JP2014149172A - New bead assay system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bead assay system for a wash-less state.SOLUTION: The present invention includes particles that are carriers for achieving a wash-less state in assay of two or more steps and that have a structure for connecting scavengers. The carriers have a pore capacity efficient to achieve a wash-less state. Another embodiment provides carriers that have a blocking index (BI) effective in achieving a wash-less state. Alternatively, the present invention provides carriers that achieve a wash-less state in assay of two or more steps, and contain 1) particles having a structure for connecting scavengers, and 2) scavengers. The scavengers and the particles directly meet or connect each other or connect via a linker. The carriers have a pore capacity effective in realizing a wash-less state.

Description

  The present invention provides a bead assay system that does not require a washing operation (washless), a short time, low cost, high sensitivity, and a simple bead assay system, and a particle or carrier (bead) used therein.

  In a commercially available bead assay system, a sample made of polystyrene beads with a reactive dye and a fluorescent dye is brought into contact with the sample, and a laser beam is irradiated to detect and measure the luminous intensity (flow cytometry). Qualitative and quantitative analysis of ingredients. It is called a multi-item simultaneous analysis method and is also called Multiple Analyte Profiling (Multiplex), and domestic and foreign manufacturers and contracted service businesses are advancing in the industry.

  However, in the conventional system, since each reaction needs to be washed for each reaction due to the background, it remains complicated.

  Patent Documents 1 to 3 disclose a method for synthesizing silica nanoparticles. Patent Document 4 and Non-Patent Document 1 disclose a bead assay technique. Patent Documents 5 to 6 disclose protein assay technology using silica particles modified with a thiol group (mercapto group). Patent Documents 7 to 11 disclose techniques for suppressing non-specific reaction and background reduction of ELISA. Patent Documents 12 to 17 disclose various immunoassay methods.

Japanese Patent No. 4572274 Japanese Patent No. 4716337 International Publication No. 2007/142316 Special table 2008-524600 gazette Special Table 2002-506517 JP-A-8-119619 Special table 2010-527005 International Publication No. 2009/025364 Special table 2009-500036 gazette International Publication No. 2005/010529 JP-A-7-218508 JP-A-5-72205 JP 2009-192386 A JP 2000-329767 A JP 2005-49276 A JP-A-8-166388 JP-A-5-264549

Luminex (registered trademark) system description (Hitachi Solutions, Philgen, Merck Millipore)

  The present inventors use particles typified by organic silica particles (particles made only from organoalkoxysilanes such as mercaptopropyltrimethoxysilane) and the like, with or without a blocking agent, and no washing operation is required. (Washless), low cost, high sensitivity bead assay system was developed.

  The bead assay by flow cytometry is becoming widely used in recent years because simultaneous multi-item measurement can be performed with a small amount of sample as compared with an ELISA measurement method using a conventional plate. R & D was conducted to realize a bead assay with high sensitivity and a wide measurement range using particles that can be washed with organic silica particles. According to the present invention, in the measurement of protein, a quantitative measurement in the range of several pg / ml to several hundred ng / ml, which is equivalent to the beads assay particles commercialized in the prior art, has been successfully performed in a washless manner. In addition to the measurement of antigen protein, we have succeeded in measuring genes via antibodies, biotinylated proteins, nucleic acids (eg, DNA, RNA, etc.). Furthermore, the reaction conditions were examined, and the measurement time was shortened and the efficiency improved. In the future, it will be possible to conduct simultaneous multi-item measurement and clinical testing of molecules important in biomedicine. In particular, the ability to perform washless measurement makes it possible to automate these measurements and inspections. The present invention provides the following. Further, it has been found that even nucleic acids and the like do not require blocking, and an assay technique that is much more efficient and simple than conventional techniques and can be easily automated can be provided.

  In one aspect, the present invention includes a carrier for achieving washless in a two or more stage assay comprising particles having a structure for binding a capture agent, wherein the carrier is used to achieve washless. A support having an effective pore volume is provided.

In one embodiment, the pore volume is 1.0 cm ³ / g or less.

  In one embodiment, the carrier has a blocking index (BI) effective to achieve washless.

  In one embodiment, the BI is 70% or more.

  In one embodiment, the particles are silica particles.

  In one embodiment, the particles are organic silica particles.

  In one embodiment, the particle can include a label. Such a label may be any entity (eg, substance, energy, electromagnetic wave, etc.) for distinguishing the target molecule or substance from others, but in one embodiment, in addition to particles Examples thereof include a radioactive label (RI (radioisotope)), a fluorescent dye, biotin, a chemiluminescent substance, and the like. The label that can be included in the particles of the present invention is preferably advantageously distinguishable from the label used for the labeling agent used in the assay.

  In one embodiment, the structure for binding the scavenger is thiol group, amino group, carboxyl group, maleimide group, N-succinimide ester group, and isocyanate group, mercaptopropyl group, aminopropyl group, thiocyanate propyl group, maleimide. A group selected from the group consisting of a group, an N-succinimide ester group, and an isocyanate group.

  In one embodiment, the particles are organic silica particles, and the structure for binding the scavenger is a thiol group, an amino group, a carboxyl group, a maleimide group, an N-succinimide ester group, and an isocyanate group, a mercaptopropyl group, A group selected from the group consisting of an aminopropyl group, a thiocyanate propyl group, a maleimide group, an N-succinimide ester group, and a group selected from the group consisting of isocyanate groups.

  In another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier according to the present invention and a capture agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a two-step or more washless assay kit comprising the carrier of the present invention and a blocking agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier of the present invention, a capture agent, and a blocking agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier according to the present invention and a linker-imparting agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a two-step or more washless assay kit comprising the carrier of the present invention, a linker-imparting agent, and a blocking agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a two-step or more washless assay kit comprising the carrier of the present invention, a linker-imparting agent, and a capture agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a two-step or more washless assay kit comprising the carrier of the present invention, a linker-imparting agent, a capture agent, and a blocking agent. Preferably, the kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In one embodiment of the kit of the present invention, the blocking agent is selected from the group consisting of skim milk, albumin, serum, surfactant, synthetic blocking agent, and mixtures thereof.

  In one embodiment of the kit of the present invention, the linker-imparting agent is a maleimide group, an N-succinimide ester group, an isocyanate group, an iodoacetyl group, a sulfo-N-succinimide ester group, a pyridyldithio group, and a phenyl azide. At least one functional group selected from the group consisting of groups, on the other hand, maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyl It includes at least one functional group selected from the group consisting of a dithio group, a phenyl azide group, and an optionally protected amino group, and optionally includes a spacer between the functional groups. Also preferably, the linker-providing agent is capable of binding to the structure of the functional group present in the particle preparation material. These linker imparting agents can be preferably used when the particles are organic silica.

