lipids, etc.) between the supports (beads) and regardless of the presence of measurement interference substances that act only when antibodies are present Can be calculated.

According to the present invention, the following solution is given to the content pointed out as a problem remaining in the method of Patent Document 2 above.

The present invention and Patent Document 2 are similar in that a capture site molecule (epitope) captured by an antibody is used for each molecule to be measured. However, as in Patent Document 2, an expensive substance such as a luciferyl peptide is used. Since the epitope can be used as it is without being synthesized, the reagent cost can be reduced. In addition, since an optical detection system is not required, a compact device configuration is possible. Furthermore, detection by a macroscopic and simpler measurement system is possible by converting the molecule to be measured and the peptide (epitope) substitution reaction into a change in the aggregation state of the fine particles without going through an enzyme reaction process.

According to the present invention, the following solution is given to the content pointed out as a problem remaining in the method of Patent Document 3 above.

In the present invention, the capture site molecule (peptide or sugar) can be continuously added to the reaction vessel or the flow path and the measurement can be continuously performed without the need to divide the sample that may contain the molecule to be measured.

FIG. 1 is a schematic diagram showing the principle of detecting a molecule to be measured with high sensitivity by a sandwich method using antibody-immobilized beads. FIG. 2 is a schematic diagram showing a phenomenon in which nonspecific adsorption, which is a problem in the detection of highly sensitive molecules to be measured by the sandwich method using beads, occurs. FIG. 3 is a schematic diagram showing a method of disaggregating only specific bead aggregation by using a site (capture site molecule) captured by an antibody. FIG. 4 is a schematic diagram showing a method of dissociating only specific bead attachment by using a site (capture site molecule) captured by an antibody. FIG. 5 is a schematic diagram showing a configuration example of an apparatus according to the present invention. FIG. 6 is a flowchart for detecting a molecule to be measured using the apparatus according to the present invention. FIG. 7 is a schematic diagram showing an example of elements constituting the bead aggregation degree measurement step when the blocking current measurement is used. A represents a cell capable of partitioning the inside of the container with a bottleneck and applying a voltage, and B represents a current measuring device. FIG. 8 is a schematic diagram showing an example of elements constituting the specific binding disaggregation step. FIG. 9 is a schematic diagram showing a flow chart of the bead aggregation degree measurement step and the specific binding disaggregation step and an instantaneous current amount decrease (blocking current) measurement in the case of using the blocking current measurement. FIG. 10 is a diagram showing the flow order in the configuration diagram showing the relationship between the bead aggregation degree measurement step and the specific binding disaggregation step when the blocking current measurement is used. FIG. 11 is a sensorgram showing the result of measuring the inhibition by the capture site molecule of the interaction with the antibody molecule by surface plasmon resonance using human cardiac troponin I (hcTnI) as a molecule to be measured.

Hereinafter, the present invention will be described in detail.

The present invention relates to a system for measuring agglutination (immunoaggregation) caused by the action of a carrier (microparticles, beads, etc.) on which an antibody specific to the molecule to be measured is immobilized on a sample containing the molecule to be measured. Nonspecific adsorption by disaggregating only aggregates derived from the molecule to be measured using a molecule containing the site (epitope) captured by the antibody (also referred to as “capture site molecule” in this specification). The present invention relates to a method for discriminating undesired aggregation such as from a molecule to be measured.

That is, the present invention relates to a method for detecting a molecule to be measured in a sample.
A first carrier immobilizing a first antibody that binds to one site of the molecule to be measured and a second carrier immobilizing a second antibody that binds to another site of the molecule to be measured are brought into contact with the sample, whereby the first carrier A step of agglomerating or adhering to the second carrier,
Measuring aggregation or adhesion between the first carrier and the second carrier;
A capture site molecule consisting of or containing the site to which the first antibody of the molecule to be measured binds and / or the site to which the second antibody binds is added to the reaction solution, whereby the molecule to be measured and the first antibody Specific binding and / or specific binding between the molecule to be measured and the second antibody is inhibited or dissociated,
Measuring aggregation or adhesion between the first carrier and the second carrier;
A step of calculating specific binding between the molecule to be measured and the first antibody and the second antibody based on the difference in the degree of aggregation or adhesion of the carrier before and after the addition of the capture site molecule.

The molecule to be measured to be detected is not particularly limited as long as it can bind to the antibody, and examples thereof include peptides, proteins and sugar chains, cells containing these on the surface, viruses and bacteria.

The target sample is not particularly limited as long as it is a sample containing the molecule to be measured or a sample that may contain the molecule to be measured, and it is a liquid, solid or gas sample, or a mixed sample thereof. Good. Specifically, the sample is a biological fluid sample (saliva, nasal discharge, blood, serum, plasma, urine, etc.), cell or tissue sample (oral swab, cancer tissue or cell, etc.), environmental sample (seawater, river water, industrial, etc.) Water, soil, etc.). The liquid sample can be used as it is, or diluted or concentrated with a solvent. The solid sample may be suspended in a solvent, homogenized by a pulverizer or the like, or a supernatant obtained by stirring with a solvent may be used. For example, a stick such as a cotton swab may be moistened with sterile water and the measurement location may be wiped, then rinsed in a solvent, and the resulting liquid may be used as a sample. Examples of the solvent include distilled water, physiological saline, phosphate buffer, Tris buffer, and the like. Furthermore, it is also possible to use a filter obtained by filtering a sample with a filter and capturing sample components (cells, bacteria, viruses, etc.). Such a filter is not particularly limited as long as it has a size that can capture a molecule to be measured.