  In one embodiment, the linker-providing agent is maleimidobutyric acid N-succinimidyl ester, N- (α-maleimidoacetoxy) -succinimide ester, N- (β-maleimidopropionic acid) hydrazide.TFA, N, N- Dicyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ε-maleimidocaproic acid, N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidocaproyloxy) Succinimide ester, N- (γ-maleimidobutyryloxy) succinimide ester 1,6-hexane-bis-vinylsulfone, N-κ-maleimidoundecanoic acid, N- (κ-maleimidoundecanoic acid) hydrazide, succinimidyl 4- (N -Maleimidomethyl) Chlohexane-1-carboxy- (6-amidocaproate), succinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- (4- N-maleimidophenyl) -butyric acid hydrazide.HCl, N-hydroxysuccinimidyl-4-azidosalicylic acid 3- (2-pyridyldithio) propionyl hydrazide, N- (p-maleimidophenyl) isocyanate N-succinimidyl (4′- Azidophenyl) 1,3′-dithiopropionate, succinimidyl 4-hydrazinonicotinate acetone hydrazone, N-succinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, succinimidyl 3- (bromo Acetamido) propionate, succinimidyl 4-formylbenzoate, succinimidyl 4-hydrazide terephthalate hydrochloride, N-succinimidyl iodoacetate N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 -Carboxylate, NHS-PEO2-maleimide, NHS-PEO4-maleimide, NHS-PEO8-maleimide, NHS-PEO12-maleimide, succinimidyl 4- (p-maleimido-phenyl) butyrate succinimidyl-6- (β- Maleimidopropionamido) hexanoate, 4-succinimidyloxycarbonylethyl-α- (2-pyridyldithio) toluenesuccinimidyl- (4-so Laren-8-yloxy) butyrate, N-succinimidyl 3- (2-pyridyldithio) propionate, ethylene glycol bis (sulfo-succinimidyl succinate), N- (ε-maleimidocaproyloxy) sulfosuccinimide ester, N -(Γ-maleimidobutyryloxy) sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N- (κ-maleimidoundecanoyloxy) sulfosuccinimide ester, sulfosuccinimidyl 6- ( 3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl (4-azido-salicylamido) hexanoate, sulfo Succinimidyl (4-azido-phenyldithio) propionate, sulfosuccinimidyl 2- [7-azido-4-methylcoumarin-3-acetamido] ethyl-1,3′-dithiopropionate, sulfosuccinimidyl- 2- (m-azido-o-nitrobenzamido) ethyl, 1,3′-sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, sulfosuccinimidyl 2- (p -Azido-salicylamido) ethyl 1,3'-dithiopropionate, sulfo-NHS- (2-6- [biotinamide] -2- (p-azidobenzamido) hexanoamido) ethyl-1,3'-dithiopro Pionate, sulfosuccinimidyl (perfluoroazidobenzamido) ethyl 1,3′-dithiopropione , Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- (p-maleimidophenyl) It is selected from the group consisting of butyrate, 1,6-bismaleimidohexane, bis-maleimidoethane, maleimide diethylene glycol, disuccinimidyl glutarate. Also preferably, the linker-providing agent is capable of binding to the structure of the functional group present in the particle preparation material. These linker imparting agents can be preferably used when the particles are organic silica.

  In another aspect, the present invention provides a two-step or more washless assay using the carrier of the present invention. Preferably, the assay of the present invention is capable of quantitative and / or qualitative measurements.

  In another aspect, the present invention provides (A) a capture agent-binding carrier obtained by directly or indirectly binding a capture agent to the carrier of the present invention; (B) the capture agent in the presence of a blocking agent. A step of contacting a sample that contains or seems to contain a test substance that specifically binds to the capture agent-binding carrier; (C) a label that specifically binds to the test substance when the test substance is not labeled; A step of further contacting a drug, or a contact with a binding agent that specifically binds to the test substance, and further contacting with a labeled agent that specifically binds to the binding agent (or a plurality of binding agents) And (D) detecting the label directly or indirectly bound to the particle and determining the detected label as the amount of the test substance in the sample. , Two or more stages To provide a Yuresuassei. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed.

  In another aspect, the present invention provides (A) a capture agent-binding carrier in which a capture agent is directly or indirectly bound to the carrier of the present invention; (B) the capture in the absence of a blocking agent. Contacting a sample that contains or appears to contain a test substance to which the agent specifically binds with the capture agent-binding carrier; (C) specifically binds to the test substance when the test substance is not labeled; A step of further contacting a labeling agent, or contacting a binding agent that specifically binds to the test substance, and further contacting a labeling agent that specifically binds to the binding agent (or further binding agent) And (D) detecting the label directly or indirectly bound to the particle, and determining the detected label as the amount of the test substance in the sample. Two or more stages To provide a shoe-less assay. In this form, the assay of the present invention can be said to be washless and blockingless. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed.

  In one embodiment, the labeling agent is a fluorescent label, and the self-fluorescence of the particles recognizes the effect of facilitating detection of the fluorescence of the fluorescent label.

  In another aspect, the present invention is a carrier for achieving washless in a two or more stage assay comprising 1) particles having a structure for binding a capture agent and 2) a capture agent, the capture agent And the particles are directly associated or bound, or linked via a linker, the carrier provides a carrier having a pore volume effective to achieve a washless.

In one embodiment, the pore volume is 1.0 cm ³ / g or less.

  In one embodiment, the carrier has a blocking index (BI) effective to achieve washless.

  In one embodiment, the BI is 70% or more.

  In one embodiment, the particles are silica particles.

  In one embodiment, the particles are organic silica particles.

  In one embodiment, the particle can include a label. In one embodiment, such a label may be any entity that distinguishes the molecule or substance of interest from others (eg, substance, energy, electromagnetic wave, etc.). Examples that may be added include radiolabels (RI (radioisotopes)), fluorescent dyes, biotin, chemiluminescent substances and the like. The label that can be included in the particles of the present invention is preferably advantageously distinguishable from the label used for the labeling agent used in the assay.

  In one embodiment, the structure for binding the scavenger is from thiol groups, amino groups, carboxyl groups, maleimide groups, N-succinimide ester groups, and isocyanate groups, mercaptopropyl groups, aminopropyl groups, thiocyanate propyl groups, A group selected from the group consisting of:

  In one embodiment, the particles are organic silica particles, and the structure for binding the scavenger is a thiol group, an amino group, a carboxyl group, a maleimide group, an N-succinimide ester group, and an isocyanate group, a mercaptopropyl group, A group selected from the group consisting of an aminopropyl group and a thiocyanate propyl group is included.

  In one embodiment, the capture agent is selected from the group consisting of proteins, peptides, avidin, streptavidin, nucleic acids, saccharides, variants thereof and conjugates thereof. In another embodiment, the capture agent is an antibody, an antigen-binding antibody fragment, an antigen, avidin, streptavidin, a nucleic acid, DNA, RNA, a sugar chain, a peptide, a ligand, a receptor, a modified form thereof, and a modified form thereof. Selected from the group consisting of fusions.

  In one embodiment, the capture agent and the particle are directly associated or bound.

  In one embodiment, the capture agent and the particle are attached via a linker.

  In one embodiment, the linker is selected from the group consisting of a maleimide group, an N-succinimide ester group, an isocyanate group, an iodoacetyl group, a sulfo-N-succinimide ester group, a pyridyldithio group, and a phenyl azide group on one side. At least one functional group, on the other hand, maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group, phenylazide group, And optionally protected thiol group, amino group, carboxyl group, at least one functional group selected from the group consisting of at least one functional group selected from, and optionally a spacer between the functional groups (Including phosphorus in this specification) Is the residue of also referred) and over imparting agent. Also preferably, this linker is bonded to the structure of a functional group present in the particle preparation material. These linkers can be preferably used when the particles are organic silica.

  In one embodiment, the linker is maleimidobutyric acid N-succinimidyl ester, N- (α-maleimidoacetoxy) -succinimide ester, N- (β-maleimidopropionic acid) hydrazide TFA, N, N-dichlorohexyl. Carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ε-maleimidocaproic acid, N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidocaproyloxy) succinimide ester N- (γ-maleimidobutyryloxy) succinimide ester 1,6-hexane-bis-vinylsulfone, N-κ-maleimidoundecanoic acid, N- (κ-maleimidoundecanoic acid) hydrazide, succinimidyl 4- (N-maleimide) Methyl) cyclohe Xan-1-carboxy- (6-amidocaproate), succinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- (4-N -Maleimidophenyl) -butyric acid hydrazide.HCl, N-hydroxysuccinimidyl-4-azidosalicylic acid 3- (2-pyridyldithio) propionyl hydrazide, N- (p-maleimidophenyl) isocyanate N-succinimidyl (4'-azide) Phenyl) 1,3′-dithiopropionate, succinimidyl 4-hydrazinonicotinate acetone hydrazone, N-succinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, succinimidyl 3- (bromoacetoa D) propionate, succinimidyl 4-formylbenzoate, succinimidyl 4-hydrazide terephthalate hydrochloride, N-succinimidyl iodoacetate N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 -Carboxylate, NHS-PEO2-maleimide, NHS-PEO4-maleimide, NHS-PEO8-maleimide, NHS-PEO12-maleimide, succinimidyl 4- (p-maleimido-phenyl) butyrate succinimidyl-6- (β- Maleimidopropionamido) hexanoate, 4-succinimidyloxycarbonylethyl-α- (2-pyridyldithio) toluenesuccinimidyl- (4-psoralen 8-yloxy) butyrate, N-succinimidyl 3- (2-pyridyldithio) propionate, ethylene glycol bis (sulfo-succinimidyl succinate), N- (ε-maleimidocaproyloxy) sulfosuccinimide ester, N- ( γ-maleimidobutyryloxy) sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N- (κ-maleimidoundecanoyloxy) sulfosuccinimide ester, sulfosuccinimidyl 6- (3 ′ -[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl (4-azido-salicylamido) hexanoate, sulfosucci Imidyl (4-azido-phenyldithio) propionate, sulfosuccinimidyl 2- [7-azido-4-methylcoumarin-3-acetamido] ethyl-1,3′-dithiopropionate, sulfosuccinimidyl- 2- (m-azido-o-nitrobenzamido) ethyl, 1,3′-sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, sulfosuccinimidyl 2- (p -Azido-salicylamido) ethyl 1,3'-dithiopropionate, sulfo-NHS- (2-6- [biotinamide] -2- (p-azidobenzamido) hexanoamido) ethyl-1,3'-dithiopro Pionate, sulfosuccinimidyl (perfluoroazidobenzamido) ethyl 1,3′-dithiopropionate, sulfo Hosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate, 1 , 6-bismaleimidohexane, Bis-maleimidoethane, maleimidodiethylene glycol, disuccinimidyl glutarate. Also preferably, this linker is bonded to the structure of a functional group present in the particle preparation material. These linkers can be preferably used when the particles are organic silica.

  In one embodiment, the carrier of the present invention utilizes a carrier in which a maleimide group, an N-succinimide ester group, an isocyanate group, or the like is bonded to the capturing agent and then bonded to particles.

  In one embodiment, the carrier of the present invention utilizes a maleimide activated neutravidin (protein with a maleimide group attached) particles.

  In one embodiment, a capture agent in which a protein or a linker is fused is used to directly associate or bind the capture agent with the particle.

  In one embodiment, the carrier of the present invention is prepared by either allowing the linker-providing agent to first undergo a binding reaction with the particle or first to the capture agent.

  In one aspect, the present invention provides a two-step or more washless assay kit comprising the carrier of the present invention and a blocking agent. Preferably, the assay kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In one embodiment, the blocking agent is selected from the group consisting of skim milk, albumin, serum, surfactant, synthetic blocking agent, and mixtures thereof.

  In one aspect, the present invention provides a two-step or more washless assay using the carrier of the present invention.

  In one aspect, the present invention provides (A) a step of providing a carrier of the present invention; (B) a sample that contains or appears to contain a test substance to which the capture agent specifically binds in the presence of a blocking agent. (C) when the test substance is not labeled, further contact with a labeled drug that specifically binds to the test substance or a binding drug that specifically binds to the test substance Is further contacted with a labeling agent that specifically binds to the binding agent (this step is skipped if there are two steps, and includes steps 3 and 4). And (D) detecting the label directly or indirectly bound to the capture agent-binding carrier, and detecting the detected label in the sample. Of the test substance in The step of determining to include, to provide a two-stage or more wash-less assay. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed.

  In one aspect, the invention includes (A) providing a carrier of the invention; (B) in the absence of a blocking agent, including or including a test substance to which the capture agent specifically binds. Contacting the sample with the carrier; (C) if the test substance is not labeled, further contacting with a labeled agent that specifically binds to the test substance, or binding specifically binding to the test substance A step of further contacting a labeled drug that specifically binds to the binding drug after contacting the drug (this process is skipped if there are two stages, and includes three and four stages) (Alternatively, a binding agent may be interposed more than once.); And (D) detecting the label directly or indirectly bound to the capture agent binding carrier, and detecting the detected label in the sample. The test substance in The step of determining the amount including, provides a two-stage or more wash-less assay. Thus, in this form, the assay of the present invention can be said to be blockingless and washless. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed.

  In one embodiment, the labeling agent is a fluorescent label, and the fluorescence of the fluorescent label is significantly higher than the autofluorescence of the particles.

  In one aspect, the present invention is a carrier for achieving washless in a two-step or more assay, comprising 1) particles having a structure that binds a capture agent, 2) a capture agent, and 3) a blocking moiety. In addition, the capture agent and the particles are directly associated or bound, or are linked via a linker, the carrier providing a carrier having a pore volume effective to achieve a washless.

In one embodiment, the pore volume is 1.0 cm ³ / g or less.

  In one embodiment, the carrier has a blocking index (BI) effective to achieve washless.

  In one embodiment, the BI is 70% or more.

  In one embodiment, the particles are silica particles.

  In one embodiment, the particles are organic silica particles.

  In one embodiment, the particle can include a label. In one embodiment, such a label may be any entity that distinguishes the molecule or substance of interest from others (eg, substance, energy, electromagnetic wave, etc.). Examples that may be added include radiolabels (RI (radioisotopes)), fluorescent dyes, biotin, chemiluminescent substances and the like. The label that can be included in the particles of the present invention is preferably advantageously distinguishable from the label used for the labeling agent used in the assay.

  In one embodiment, the structure for binding the scavenger is from thiol groups, amino groups, carboxyl groups, maleimide groups, N-succinimide ester groups, and isocyanate groups, mercaptopropyl groups, aminopropyl groups, thiocyanate propyl groups, A group selected from the group consisting of:

  In one embodiment, the particles are organic silica particles, and the structure for binding the scavenger is a thiol group, an amino group, a carboxyl group, a maleimide group, an N-succinimide ester group, and an isocyanate group, a mercaptopropyl group, A group selected from the group consisting of an aminopropyl group and a thiocyanate propyl group is included.

  In one embodiment, the surface structure of the particles of the present invention includes specific surface functional groups.

  In one embodiment, the capture agent is an antibody or an antigen-binding antibody fragment.

  In one embodiment, the capture agent and the particle are directly associated or bound.

  In one embodiment, the capture agent and the particle are attached via a linker.

  In one embodiment, the linker is selected from the group consisting of a maleimide group, an N-succinimide ester group, an isocyanate group, an iodoacetyl group, a sulfo-N-succinimide ester group, a pyridyldithio group, and a phenyl azide group on one side. At least one functional group, on the other hand, maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group, phenylazide group, And optionally a residue of at least one functional group selected from the group consisting of protected amino groups, optionally including a spacer between the functional groups. Also preferably, this linker is bonded to the structure of a functional group present in the particle preparation material. These linkers can be preferably used when the particles are organic silica.