The purpose of the present invention is to detect a molecule to be measured in a sample. However, “detection” is not only the detection of the presence or absence of a molecule to be measured in a sample, but also quantitative measurement of the molecule to be measured. Detection (ie, determining the amount or concentration), and determining the abundance ratio of the molecule to be measured.

The principle of the present invention is shown in FIGS.

FIG. 1 shows the principle of detecting a molecule to be measured by aggregation of antibody-immobilized beads. Here, a bead 1 on which an antibody that recognizes and binds to one site of the molecule to be measured is immobilized and a bead 2 on which an antibody that recognizes and binds to another site of the molecule to be measured is immobilized are shown. When the molecule to be measured is present, the beads 1 and 2 are simultaneously bonded to the molecule to be measured and aggregate. By measuring this aggregation, it becomes possible to detect the molecule to be measured.

However, as shown in FIG. 2, when antibody-immobilized beads are allowed to act on a sample solution containing a molecule to be measured, bead aggregation caused by the molecule to be measured (shown as “antigen recognition pair” in FIG. 2), Both bead agglomeration that does not originate from the measurement molecule are mixed. As the bead aggregation not caused by the molecule to be measured, bead aggregation due to nonspecific adsorption between the antibodies (shown as “nonspecific adsorption pair” in FIG. 2), the antibody binds by recognizing an antigen different from the molecule to be measured. Bead aggregation (shown as “non-antigen recognition pair” in FIG. 2). Therefore, the molecule to be measured cannot be accurately detected simply by measuring the bead aggregation.

In the present invention, as shown in FIG. 3, the molecule to be measured is added by adding the capture site molecule to a reaction solution in which bead aggregation caused by the molecule to be measured and bead aggregation not caused by the molecule to be measured are mixed. Only the bead aggregation resulting from the can be disaggregated. As a result, the difference between the aggregation degree measured before adding the capture site molecule (aggregation due to specific binding + nonspecific aggregation) and the aggregation degree measured after addition (nonspecific aggregation) is measured. It is possible to calculate the bead aggregation due to the molecule and detect the molecule to be measured.

As another embodiment, FIG. 4 shows the principle of detecting a molecule to be measured based on a sandwich method using antibody-immobilized beads and an antibody-immobilized substrate. Here, a bead on which an antibody that recognizes and binds to one site of a molecule to be measured is immobilized and a substrate on which an antibody that recognizes and binds to another site of the molecule to be measured is immobilized are shown. As shown in FIG. 4A, when a molecule to be measured is present, the antibody immobilized on the bead and the antibody immobilized on the substrate are simultaneously bound to the molecule to be measured, and the bead adheres to the substrate. By measuring this adhesion, it becomes possible to detect the molecule to be measured.

However, as shown in FIG. 4 (A), when a sample solution containing a molecule to be measured is allowed to act on an antibody-immobilized bead and an antibody-immobilized substrate, the beads attached due to the molecule to be measured and those caused by the molecule to be measured Both non-bead attachments are mixed. Examples of the bead attachment not caused by the molecule to be measured include bead attachment by non-specific adsorption between antibodies, and bead attachment in which an antibody different from the molecule to be measured is recognized and bound to the antibody. Therefore, the molecule to be measured cannot be accurately detected simply by measuring the adhesion of the beads to the substrate.

Subsequently, as shown in FIGS. 4B and 4C, by adding the capture site molecule to the substrate, it is possible to dissociate only the beads attached due to the molecule to be measured. As a result, the difference between the degree of adhesion measured before adding the capture site molecule (adhesion due to specific binding + nonspecific adhesion) and the degree of adhesion measured after addition (nonspecific adhesion) It is possible to detect the measurement target molecule by calculating the bead adhesion caused by the molecule.

Therefore, first, in the method according to the present invention, a first carrier that fixes a first antibody that binds to one site of a molecule to be measured and a second carrier that fixes a second antibody that binds to another site of the molecule to be measured. prepare.

The first antibody and the second antibody differ depending on the type of molecule to be measured, and may be commercially available or may be prepared by methods known in the art. It is preferable that the antibody specifically recognizes the molecule to be measured, binds to the molecule to be measured stably in a solution, and maintains the state. Moreover, it is preferable that the binding site (epitope) with the molecule to be measured is known for binding with a capture site molecule described later.

The antibody may be a polyclonal antibody or a monoclonal antibody as long as it has a specific binding ability to the molecule to be measured. Further, it may be in any form such as a complete antibody, an antibody fragment (Fav fragment, single chain Fv fragment (scFv), etc.), a domain antibody and the like. The preparation of such antibodies is known in the art. By selecting the epitope and determining the type of antibody according to the target molecule to be measured, it is possible to detect the amount or concentration of the molecule to be measured with higher sensitivity.

Since the present invention is based on the sandwich method, it is desirable that the first antibody and the second antibody bind to the molecule to be measured simultaneously. Alternatively, when the molecule to be measured is a homomultimer, the first antibody and the second antibody may be the same, and two or more antibody molecules are simultaneously formed on the homomultimer that is the molecule to be measured. It is desirable to bond stably. It is desirable to confirm that the first antibody and the second antibody are simultaneously bound to the molecule to be measured before actually detecting the molecule to be measured.

Furthermore, since the antibody used in the method according to the present invention stably binds to the molecule to be measured in a solution, its binding equilibrium constant is desirably about 10 ⁻⁹ M or less.

The carrier can be of any material and shape as long as it is a carrier generally used in immunoassays.