  In one embodiment, the linker is maleimidobutyric acid N-succinimidyl ester, N- (α-maleimidoacetoxy) -succinimide ester, N- (β-maleimidopropionic acid) hydrazide TFA, N, N-dichlorohexyl. Carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ε-maleimidocaproic acid, N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidocaproyloxy) succinimide ester N- (γ-maleimidobutyryloxy) succinimide ester 1,6-hexane-bis-vinylsulfone, N-κ-maleimidoundecanoic acid, N- (κ-maleimidoundecanoic acid) hydrazide, succinimidyl 4- (N-maleimide) Methyl) cyclohe Xan-1-carboxy- (6-amidocaproate), succinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- (4-N -Maleimidophenyl) -butyric acid hydrazide.HCl, N-hydroxysuccinimidyl-4-azidosalicylic acid 3- (2-pyridyldithio) propionyl hydrazide, N- (p-maleimidophenyl) isocyanate N-succinimidyl (4'-azide) Phenyl) 1,3′-dithiopropionate, succinimidyl 4-hydrazinonicotinate acetone hydrazone, N-succinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, succinimidyl 3- (bromoacetoa D) propionate, succinimidyl 4-formylbenzoate, succinimidyl 4-hydrazide terephthalate hydrochloride, N-succinimidyl iodoacetate N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 -Carboxylate, NHS-PEO2-maleimide, NHS-PEO4-maleimide, NHS-PEO8-maleimide, NHS-PEO12-maleimide, succinimidyl 4- (p-maleimido-phenyl) butyrate succinimidyl-6- (β- Maleimidopropionamido) hexanoate, 4-succinimidyloxycarbonylethyl-α- (2-pyridyldithio) toluenesuccinimidyl- (4-psoralen 8-yloxy) butyrate, N-succinimidyl 3- (2-pyridyldithio) propionate, ethylene glycol bis (sulfo-succinimidyl succinate), N- (ε-maleimidocaproyloxy) sulfosuccinimide ester, N- ( γ-maleimidobutyryloxy) sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N- (κ-maleimidoundecanoyloxy) sulfosuccinimide ester, sulfosuccinimidyl 6- (3 ′ -[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl (4-azido-salicylamido) hexanoate, sulfosucci Imidyl (4-azido-phenyldithio) propionate, sulfosuccinimidyl 2- [7-azido-4-methylcoumarin-3-acetamido] ethyl-1,3′-dithiopropionate, sulfosuccinimidyl- 2- (m-azido-o-nitrobenzamido) ethyl, 1,3′-sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, sulfosuccinimidyl 2- (p -Azido-salicylamido) ethyl 1,3'-dithiopropionate, sulfo-NHS- (2-6- [biotinamide] -2- (p-azidobenzamido) hexanoamido) ethyl-1,3'-dithiopro Pionate, sulfosuccinimidyl (perfluoroazidobenzamido) ethyl 1,3′-dithiopropionate, sulfo Hosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- (p-maleimidophenyl) butyrate, 1 , 6-bismaleimidohexane, bis-maleimidoethane, maleimidodiethylene glycol, disuccinimidyl glutarate or a residue thereof. Also preferably, this linker is capable of binding to the structure of the functional group present in the particle preparation material. These linkers can be preferably used when the particles are organic silica.

  In one embodiment, the blocking moiety is selected from the group consisting of skim milk, albumin, serum, surfactant, synthetic blocking agent, and mixtures thereof.

  In one aspect, the present invention provides a kit for a two or more washless assay comprising the carrier of the present invention. Preferably, the assay kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides a two-step or more washless assay using the carrier of the present invention. Preferably, the assay kit of the present invention is capable of quantitative measurement and / or qualitative measurement.

  In another aspect, the present invention provides (A) a step of providing a carrier of the present invention; (B) a step of contacting a sample suspected of containing or including a test substance to which the capture agent specifically binds, with the carrier. (C) when the test substance is not labeled, further contact with a labeled agent that specifically binds to the test substance, or after contacting a binding agent that specifically binds to the test substance, A step of further contacting a labeled agent that specifically binds to the binding agent (this step is skipped if there are two steps, and includes steps 3 and 4) (or further binding And (D) detecting the label directly or indirectly bound to the capture agent-binding carrier, and detecting the detected label in the amount of the test substance in the sample. Including the step of determining, To provide a more stages of the wash-free assay.

  Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed.

  In one embodiment, the labeling agent is a fluorescent label, and the fluorescence of the fluorescent label is significantly higher than the autofluorescence of the particles.

  Alternatively, in another aspect, a washless assay carrier comprising 1) particles, the particles comprising 1-1) a core 1-2) surface structure, wherein the carrier comprises a washless assay A carrier having a blocking index (BI) effective for realizing the above is provided.

  It is understood that the present invention includes those in which the above features are further used in any combination. Preferred embodiments of the present invention will be shown below, but those skilled in the art can appropriately implement the embodiments and the like from the description of the present invention and well-known and common techniques in the field, and the effects and effects of the present invention can be easily achieved. It should be recognized to understand.

  The present invention realizes an assay that does not require a washing step, particularly an assay by flow cytometry, which could not be achieved conventionally. Quantitative measurements in the range of a few pg / ml to several hundred ng / ml, comparable to the bead assay particles commercialized in the prior art, can be performed in a washless manner.

FIG. 1 shows the measurement reaction on the surface of the organic silica particles. Two-stage reaction (left figure), three-stage (middle figure), and four-stage (right figure) sandwich reaction for antibody binding evaluation. It is a schematic diagram of the internal structure of the organic silica particle of this invention. FIG. 2 shows an electron microscopic image of organosilica particles for bead assay. Bar indicates 10 μm. Photographing was performed with an electron microscope. The whole image was taken with a scanning electron microscope (JEOL, JCM5700), and the upper right photo was taken with a transmission electron microscope (Hitachi, H7650). FIG. 3 shows antibody binding to the particle surface. This is an example in which an amino group of an antibody is bonded to a thiol group of silica particles. The figure below shows 4-maleimidobutyric acid N-succinimide ester as a linker. FIG. 4 shows an evaluation example of antibody binding activity. A primary evaluation system (FIG. 1 left) by a two-stage assay was established, and the antibody binding method, blocking conditions were examined, and the affinity of the antibody for the antigen was evaluated. Various anti-BSA antibodies were bound to the particle surface. Various antibodies such as mouse monoclonal antibodies, rabbit and sheep polyclonal antibodies were examined. One hour after addition of various concentrations of FITC-labeled BSA, measurement was performed by flow cytometry. The mouse monoclonal antibody (♦) can only measure from 100 ng / ml to 10 ng / ml, whereas the rabbit polyclonal antibody (●) can measure a wide range from 100 ng / ml to 2.5 ng / ml. . The primary evaluation made it possible to select an antibody with high binding activity suitable for sandwich measurement. In addition, this evaluation can be applied as a simple evaluation method for various antigen-antibody reactions. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 5 is a schematic diagram of particles in which BSA is bonded to particles in which an anti-BSA antibody is bonded to the particle surface. FIG. 6 shows the results of examining the reaction time. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). The reaction time was 0.5 hours (●), 1 hour (■), and 2 hours (♦). FIG. 7 shows the results of examining the reaction temperature. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). The temperature was 37 ° C. (●) and 25 ° C. (▲). FIG. 8 shows an example of BSA sandwich measurement (1) in Example 7. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 9 shows a schematic diagram of the sandwich assay of Example 7. The schematic diagram of the particle | grains which fluorescent-labeled anti- BSA antibody (3) couple | bonded after BSA (2) couple | bonds with the particle | grains (1) which couple | bonded the anti- BSA antibody with the thiol organic silica particle surface is shown. SH on the particles indicates the terminal structure of the organic component on the surface of the organic silica particles, and the schematic diagram indicates that the particles are thiol organic silica particles. The right side of the particle shows an antibody conjugated with SH and a reaction model of a sandwich assay. FIG. 10 shows a sandwich measurement of the antibody of Example 8. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 11 shows a schematic diagram of the sandwich assay of Example 9. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 12 shows a schematic diagram of the sandwich assay of Example 9. The schematic diagram of the particle | grains which the streptavidin (3) labeled with PE was couple | bonded after the piotin labeled anti-sheep IgG antibody (2) couple | bonded with the sheep anti- BSA antibody (1) couple | bonded with the particle | grain surface is shown. SH on the particles indicates the terminal structure of the organic component on the surface of the organic silica particles, and the schematic diagram indicates that the particles are thiol organic silica particles. The right side of the particle shows an antibody conjugated with SH and a reaction model of a sandwich assay. FIG. 12A shows the SSC and FL2 profiles of the bead assay of Example 9 in (A) and (C), respectively. In (A), the region of the particle scattered light is designated by R8. SSC and FL2 gated in region R8 are shown in FIGS. The X-axis of each profile indicates the intensity of scattered light (SSC-H) and fluorescence (FL2-H), and the Y-axis indicates the particle count value (counts) at each intensity. (D) The geometric mean (GeoMean) of the particle fluorescence in the profile was 1938.97, and this value was defined as the value of the particle fluorescence bound with the streptavidin labeled with PE. FIG. 13 shows the results of quantification of a biotin-labeled antibody that binds to Neutravidin in Example 10. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 14 shows the results of quantification of the amount of adsorbed binding of the anti-GST antibody of Example 11. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 15 shows a comparison of the experimental example of Example 12 with and without cleaning. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). A triangle indicates washless, and a circle indicates that there is a wash. FIG. 16 shows the results of experiments on the necessity of washing in the assay using Bio-Plex of Example 13. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). The left shows washing and the right shows no washing. FIG. 17 shows the results of the four-step reaction of Example 14. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 18 shows the results of measurement using the multistage antigen-antibody reaction of Example 15. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 19 shows the results of measurement using the antigen-antibody reaction of Example 16 and avidin-biotin binding. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents protein concentration (pg / ml). FIG. 20 illustrates binding via biotin as an example of a capture agent-bound carrier in which a capture agent is indirectly bound to the carrier of the present invention. FIG. 21 shows the results of a multi-step assay in which the binding of the particles of Example 23 and the capture agent is adsorption. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (pg / ml). FIG. 22 shows the results of washless one-step measurement of Example 24. FIG. 22 shows the case of using 10 μl of Bio-rad Bio-Plex COOH beads (0.8 × 10 ⁷ / ml) not subjected to blocking treatment or the like. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (pg / ml). FIG. 23 shows the results of the washless one-step measurement of Example 24. FIG. FIG. 23 shows the case where 5 μl of thiol organic silica particles are used. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (pg / ml). FIG. 24 shows the results of an assay using Sephadex G-50. FIG. 24 is the result of a one-step assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (pg / ml). FIG. 25 shows the results of an assay using Sephadex G-50. FIG. 24 is the result of a two-stage assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (pg / ml). FIG. 26 shows the results of an assay using Silica Gel. FIG. 26 is the result of a one-step assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (ng / ml). FIG. 27 shows the results of an assay using Silica Gel. FIG. 27 is the result of a two-stage assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (ng / ml). FIG. 28 shows the results of an assay using rhodamine red-containing thiol organosilica particles. FIG. 28 is the result of a two-stage assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (ng / ml). FIG. 29 shows the results of an assay using epoxy organosilica particles. FIG. 29 shows the results of a one-step assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (ng / ml). FIG. 30 shows the results of an assay using epoxy organosilica particles. FIG. 30 is the result of a two-stage assay. The vertical axis represents fluorescence intensity (GeoMean), and the horizontal axis represents antibody concentration (ng / ml). FIG. 31 shows a schematic diagram of the binding of the nucleotide sequence having an amino group and the thiol group of the particle by 4-maleimidobutyric acid N-succinimidyl ester of the particles used in Example 31. FIG. 32 shows a schematic diagram in which binding particles bound to nucleotide A bind to nucleotides complementary to target fluorescently labeled nucleotide A in the assay of Example 31.

  The present invention will be described below. Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. Therefore, it is understood that modifiers such as singular articles (eg, “a”, “an”, “the” in the case of English, etc.) also include the plural concept unless otherwise stated. Should. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified. Thus, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

(Definition of terms)
Listed below are definitions of terms particularly used in the present specification.

  In the present specification, the “washless” assay means that, in an assay such as a sandwich assay, a desired assay result can be obtained without a washing step after contacting a reagent with a sample. As a typical example, for example, (A) a step of providing a capture agent-binding carrier obtained by binding a capture agent to a carrier for achieving the washless of the present invention; (B) in the presence of a blocking agent, Contacting a sample that contains or appears to contain a test substance to which the capture agent specifically binds with the capture agent-binding carrier; (C) when the test substance is not labeled, specifically binds to the test substance Or a step of contacting a binding agent that specifically binds to the test substance, and further contacting a labeling agent that specifically binds to the binding agent (or further binding agent) And (D) detecting the label directly or indirectly bound to the particle, and determining the detected label as the amount of the test substance in the sample. Inclusion method In this regard, the ability to detect and / or judge in the step (D) can be given even if the washing step is not included after the steps (B), (C), etc., but is not limited thereto. Not. And it is not necessary to use the centrifuge, filter membrane, filter plate, and aspirator required for the wash, and the assay does not lose the beads associated with the wash. (One kind of operation assay), but is not limited thereto. Alternatively, the drug may include an antibody or an antibody fragment, but other biomolecules such as DNA may be used in addition to the antibody. Preferably, achievement of washless is achieved by an assay of two steps or more. For example, in the case of a two-step assay, the labeling agent usually binds specifically to the capture agent. However, it will be understood that usually more than two stages will result in unlabeled drug, and the multi-stage final stage will be labeled drug. In the washless assay of the present invention, antigen, antibody (both monoclonal antibody and polyclonal antibody), receptor, hapten, enzyme, protein, peptide, nucleic acid, drug, hormone, chemical, polymer, pathogen, toxin, aptamer, Various analytes can be detected and analyzed in a broad sense that can be a displayed microorganism such as a phage, or a variant thereof, or a combination or conjugate thereof. These analytes can be antibodies, antigen-binding antibody fragments, antigens, avidin, streptavidin, nucleic acids, DNA, RNA, sugar chains, peptides, ligands, receptors, variants thereof and fusions thereof .

  As used herein, “stage” for an assay refers to the number of agents or factors (including capture agents) involved up to the label bound to the carrier of the present invention. Typically, the stage can be represented by the number of steps of the binding reaction to the particle. For example, in the schematic diagram of FIG. 1, in the leftmost two stages, the capture agent binds to the particle. (First step), the test substance labeled with this binds (second step). In the third stage, the capture agent binds to the particles (first stage), the test substance binds to this (second stage), and the labeled binding agent binds to the test substance (third stage). In step 4, the capture agent binds to the particles (first step), the test substance binds to this (second step), and the binding agent binds to the test substance (third step). (4th stage) The number of these bonds can be counted (or the binding agent may be further intervened several times). Examples of two-step assays include protein / dot blotting, nucleic acid dot blotting, phage dot blotting, gene (DNA, RNA) hybridization (hybridization), examples of three-step and four-step assays include sandwich assays, enzyme ligation Examples include an immunosorbent assay (Enzyme-Linked Immunosorbent Assay; ELISA), an enzyme antibody method, Western blotting, radioimmunoassay, an enzyme-linked immunospot (ELISpot) assay, and the like. In the assay of the present invention, DNA and the like can be measured in addition to antibodies (for example, a DNA chip). In this case, a step of further contacting the DNA chip is included. In the case of a two-step assay, it is a labeled agent that specifically binds to the capture agent, but usually it is an unlabeled agent if there are three or more steps. And the multistage final stage is the labeling drug.