For example, the material of the carrier is not particularly limited as long as it is a carrier generally used in this technical field. Specifically, metals such as precious metals (gold, silver, platinum, palladium, rhodium, iridium, ruthenium, etc.), copper, aluminum, tungsten, molybdenum, chromium, titanium, nickel, etc .; stainless steel, hastelloy, inconel, monel, duralumin, etc. Alloys: Electrodes of semiconductor elements (transistors, FETs, etc.); Silicon; Glass materials such as glass, quartz glass, fused silica, synthetic quartz, alumina, sapphire, ceramics, forsterite and photosensitive glass; polyester, polystyrene, polyethylene , Polypropylene, nylon, acrylic, polycarbonate, polyethylene terephthalate (PET), polyurethane, phenolic resin, melamine resin, epoxy resin and polyvinyl chloride; agarose, dextran, cellulose Scan, polyvinyl alcohol, nitrocellulose, latex and the like.

The shape of the carrier is not particularly limited, and examples thereof include substrates (for example, titer plates, porous or pore arrays, microchannels, flat plates, films, etc.) and fine particles (for example, magnetic particles, beads, etc.). Fine particles having an appropriate size can be used. For example, fine particles having a diameter of 1 nm to 50 μm, preferably 10 nm to 10 μm are used.

The carrier to be used is selected appropriately depending on the type of molecule to be measured and the type of measurement method described below. For example, when detecting a molecule to be measured by agglutination of beads, both the first carrier and the second carrier are made into fine particles, and the antibody immobilized on the first carrier and the antibody immobilized on the second carrier simultaneously become molecules to be measured. By bonding, the first carrier and the second carrier which are fine particles are aggregated. On the other hand, when one of the first carrier and the second carrier is a fine particle and the other is a substrate, the antibody immobilized on the first carrier and the antibody immobilized on the second carrier are simultaneously bound to the molecule to be measured, A carrier that is a fine particle adheres to a carrier that is a substrate.

Immobilization of an antibody on a carrier is also known in the art and is not particularly limited. For example, the antibody can be immobilized on a carrier by covalent bond, ionic bond, or physical adsorption.

Subsequently, the first carrier immobilizing the first antibody and the second carrier immobilizing the second antibody are brought into contact with the sample. “Contact” means that a molecule to be measured existing in a sample can be brought into close proximity so that the first antibody and the second antibody can bind to each other. Operations such as mixing the solution containing the solution and flowing the solution containing the sample and the carrier on which the antibody is immobilized on the substrate carrier on which the antibody is immobilized are included. The contact conditions may be those usually employed in the field of immune reactions. For example, the contact is performed at 20 to 45 ° C. for 1 minute to 4 hours, preferably at 25 to 37 ° C. for 1 minute to 1 hour. .

By the above operation, the molecule to be measured in the sample is bound to the first antibody and the second antibody. Further, the first carrier and the second carrier are aggregated or adhered.

Next, the aggregation or adhesion of the first carrier and the second carrier is measured. The measurement of the aggregation or adhesion of the carrier can be performed by a method known in the art, and can be appropriately selected according to the type and shape of the carrier used. For example, when both the first carrier and the second carrier are fine particles, the aggregation of the carrier can be measured by, for example, a blocking current of a fine channel. In the case where one of the first carrier and the second carrier is a fine particle and the other is a substrate, adhesion between the first carrier and the second carrier, for example, using the substrate as a measurement substrate, surface plasmon resonance method It can be measured by a quartz crystal microbalance method, surface scattering light measurement, or a field effect transistor.

Subsequently, a capture site molecule consisting of or including the site to which the first antibody binds and / or the site to which the second antibody binds is added to the reaction solution. Thereby, the specific binding between the molecule to be measured and the first antibody and / or the specific binding between the molecule to be measured and the second antibody is inhibited or dissociated. As a result, the first carrier and the second carrier are disaggregated, or the second carrier attached to the first carrier is dissociated.

The site to which the first antibody of the molecule to be measured binds and / or the site to which the second antibody binds (the site captured by the antibody) is preferably an epitope on the molecule to be measured to which these antibodies bind. Thus, capture site molecules can be oligopeptides, monosaccharides and polysaccharide molecules consisting of or containing such epitopes. Preferably, the capture site molecule is a peptide or sugar molecule consisting of the antigen used in the production of the antibody.

Alternatively, the capture site molecule may consist of or include a part of such an epitope as long as the ability to bind to the antibody is retained. Further, the capture site molecule may be a peptide or sugar molecule in which a part of the amino acid or sugar of the epitope is modified as long as the binding ability to the antibody is maintained. Here, the modification includes when the epitope is a sugar molecule, as long as the antibody-binding ability is maintained, a part of the functional group is substituted with another functional group, or the sugar molecule is a polysaccharide. A part of the monosaccharide unit is replaced with another monosaccharide. On the other hand, when the epitope is an oligopeptide, the modification means that a part of the functional group on the amino acid is substituted with another functional group or the amino acid sequence of the oligopeptide as long as the antibody binding ability is maintained. Some (one or several) amino acids may be substituted with other amino acids.

The capture site molecule is added to the reaction solution containing the sample, the first carrier and the second carrier. After the addition, the reaction is carried out at a temperature and time at which disaggregation or dissociation of adhesion occurs, for example, at 20 to 45 ° C. for 1 minute to 4 hours, preferably at 25 to 37 ° C. for 1 minute to 1 hour.

Next, the aggregation or adhesion of the first carrier and the second carrier is measured again. Preferably, the measurement is performed by the same method as the first measurement method described above.

Then, the specific binding between the molecule to be measured and the first and second antibodies is calculated by determining the difference in the degree of aggregation or adhesion of the carrier before and after the addition of the capture site molecule. Thereby, the molecule to be measured present in the sample can be detected.

In the method according to the present invention, only the aggregation of the carrier due to the binding to the molecule to be measured is disaggregated (or the adhesion of the carrier is dissociated), and the number thereof is calculated. Regardless of the amount of aggregation or adhesion (not due to the measurement molecule), only the aggregation or adhesion of the carrier due to the binding to the molecule to be measured can be calculated.

The method according to the present invention can be carried out easily and simply by using an apparatus and / or kit composed of appropriate elements.
An apparatus 1 for detecting a molecule to be measured in a sample according to the present invention, for example, as shown in FIG.
Reaction unit 2 for performing a reaction in which a first carrier immobilizing a first antibody that binds to one site of a molecule to be measured and a second carrier immobilizing a second antibody that binds to another site of the molecule to be measured are brought into contact with the sample. When,
A sample introduction unit 3 for introducing a sample into the reaction unit 2;
A reagent supply unit 4 for supplying the first carrier and the second carrier to the reaction unit 2;
A reagent supply unit 4 for supplying the reaction site 2 with a capture site molecule comprising or including a site to which the first antibody of the molecule to be measured binds and / or a site to which the second antibody binds;
A measuring unit 5 for measuring aggregation or adhesion between the first carrier and the second carrier;
And a calculation unit 6 for analyzing the measurement result obtained by the measurement unit 5.

Here, the reaction unit 2 is a region that performs a reaction between a molecule to be measured as an antigen and an antibody, and / or a reaction between an antibody and a capture site molecule. For example, a single or multiwell plate, petri dish, dish, Tube, cell, etc. It is preferable that the reaction part 2 can maintain the temperature and conditions suitable for each reaction.

As long as the sample introduction unit 3 and the reagent supply unit 4 can introduce a sample into the reaction unit 2 and supply reagents (first carrier, second carrier, and capture site molecule), respectively, this technical field. And can be any known one.

As described above, the measurement unit 5 includes, for example, a device for measuring the blocking current of the fine channel when measuring the aggregation of the carrier. On the other hand, when measuring the adhesion of the carrier, the measuring unit 5 includes a device for measuring by, for example, a surface plasmon resonance method, a crystal oscillator microbalance method, a surface scattered light measurement, or a field effect transistor. The measuring unit 5 is not particularly limited as long as it is a device or an apparatus that can measure the aggregation or adhesion of the carrier.

The calculation unit 6 analyzes the measurement result obtained from the measurement unit 5 and includes, for example, a data analysis unit including software and a computer for processing the measurement result. The calculation unit 6 refers to the measurement result obtained from the measurement unit 5 with reference to data stored in the storage unit 7 (for example, data such as a calibration curve), so that the molecule to be measured, the first antibody, the second antibody, Specific binding amount, that is, the amount or concentration of the molecule to be measured in the sample is calculated. The calculation unit 6 can include, for example, a measurement result display part, a unit for analyzing the measurement result, a computer unit, and the like.

FIG. 6 shows a flowchart for carrying out a method for detecting a molecule to be measured in a sample using the apparatus according to the present invention. First, the reagent supply unit 4 is filled with a reagent, that is, a first carrier, a second carrier, and a capture site molecule. The sample introduction unit 3 is filled with a sample to be detected for the presence or amount of the molecule to be measured.

Next, the sample is introduced from the sample introduction unit 3 to the reaction unit 2, and the first carrier and the second carrier are supplied from the reagent supply unit 4 to the reaction unit 2. Reaction is performed with the first antibody immobilized on the first carrier and the second antibody immobilized on the second carrier. Thereafter, the aggregation or adhesion of the carrier in the reaction solution is measured in the measurement unit 5. The measurement result is stored in the storage unit 7.

Subsequently, the capture site molecule is supplied from the reagent supply unit 4 to the reaction unit 2, and the reaction unit 2 reacts the capture site molecule with the first antibody and / or the second antibody. As a result, among the aggregation or attachment of the carrier in the reaction solution, the aggregation or attachment derived from the specific binding between the molecule to be measured and the antibody is disaggregated or dissociated. Next, the aggregation or adhesion of the carrier in the reaction solution is measured in the measurement unit 5. In the calculation unit 6, the measurement result before addition of the capture site molecule is compared with the measurement result after addition of the capture site molecule, and if necessary, using data such as a calibration curve stored in the storage unit 7, Perform analysis.

In the calculation unit 6, detection data relating to the presence or amount of the molecule to be measured in the sample is acquired from the aggregation or adhesion resulting from the specific binding between the molecule to be measured and the antibody. If necessary, the data obtained in the measurement result display part is displayed.

These procedures may be automatically processed by the control unit 8, for example. The control unit 8 may be independent, or may be integrated with the calculation unit 6 and / or the storage unit 7.

Further, a kit for detecting a molecule to be measured in a sample according to the present invention,
A first carrier immobilizing a first antibody that binds to one site of a molecule to be measured;
A second carrier immobilizing a second antibody that binds to another site of the molecule to be measured;
The molecule to be measured includes a site to which the first antibody binds and / or a site to which the second antibody binds or a capture site molecule containing the site.

The kit according to the present invention may contain other elements suitable for immunoassay by the sandwich method, such as a dilution buffer and a washing buffer. The kit according to the present invention may include a manual describing a procedure and protocol for detecting a molecule to be measured in a sample, a table indicating a reference value or a reference range used in detection, and the like.

The components included in the kit may be provided individually or may be provided in a single container. Preferably, the kit according to the present invention can be used in such a manner that all the components necessary for carrying out the above-described method for detecting a molecule to be measured in a sample can be used immediately. Include as a component.