  In the present specification, the term “particle” is used in the broadest sense in the field, and refers to a fine existence constituting a substance. For example, the particle that can be used in the present invention has a diameter of less than 1 millimeter. Yes, when using commercialized flow cytometry, it is preferably a size in the range of about 0.1-100 micrometers (μm) in diameter. The microparticles can be of any size, but the preferred size is about 1-50 μm, more preferably about 2-30 μm, even more preferably about 3-15 μm, and even more preferably about 2-10 μm. It is. Alternatively, currently used flow cytometry instruments range from about 100 nanometers (nm) to about 100,000 nm in invention diameters that can be used in the present invention. The optimal preferred diameter is between about 100 and about 40,000 nm, preferably between about 1000 and about 10,000 nm, more preferably between about 2000 and about 5000 nm. It should be noted that the appropriate particle size also depends on the performance of the flow cytometry device, so this value can vary as the flow cytometry device is modified.

  As used herein, “capture agent” refers to any agent or factor (both agents in English) that can capture a test substance for detection in a certain assay. Representative examples of such capturing agents include, but are not limited to, proteins such as antibodies, nucleic acid molecules such as DNA, lipid molecules, and other small molecules.

As used herein, “antibody” broadly refers to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, chimeric antibodies, and anti-idiotype antibodies, and fragments thereof such as Fv fragments, Fab ′ fragments, F (ab ′). ₂ and Fab fragments, as well as other recombinantly produced conjugates or functional equivalents (eg, chimeric, humanized, multifunctional, bispecific or oligospecific antibodies, single chain Antibody, scFv, diabody, sc (Fv) ₂ (single chain (Fv) ₂ ), scFv-Fc). In addition, such antibodies may be covalently linked or recombinantly fused to enzymes such as alkaline phosphatase, horseradish peroxidase, alpha galactosidase, and the like. The antibody used in the present invention may be bound to a target, and its origin, type, shape, etc. are not limited. Specifically, known antibodies such as non-human animal antibodies (eg, mouse antibodies, rat antibodies, camel antibodies), human antibodies, chimeric antibodies, and humanized antibodies can be used. In the present invention, monoclonal or polyclonal antibodies can be used as antibodies, but monoclonal antibodies are preferred. The binding of the antibody to the protein to the subject is preferably specific binding. Antibodies with improved affinity due to amino acid mutations and the like can also be applied.

  As used herein, “antigen” refers to any substrate that can be specifically bound by an antibody molecule. As used herein, “immunogen” refers to an antigen capable of initiating lymphocyte activation that produces an antigen-specific immune response. As used herein, “epitope” or “antigenic determinant” refers to a site in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well known techniques once the primary sequence of the nucleic acid or amino acid is provided.

  Methods for detecting antigen-antibody reactions are well known to those skilled in the art. For example, a target object can be detected by an immunological method. Immunological methods include cell tissue samples that have been appropriately treated, such as cell separation and extraction procedures, immunohistochemical staining, enzyme immunoassay, western blotting, agglutination, competition, etc. A known method such as a method or a sandwich method can be applied. The immunohistochemical staining method can be performed by, for example, a direct method using a labeled antibody, an indirect method using a labeled antibody against the antibody, or the like. As the labeling agent, known labels such as fluorescent substances, radioactive substances, enzymes, metals, and dyes can be used.

  As used herein, “means” refers to any tool that can achieve a certain purpose (eg, detection, diagnosis, treatment), and “capturing means” refers to a means that can capture a certain object. The “detecting means” means means capable of detecting a certain object based on a label or the like.

  Any detection means can be used in the assay of the present invention. For example, the means is a flow cytometry, cell sorter, microchip, flow cell, bead array, or other apparatus capable of measuring the fluorescence of one particle. In addition, mass spectrometer, nuclear magnetic resonance analyzer, X-ray analyzer, SPR, chromatography (eg, HPLC, thin layer chromatography, gas chromatography), immunological means (eg, Western blotting, EIA (enzyme immunoassay) ), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay)), biochemical means (eg, pI electrophoresis, Southern blotting, two-dimensional electrophoresis), electrophoresis instrument, chemical analysis instrument, two-dimensional fluorescence Differential electrophoresis (2DE-DIGE), isotopic labeling (ICAT), tandem affinity purification method (TAP method), physical means, can be utilized such as laser microdissection and combinations thereof.

  As used herein, “diagnosis” refers to identifying various parameters related to a disease, disorder, condition, etc. in a subject and determining the current state or future of such a disease, disorder, condition. By using the carrier, assay, method, kit, and the like of the present invention, the state in the body can be examined, and such information can be used to diagnose a disease, disorder, or condition in a subject.

  In the present specification, the “structure that binds a capture agent” means a direct association such as adsorption through a hydrophobic bond, a hydrogen bond, an electrostatic bond or the like, or a bond (covalent bond) such as a disulfide bond or an amide bond, or , Refers to any structure that allows binding through a linker, and further to a structure in which a capture agent binds to particles. Examples of functional groups that give such a structure include, for example, when organic silica particles are used, thiol groups, amino groups, carboxyl groups, maleimide groups, N-succinimide ester groups, and isocyanate groups, mercaptopropyl groups, A group selected from the group consisting of an aminopropyl group, a thiocyanate propyl group, a maleimide group, an N-succinimide ester group, and an isocyanate group can be exemplified, but not limited thereto.

  In the present specification, the “linker” refers to a part of a substance having a function of binding a certain substance to another substance. Further, in this specification, a drug that imparts a “linker” is referred to as a “linker imparting agent”. When the linker-imparting agent is reacted with the two substances to be bound to each other and binding occurs, the resulting binding moiety becomes a “linker”. Accordingly, the “linker” can be referred to as a residue (part) of a linker-imparting agent. In the present specification, for convenience of explanation, “linker” may be referred to including a linker-imparting agent in a broad sense, but those skilled in the art can recognize and understand these differences. In the present invention, the capture agent and the particles can be bound via a linker. In one embodiment, the linker-imparting agent used in the present invention is a maleimide group, an N-succinimide ester group, an isocyanate group, an iodoacetyl group, a sulfo-N-succinimide ester group, a pyridyldithio group, and a phenyl azide. At least one functional group selected from the group consisting of groups, on the other hand, maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyl Polyethylene glycol (PEO) of various lengths including at least one functional group selected from the group consisting of a dithio group, a phenyl azide group, and an optionally protected amino group, if necessary, between the functional groups Can include those containing spacers But not limited to these. Thus, it is understood that the linkers that can be used in the present invention can use the residues of examples of these linker-providing agents.