Hereinafter, the present invention will be described in more detail by way of examples. However, the technical scope of the present invention is not limited to the following examples. It will be apparent to those skilled in the art that various embodiments based on the inventive concepts described herein are possible.

[Example 1] Specific Example of Method and Apparatus In one embodiment, the method according to the present invention is performed by the following elements A and B: [1] a bead aggregation measurement step and elements D to F [2] A specific binding disaggregation step is performed. This will be described with reference to FIGS.

[1] Bead aggregation degree measuring step [1] Bead aggregation degree measuring step of the method according to the present invention comprises the following elements (FIG. 7). For example, it can be set as the well-known structure shown by the nonpatent literature 2. FIG.

A) A cell in which the inside of a container is partitioned by a bottleneck and voltage can be applied before and after that, and current can be measured simultaneously (also referred to as “cell A partitioned by bottleneck”).

B) When the beads pass through the bottleneck by the applied DC voltage, a current measuring device ("current measuring device B") that can observe an instantaneous current decrease (blocking current) having a magnitude corresponding to the volume of the passing beads Corresponds to the measuring part).

In the [1] bead aggregation measurement step of the method according to the present invention, the “cell A partitioned by a bottleneck” receives a sample solution containing or possibly containing a molecule to be measured, and is a single particle or aggregate. In order to include many beads in a state, it is necessary to have a sufficient capacity compared to the bead volume. Specifically, a container having a volume in the range of several microliters to several milliliters is desirable.

The cell A partitioned by the Kushiro is divided at the center by the partition including the Kushiro, and it is desirable that the diameter of the Kushiro is the same as the diameter of the beads used in the present invention. It is desirable that the diameter be the same as or twice the diameter of the beads used in the invention.

Cell A partitioned by a bottleneck has a configuration in which electrodes are arranged before and after the partition, and the electrodes can be electrically connected to the solution inside the cell. Further, the cell A partitioned by a bottleneck has an electric power source that can arrange an electrode before and after the partition and can apply a voltage to the divided cell inside through the electrode.

The cell A partitioned by a bottleneck requires an electric power source that can arrange an electrode before and after the partition, and can apply a voltage to the divided cell inside through the electrode. A “current measuring device B” capable of measuring a current value can be connected.

In the [1] bead aggregation degree measuring step of the method according to the present invention, the “current measuring device B” is a method of measuring a minute current change before and after passing through the beads and a difference in minute current change that varies depending on the difference in the degree of aggregation of the beads. Sufficient sensitivity and accuracy to measure

In the [1] bead aggregation measurement step of the method according to the present invention, the cell A partitioned by a bottleneck is filled with a buffer suitable for performing an antigen-antibody reaction. A current measuring device B that can measure a current generated through a bottleneck by applying a DC voltage before and after the partition is disposed. It is generated by introducing beads into one of the partitions in the cell and applying a DC voltage so that the beads pass through the bottleneck and the current path is blocked when the beads pass through the bottleneck of the divider. Measure the instantaneous current phenomenon (blocking current). Since this blocking current amount increases according to the degree of aggregation of beads, the degree of aggregation of beads can be calculated.

FIG. 7 is a diagram showing an outline of the bead aggregation degree measuring step. First, an antibody-immobilized bead is allowed to act on a sample solution containing or possibly containing a molecule to be measured in the upstream cell of cell A, and a part of the beads is aggregated, and a voltage is applied to the downstream cell through a bottleneck. Allow the beads to flow to the side. During this process, the current measurement device B measures the instantaneous decrease in current amount (current decrease peak). After the deagglomeration reaction, the current drop peak measurement is performed with a current measuring device by the same process.

[2] Specific binding disaggregation step The [2] specific binding disaggregation step of the method according to the present invention comprises the following elements (FIG. 8).

C) A molecule consisting only of or containing a capture site (peptide molecule or sugar chain molecule serving as an epitope) where the molecule to be measured is captured by an antibody (referred to as “capture site molecule”).

In one embodiment, the capture site molecule used in the present invention is desirably the epitope (peptide or sugar molecule) itself used in producing the antibody molecule. As a typical example, a glycoprotein that is an antigen (corresponding to a molecule to be measured) or a glycomolecule that is a part of a protein, or an oligopeptide, which is injected into an animal body that produces an antibody. Epitopes can be used as capture site molecules. The capture site molecule used in the present invention may be a part of an epitope (peptide or sugar molecule) as long as the binding ability to the antibody is maintained. In addition, as long as the capture site molecule retains the ability to bind to an antibody, some of the amino acids of the epitope may be modified. Here, the modification includes when the epitope is a sugar molecule, as long as the antibody-binding ability is maintained, a part of the functional group is substituted with another functional group, or the sugar molecule is a polysaccharide. A part of the monosaccharide unit is replaced with another monosaccharide. On the other hand, when the epitope is an oligopeptide, the modification means that a part of the functional group on the amino acid is substituted with another functional group or the amino acid sequence of the oligopeptide as long as the antibody binding ability is maintained. Some (one or several) amino acids may be substituted with other amino acids.

D) Two or more types of antibody molecules (corresponding to the first antibody and the second antibody) that recognize and bind (capture) the molecule to be measured at two or more different sites.

It is desirable that the antibody molecule used in the present invention can simultaneously bind to the molecule to be measured. However, when the molecule to be measured is a homomultimer, the number of antibody molecules may be one, and two or more antibody molecules bind to one molecule of the molecule to be measured.