  Specific examples of the linker-imparting agent include maleimidobutyric acid N-succinimidyl ester, N- (α-maleimidoacetoxy) -succinimide ester, N- (β-maleimidopropionic acid) hydrazide.TFA, N, N-dichlorocarbodiimide 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ε-maleimidocaproic acid, N- (ε-maleimidocaproic acid) hydrazide, N- (ε-maleimidocaproyloxy) succinimide ester, N- (γ-maleimidobutyryloxy) succinimide ester 1,6-hexane-bis-vinylsulfone, N-κ-maleimidoundecanoic acid, N- (κ-maleimidoundecanoic acid) hydrazide, succinimidyl 4- (N-maleimidomethyl) ) Cyclohexa -1-carboxy- (6-amidocaproate), succinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- (4-N -Maleimidophenyl) -butyric acid hydrazide.HCl, N-hydroxysuccinimidyl-4-azidosalicylic acid 3- (2-pyridyldithio) propionyl hydrazide, N- (p-maleimidophenyl) isocyanate N-succinimidyl (4'-azide) Phenyl) 1,3′-dithiopropionate, succinimidyl 4-hydrazinonicotinate acetone hydrazone, N-succinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, succinimidyl 3- (bromoacetamide) Lomoacetamido) propionate, succinimidyl 4-formylbenzoate, succinimidyl 4-hydrazide terephthalate hydrochloride, N-succinimidyl iodoacetate N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4- (N-maleimidomethyl) cyclohexane 1-carboxylate, NHS-PEO2-maleimide, NHS-PEO4-maleimide, NHS-PEO8-maleimide, NHS-PEO12-maleimide, succinimidyl 4- (p-maleimido-phenyl) butyrate succinimidyl-6- (β -Maleimidopropionamido) hexanoate, 4-succinimidyloxycarbonylethyl-α- (2-pyridyldithio) toluenesuccinimidyl- ( -Soralen-8-yloxy) butyrate, N-succinimidyl 3- (2-pyridyldithio) propionate, ethylene glycol bis (sulfo-succinimidyl succinate), N- (ε-maleimidocaproyloxy) sulfosuccinimide ester, N- (γ-maleimidobutyryloxy) sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N- (κ-maleimidoundecanoyloxy) sulfosuccinimide ester, sulfosuccinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamide) hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl (4-azido-salicylamide) hexanoate Sulfosuccinimidyl (4-azido-phenyldithio) propionate, sulfosuccinimidyl 2- [7-azido-4-methylcoumarin-3-acetamido] ethyl-1,3′-dithiopropionate, sulfosk Cinimidyl-2- (m-azido-o-nitrobenzamido) ethyl, 1,3'-sulfosuccinimidyl 6- (4'-azido-2'-nitrophenylamino) hexanoate, sulfosuccinimidyl 2- (p-azido-salicylamido) ethyl 1,3′-dithiopropionate, sulfo-NHS- (2-6- [biotinamide] -2- (p-azidobenzamido) hexanoamido) ethyl-1,3 '-Dithiopropionate, sulfosuccinimidyl (perfluoroazidobenzamido) ethyl 1,3'-dithiopropiate Onate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- (p-maleimidophenyl) Commercially available products such as butyrate, 1,6-bismaleimidohexane, bis-maleimidoethane, maleimide diethylene glycol, disuccinimidyl glutarate, etc. can be used, but not limited to these, and synthesized ones Can be used. A coupling method using click chemistry can also be applied. Thus, it is understood that the linkers that can be used in the present invention can use the residues of examples of these linker-providing agents.

  Examples of the “structure in which the scavenger binds to the particles” include, for example, a linker-providing agent that can bind to the scavenger, for example, maleimide group, N-succinimide ester group, isocyanate group, iodoacetyl group, sulfo- Including at least one functional group selected from the group consisting of an N-succinimide ester group, a pyridyldithio group, and a phenyl azide group, on the other hand, a maleimide group, an N-succinimide ester group or an isocyanate group, a hydrazide group, a carboxy group, A structure formed by bonding an iodoacetyl group, a sulfo-N-succinimide ester group, a pyridyldithio group, or a phenylazide group to a particle is also included.

  Specifically, maleimide activated neutravidin, bovine serum albumin introduced with a maleimide group. (BSA), a protein in which a maleimide group is introduced, a structure formed by binding a nucleic acid, etc. to the particle.

  In the present invention, the capture agent and the particles may be directly associated or bound. In this specification, “association” refers to a state in which the capture agent and the particles are integrated by a force other than a covalent bond, and “bond” refers to a state in which they are generally integrated by a covalent bond. A bond that does not involve a linker is referred to as a direct bond.

As used herein, “total pore volume” refers to the volume of pores in a particle or carrier. In this specification, “pore volume (pore volume)”. Used interchangeably. The total volume of all pores per constant weight of material is the total pore volume, which is expressed in cm ³ / g or the like. There are many small holes called pores on the solid surface, and the size, structure, and amount of the pores are closely related to physical properties (strength, adsorptivity, reactivity, density, catalysis). The pore volume can be calculated by a mercury intrusion method (mercury intrusion porosimeter) or a gas adsorption method. In the gas adsorption method, a specific surface area and pore distribution can be determined from the amount of adsorbed gas molecules by adsorbing a gas (such as nitrogen) having a known molecular cross-sectional area and properties on the solid surface. For measurement of pore volume, etc., http://www.an.shimadzu.co.jp/powder/lecture/practice/p02/lesson14.htm; http://www.nippon-bel.co.jp/tech /seminar17.html;
You can visit http://www.jsac.or.jp/bunseki/pdf/bunseki2009/200907kaisetsu.pdf.

  As used herein, “particle size” is an index indicating the size of a particle to be measured, and can be expressed by the diameter of the particle. This “particle size” can be measured and determined by various techniques. For example, the particle size can also be determined by using a transmission electron microscope. A preferable particle size for the particles used in the carrier of the present invention can be 2 to 8 μm, and this depends on the performance of the measuring device (flow cytometry). In the present invention, 3 to 5 μm can be used.

As used herein, “effective pore volume to achieve washless” refers to a pore volume sufficient to achieve a washless assay. In the prior art, a washless assay has not been realized. This is because the labeling substance not directly or indirectly bound to the test substance enters the pores in the particle and is measured as the fluorescence of the particle in the same manner as the labeling substance bound to the particle. Therefore, it is necessary to wash out the labeling substance that has entered the pores by washing. Without wishing to be bound by theory, a particle with a pore volume below a certain level will achieve a washless because the background will drop to the extent that it will not substantially interfere with the evaluation of the assay results. It is demonstrated in the present invention that this can be done. The pore volume for achieving such washless is 1 cm ³ / g or less, 0.5 cm ³ / g or less, 0.4 cm ³ / g or less, 0.3 cm ³ / g or less, 0.2 cm ³ / g or less, 0.1 cm ³ / g or less, 0.05 cm ³ / g or less, 0.04 cm ³ / g or less, 0.03 cm ³ / g or less, 0.0 cm ³ / g or less, 0.0 cm ³ / g The following can be mentioned, but it is not limited to them. It is preferable that it is 0.02 cm < ³ > / g or less.

  In the present specification, the “carrier” (or carrier) refers to any substance or support that shows the ability to bind other substances and serves as a foundation for fixing the other substances. The carrier itself is preferably chemically stable and does not impede the intended operation.

In this specification, the “blocking index (BI)” is an index indicating the degree of binding of the blocking agent, and is calculated by the following formula.
Blocking Index (BI) =
((Particle fluorescence without blocking treatment)-(Particle fluorescence with blocking treatment))
/ (Particle fluorescence without blocking treatment) x 100 (%).