The antibody molecule used in the present invention is preferably one that specifically recognizes the molecule to be measured, binds to the molecule to be measured stably in a solution, and maintains the state.

The antibody molecule used in the present invention is preferably a pair of two or more antibody molecules capable of simultaneously recognizing and binding two or more sites on the molecule to be measured. However, when the molecule to be measured is a homomultimer, one type of antibody molecule that recognizes and binds to one site on the molecule to be measured can be used. It is preferable that two or more antibody molecules can be stably and simultaneously bound on the body.

Since the antibody molecule used in the present invention stably binds to the molecule to be measured in a solution, its binding equilibrium constant is desirably about 10 ⁻⁹ M or less.

In addition, it is desirable that the antibody molecule used in the present invention is one that stably binds to a molecule to be measured in a solution while being immobilized on a bead and maintains that state.

E) Beads in which two or more types of antibody molecules are independently fixed to separate beads or mixed and fixed (also referred to as “antibody-fixed beads”, which corresponds to a first carrier and a second carrier).

When the molecule to be measured is a homomultimer, the bead used in the present invention may be one kind of bead on which one kind of antibody molecule is immobilized.

In the beads used in the present invention, the immobilized antibody specifically recognizes the molecule to be measured and aggregates by binding to the molecule to be measured stably in a solution. It is desirable that it be changed according to the concentration of.

The bead diameter of the beads used in the present invention is desirably large enough to antagonize the bottleneck of the cell partitioned by the bottleneck.

In addition, the bead diameter of the beads used in the present invention is desirably large enough to be easily migrated by an applied voltage in a cell partitioned by a bottleneck. Specifically, it is desirable that the diameter is about several tens of nanometers to several micrometers.

The material of the beads used in the present invention is preferably a material having a specific gravity that can be easily migrated by an applied voltage in a cell partitioned by a bottleneck. However, if the specific gravity is lighter than that of the buffer solution, it may receive buoyancy and may adhere to the walls in the cell.

FIG. 8 is a diagram showing an outline of the specific binding disaggregation process. In the process of (1) to (2), the antibody-immobilized beads (E) are allowed to act on the sample solution containing or possibly containing the molecule to be measured, thereby producing a bead aggregation state. In the figure, within the dotted box on the left side, one capture site molecule (semi-circle) and another capture site molecule (triangle) complement the antibody binding (capture) shape that recognizes the site on each measured molecule. Indicates that it is designed to be In the process from (2) to (3), a large excess amount of the capture site molecule (C) is added to the beads (E), and the aggregated beads are deaggregated (by heating and shaking).

In the [2] specific binding disaggregation step of the method according to the present invention, the beads are aggregated by allowing the beads to which the antibody molecule (D) has been immobilized in advance to act on a sample solution containing or possibly having a molecule to be measured. And then the capture site molecule (C) is allowed to act to dissociate only the aggregate caused by the molecule to be measured, and the dissociation state before and after the action of C is compared to obtain the antigen antibody Extract only interactions.

In the present invention, for a solution containing beads aggregated by first acting on a molecule to be measured, [1] the degree of bead aggregation is calculated using the bead aggregation degree measurement step, and then [2] specific binding for the recovered solution. In the disaggregation step, only the bead aggregation resulting from the antigen-antibody interaction is disaggregated, and finally [1] the bead aggregation degree measurement step is used again to calculate the bead aggregation degree, and with the difference from the initial aggregation degree, The amount of antigen-antibody interaction is calculated.

FIG. 9 shows a flowchart of the specific embodiment described above. In the drawing, the schematic diagram of the measurement result of the current drop peak during the first current measurement and the second current measurement is shown in the right box. Here, during the first current measurement, many large peaks (the sum of bead aggregation due to the molecule to be measured and bead aggregation due to non-specific adsorption, etc.) are often observed, and the disaggregation reaction due to the addition of the capture site molecule At the later second current measurement, a large peak decreases. This decrease number indicates the number of beads aggregated due to the molecule to be measured.

FIG. 10 is a diagram showing the flow order in the configuration diagram showing the relationship between the bead aggregation degree measurement step and the specific binding disaggregation step when the blocking current measurement is used.

In a specific embodiment, a sufficiently larger number of antibody-immobilized beads (E) than the expected number of molecules to be measured are allowed to act on a sample solution that previously contains or possibly contains the molecules to be measured. The beads are aggregated. At this time, due to factors such as nonspecific adsorption, bead aggregation that is not caused by the molecule to be measured also occurs. This solution is introduced into one of the cells (A) partitioned by a bottleneck, and a voltage is applied so that the beads pass from one cell to the other so that the bottles pass through the bottleneck. At this time, the first current measurement (3) is performed. That is, the instantaneous current drop phenomenon which changes according to the degree of aggregation of the beads when passing the beads is continuously measured using the current measuring device (B).

The momentary current drop phenomenon (current drop peak) that occurs at this time varies in proportion to the presence or absence of the bead aggregation that passed when the phenomenon was observed and the degree of aggregation. The larger the number of large peaks, the larger the number of bead aggregations. Therefore, the sum of the bead aggregation caused by the molecule to be measured and the bead aggregation due to nonspecific adsorption and other factors (not caused by the molecule to be measured) is larger. .

When the current lowering phenomenon has ended, the solution containing the aggregated beads that have moved to the other cell is taken out, a capture site molecule (C) is added, and a reaction is performed to deaggregate only the aggregated beads by the molecule to be measured.

When adding a capture site molecule (C) and performing a reaction for deaggregating only the aggregated beads of the molecule to be measured, it is also desirable to increase the temperature to promote the reaction or to accelerate the reaction by stirring.

In anticipation of the end of the deagglomeration reaction, the same solution is again introduced into one cell of the cell (A) partitioned by a bottleneck, and a voltage is applied so that the beads pass from one cell to the other cell so as to pass through the bottleneck. I will let you. At this time, the second current measurement is performed (7). That is, the instantaneous current drop phenomenon which changes according to the degree of aggregation of the beads when passing the beads is continuously measured using the current measuring device (B).

The large peak of the instantaneous current drop phenomenon (current drop peak) that occurs at this time is after the reaction to disaggregate only the aggregated beads by the molecule to be measured. It is mainly due to bead aggregation.

The difference in the number of large peaks between the first current measurement and the second current measurement represents aggregation caused by the molecule to be measured (8).

In this method, only aggregated beads by the molecule to be measured are disaggregated and the number is calculated, so the molecule to be measured regardless of the number of bead aggregations due to factors such as non-specific adsorption (not due to the molecule to be measured) The number of aggregated beads can be calculated.

In another embodiment, when the first current measurement (3) is completed, the capture site molecule (C) is added to the cell without removing the solution containing the aggregated beads that have moved to the other cell, A reaction for deaggregating only the aggregated beads by the molecule to be measured is performed.

When the end of the deagglomeration reaction is expected, a reverse voltage is applied so that the beads pass through the bottleneck toward the first added cell from the same cell, and the beads are moved to the original cell. At this time, the second current measurement is performed (7).

Also in this embodiment, the difference in the number of large peaks between the first current measurement and the second current measurement represents aggregation caused by the molecule to be measured (8).

In this embodiment, in order to promote the deagglomeration reaction in the cell, it is desirable to have a function capable of adjusting the cell temperature to the high temperature side.

In a preferred embodiment, it is desirable that the sample containing or possibly containing the molecule to be measured is serum or plasma in order to avoid excessive nonspecific adsorption.

By using the apparatus and method according to the present invention, for example, when the molecule to be measured is a molecule that is specifically secreted by cancer cells, it can be applied to early diagnosis and prognosis of cancer and monitoring of cancer progression. It becomes possible.

[Example 2] Surface plasmon resonance (SPR) experiment showing the antibody molecule binding inhibitory effect of the molecule to be measured by the capture site molecule Human cardiac toroponin I (hcTnI) and its amino acid sequence from the 41st to the 49th The IgG antibody 19C7 produced using the amino acids up to and including the epitope sequence oligopeptide (Ile-Ser-Ala-Ser-Arg-Lys-Leu-Gln-Leu (SEQ ID NO: 1); ISApep) was prepared. Biotinylated IgG antibody 19C7 is immobilized on GE Healthcare Biacore sensor chip SA, and a constant concentration of hcTnI solution is allowed to act. The molecular weight to be reacted was found to decrease, and the inhibition of the interaction between the molecule to be measured (hcTnI) and the antibody molecule (IgG antibody 19C7) by the capture site molecule (ISApep) was confirmed.

The measurement was performed using GE Healthcare Biacore X100. The sensor chip SA on which the IgG antibody 19C7 was immobilized was prepared according to the instructions of GE Healthcare. The sensor chip SA on which the IgG antibody 19C7 was fixed was mounted on the Biacore X100, and sufficiently equilibrated with HBS-EP + buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v Surfactant P20). The hcTnI solution adjusted to 0.1 μM using HBS-EP + buffer at a flow rate of 10 μL / min was added thereto for 90 seconds, and the sensorgram was measured. Next, a solution obtained by adding 1 μM ISApep to the hcTnI solution adjusted to 0.1 μM was added for 90 seconds, and the sensorgram was measured. Further, a solution obtained by adding 100 μM ISApep to the hcTnI solution adjusted to 0.1 μM was added for 90 seconds, and the sensorgram was measured. Finally, a solution obtained by adding 1 mM ISApep to the hcTnI solution adjusted to 0.1 μM was added for 90 seconds, and the sensorgram was measured.

FIG. 11 shows the sensorgrams overwritten for the above four trials. As the added capture site molecule (ISApep) increases in concentration, the height of the sensorgram indicating the interaction between the molecule to be measured (hcTnI) and the antibody molecule (IgG antibody 19C7) decreases, that is, the capture site. It shows an antagonistic inhibitory action by a molecule (ISApep).

[Example 3] Aggregate formation inhibition experiment using a capture site (epitope) peptide for the formation of bead aggregates by antigen binding to antibody molecule physical adsorption beads. Using antibody-immobilized beads, an antigen sandwich bead aggregate formation experiment was performed by antigen-antibody molecule interaction.

When the specific gravity of a polystyrene bead that is 0.2% w / v in the storage solution is 1.0, the concentration of particles having a diameter of 83 nm is 10.7 nM. 100 μL of each antibody stock solution was mixed and mixed therewith, and 0, 2, 6, 20, 60 μL of the cTnI solution diluted with the dispersion medium stock solution and adjusted to 50 nM was mixed and shaken at 37 ° C. for 60 minutes. When the cTnI solution is mixed with 0, 2, 6, 20, 60 μL, the number of antigen molecules relative to the number of beads is approximately as follows: 1: 0 (+0 μL), 1: 0.1 (+2 μL), 1 : 0.3 (+6 μL), 1: 1 (+20 μL), 1: 3 (+60 μL). The bead mixture after the reaction was subjected to a particle size measurement experiment using a dynamic light scattering measurement device (LB550, HORIBA, Ltd.). As a result, a 2-bead aggregation state of 80% <under the mixing condition of 1: 3 (+60 μL) was obtained. (If 100% aggregates 2 beads, the diameter should increase 2 ^ 1/3 = 25%, and the diameter increases by about 20% from the table below).

2. The capture site (epitope) peptide was added to the above-mentioned antigen sandwich bead aggregate formation to inhibit aggregate formation.

When the specific gravity of a polystyrene bead that is 0.2% w / v in the storage solution is 1.0, the concentration of particles having a diameter of 83 nm is 10.7 nM. 100 μL of each antibody stock solution was taken and mixed, and 60 μL of the cTnI solution diluted with the dispersion medium stock solution and adjusted to 50 nM was mixed and shaken at 37 ° C. for 60 minutes. When 60 μL of cTnI solution is mixed, the number of antigen molecules relative to the number of beads is 1: 3. At this time, the formation of the two-bead aggregation system is maximized (80% <, (Table 1)). Here, an epitope peptide solution was mixed as a capture site molecule, and it was observed whether or not aggregate formation inhibition occurred competitively depending on the concentration of the epitope peptide.

The epitope peptide solution diluted to 1 μM with a dispersion medium storage solution was mixed in order with 0, 1, 10, 100 μL and shaken at 37 ° C. for 60 minutes. When the cTnI solution is mixed with 0, 1, 10, 100 μL, the number of antigen molecules relative to the number of beads is approximately as follows: 1: 0 (+0 μL), 1: 1 (+1 μL), 1:10 (+10 μL) ), 1: 100 (+100 μL).

As a result, although a decrease in diameter increased by 20% was not observed under mixing conditions of 1: 0 (+0 μL) and 1: 1 (+1 μL), it decreased to about 10% increase at 1:10 (+10 μL), and 1: 100 (+100 μL) almost returned to the original diameter.

The apparatus and method according to the present invention are useful for detecting a molecule to be measured in a sample with high sensitivity and accuracy, and can be applied to medical, pharmaceutical, food, environmental and other fields including early diagnosis of diseases and the like. It is.

Claims (15)

1. An apparatus for detecting a molecule to be measured in a sample,
   A reaction unit for performing a reaction in which a first carrier immobilizing a first antibody that binds to one site of a molecule to be measured and a second carrier immobilizing a second antibody that binds to another site of the molecule to be measured are brought into contact with a sample; ,
   A sample introduction part for introducing the sample into the reaction part;
   A reagent supply unit for supplying the first carrier and the second carrier to the reaction unit;
   A reagent supply unit for supplying a capture site molecule comprising or including a site to which the first antibody of the molecule to be measured binds and / or a site to which the second antibody binds to the reaction unit;
   A measurement unit for measuring aggregation or adhesion between the first carrier and the second carrier;
   An apparatus comprising: a calculation unit that analyzes a measurement result obtained by the measurement unit.
2. The apparatus according to claim 1, wherein both the first carrier and the second carrier are fine particles.
3. The apparatus according to claim 2, wherein the measurement unit includes a device that measures a blocking current of a fine channel.
4. The apparatus according to claim 2, wherein the fine particles have a diameter of 10 nm to 10 μm.
5. The apparatus according to claim 1, wherein one of the first carrier and the second carrier is a fine particle, and the other is a substrate.
6. The apparatus according to claim 5, wherein the measurement unit includes a device for measuring by a surface plasmon resonance method, a crystal oscillator microbalance method, a surface scattered light measurement, or a field effect transistor.
7. A first carrier immobilizing a first antibody that binds to one site of the molecule to be measured and a second carrier immobilizing a second antibody that binds to another site of the molecule to be measured are brought into contact with the sample, whereby the first carrier A step of agglomerating or adhering to the second carrier,
   Measuring aggregation or adhesion between the first carrier and the second carrier;
   A capture site molecule consisting of or containing the site to which the first antibody of the molecule to be measured binds and / or the site to which the second antibody binds is added to the reaction solution, whereby the molecule to be measured and the first antibody Specific binding and / or specific binding between the molecule to be measured and the second antibody is inhibited or dissociated,
   Measuring aggregation or adhesion between the first carrier and the second carrier;
   Including the step of calculating specific binding between the molecule to be measured and the first antibody and the second antibody based on the difference in the degree of aggregation or adhesion of the carrier before and after the addition of the capture site molecule. A method for detecting a molecule to be measured.
8. The method according to claim 7, wherein both the first carrier and the second carrier are fine particles.
9. The method according to claim 8, wherein the aggregation of the first carrier and the second carrier is measured by a blocking current of a fine channel.
10. The method according to claim 8, wherein the fine particles have a diameter of 10 nm to 10 µm.
11. The method according to claim 7, wherein one of the first carrier and the second carrier is a fine particle, and the other is a substrate.
12. The adhesion between the first carrier and the second carrier is measured by a surface plasmon resonance method, a crystal oscillator microbalance method, a surface scattered light measurement, or a field effect transistor using the substrate as a measurement substrate. The method described.
13. The method according to claim 7, wherein the molecule to be measured is selected from the group consisting of peptides, proteins, sugar chains, cells, viruses, and bacteria.
14. The method according to claim 7, wherein the capture site molecule is selected from the group consisting of monosaccharide and polysaccharide molecules, and oligopeptides.
15. A kit for detecting a molecule to be measured in a sample,
    A first carrier immobilizing a first antibody that binds to one site of a molecule to be measured;
    A second carrier immobilizing a second antibody that binds to another site of the molecule to be measured;
    A kit comprising: a capture site molecule comprising or including a site to which the first antibody of the molecule to be measured binds and / or a site to which the second antibody binds.

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