  In this specification, “blocking index (BI) effective for realizing washless” means 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more 99% or more, preferably 85% or more. Although not wishing to be bound by theory, (1) the block “efficiency” of the blocking agent to the carrier is good, or (2) the pore volume of the carrier is small and the labeling substance does not enter the pores, or Both of them are considered to be characteristics of the present invention, for example, washless or increase in sensitivity. The blocking index is achieved by the presence of blocking portions at a certain density on the particles. It was found in the process of the present invention that the commercially available latex beads (1) have a poor block “efficiency” of the blocking agent and that the pore volume of the carrier is large and the labeling substance easily enters the pores. When a fluorescence-labeled antibody that should not originally bind to latex beads is reacted and measured in a washless manner, fluorescence much higher than that of particles can be confirmed. Subsequently, when washing was performed according to the assay protocol for commercially available latex beads, it was confirmed that the fluorescence from the particles decreased according to the number of washings. In a typical example, the blocking index was changed from 16.6% to 50.3 after 3 washes. Rose to%. However, after several washes, the fluorescence did not drop to the background level. As a result, commercially available latex beads have both non-specific and gentle adsorption / absorption of fluorescently labeled antibodies (contained in the surface layer and inside like a sponge) and tight binding on the particle surface. It is possible. Further, the same blocking agent as that of the organic silica particles of the present invention was reacted with commercially available latex beads and measured in the same manner as described above. However, although some blocking effect was recognized, there was no effect as seen in the organic silica particles. From this, it is understood that it is related to the binding mode with the blocking agent, the surface structure, etc. instead of or in addition to the pore volume. Although not wishing to be bound by theory, it does not bind to a non-target object (although the selective property binds to the target object) (the binding to the target object is the measurement result itself). The characteristic that does not enter the carrier is the blocking index, and particles having characteristics that can suppress nonspecific binding and nonspecific content in flow cytometry measurement are provided. Commercially available particles have low selection characteristics, and non-specific binding can be excluded by washing, but without washing, the labeling substance enters the particles (infiltration), which affects the measurement.

  As an advantage over commercially available latex beads, bacterial growth often occurs during storage in latex beads. As a typical example, the silica particles are basically an inorganic material, so that bacterial growth does not occur. Therefore, an effect that the necessity of adding an antibacterial agent or the like is low is also recognized. As an example, it has been demonstrated that bacterial growth does not occur even in particles stored at room temperature for half a year.

  Examples of experimental methods for binding the capture agent to the particle surface include the following.

(1) The dry weight of particles such as organic silica particles having an appropriate average particle diameter is measured. The specific gravity of the particles is calculated, and an appropriate number of particles is calculated and dispersed in distilled water. after that,
(2) Next, a linker-imparting agent is bound,
(3) Next, a capture agent (for example, an antibody or the like) is bound, the particles are washed, dried, and weighed. The weight difference between (3) and (1) is the capture agent (antibody) binding amount. Measure the increase. Calculate the molecular weight of the scavenger, and calculate the number of scavengers per 1 mg of particles and the number of binding of scavengers per particle. It can be confirmed whether there is a binding of the number of capture agents (for example, one antibody per about 100 square nm) per unit area (for example, 100 square nm).

  (4) Next, a blocking agent is bound, dried and weighed. The weight difference from (3) is the blocking agent binding amount. The increase amount is calculated, and the binding amount per unit weight (for example, 1 mg) of the particles is calculated. The effect of the blocking agent can be confirmed by the difference in weight when the washing operation is performed.

  Further, the amount of protein bound to the particles by adsorption can be measured without using a linker-imparting agent. Check the increase in this case. Then, for the scavenger, the number of scavengers per unit particle weight (for example, 1 mg), the number of scavengers per particle, and the binding of the scavenger per particle surface area unit area (for example, 1 square nm) are calculated. be able to.

  A target molecule (for example, BSA (molecular weight 66 kDa)) can be used to measure the amount of protein bound to particles by adsorption without using a linker. In this case, the number of protein molecules per particle unit weight (for example, 1 mg), the number of target molecule bonds per particle, and the number of target molecule bonds per particle surface area unit area (for example, 1 square nm) can be calculated. it can.

  In this way, in the present invention, it can be confirmed that a large amount of the capturing agent can be bound to the surface of the particles regardless of the presence or absence of the linker. In the present invention, the density of the scavenger on the particle surface may be more prominent than the conventional one. Depending on the molecular weight and tertiary structure of the capture agent, the position of the structure that correlates with the capture activity, etc. When the diameter is 15 nm, the particle surface area is at least 0.1, preferably at least 0.3, more preferably at least 1, per 100 square nm. It is known that the binding activity of the capture agent varies depending on characteristics such as the surface potential of the capture agent, surface functional groups, secondary structure, and tertiary structure, and the method of binding to the particles. The binding between the organic silica particles and the scavenger can be changed as necessary, such as adsorption or binding with a linker. Usually, a blocking agent is loosely bound to particles and antibody proteins, and this gentle binding is considered to work for no washing and blocking effect.

  As used herein, “blocking agent” refers to any agent for inhibiting non-specific binding in an assay based on specific binding. Examples of blocking agents in bioassays include, but are not limited to, skim milk, albumin, gelatin, serum, surfactants, synthetic blocking agents, and mixtures thereof. Examples of synthetic blocking agents include, but are not limited to, those used in the Examples, such as 4.0% Block Ace (DS Pharma Biomedical). A blocking agent is referred to as a “blocking moiety” when bound to and integrated with a particle.

As used herein, “silica compound”, “silane compound”, “silane derivative”, and “silicon compound” are used interchangeably and refer to a compound having silicon Si at its center, In producing preferred silica particles, particularly organic silica particles, as particles of the invention, it is intended to serve as a source for providing silicon to the particles. Examples thereof include compounds provided in the form of, for example, SiR ₁ R ₂ R ₃ R ₄ (wherein R ₁ , R ₂ , R ₃ , and R ₄ are each an arbitrary organic group). Are mercaptopropyltrimethoxysilane, mercaptopropyltriethoxysilane, mercaptopropylmethyldimethoxysilane, trimethoxy [2- (7-oxabicyclo [4.1.0] -hept-3-yl) ethyl] silane, thiocycyanato Organoalkoxysilanes such as propyltriethoxysilane and acryloxypropyltrimethoxysilane, and compounds having properties equivalent to their physical and chemical properties.
Mercaptopropyltrimethoxysilane

Mercaptopropyltriethoxysilane

Trimethoxy [2- (7-oxabicyclo [4.1.0] -hept-3-yl) ethyl] silane (2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane)

  As used herein, “thiol organic silica particles” refers to particles containing thiol groups on the surface or inside, preferably on the surface, and are organic silica particles synthesized from mercaptopropyltrimethoxysilane, mercaptopropyl Examples include, but are not limited to, organic silica particles synthesized from triethoxysilane or organic silica particles synthesized from mercaptopropylmethyldimethoxysilane. In one embodiment, the lattice of the particle of the present invention showing an internal structure as a primary particle has a network-like three-dimensional structure via chemical bonds typified by Si—O—, Si—C— and the like. Have. Such a structure is illustrated in FIGS. 1-2.

  As used herein, “organic silica particles” refers to particles having an organic functional group among particles formed of a substance containing silica (silicon), and are “inorganic” silica particles in which no organic functional group remains. This is a concept for “silica particles”, which is synthesized from a silica compound (organoalkoxysilane) containing an organic component and remains in particles (for example, the surface and / or inside) in which at least one organic functional group is bonded to a silicon atom. This refers to silica particles. (Please review the definitions carefully.) The structure of a typical organic silica particle of the present invention is as shown in FIG. As one explanation, the organic silica particles are those in which thiol groups are preferably bonded to particles composed of silicon, oxygen, carbon, and hydrogen. In the present specification, the organic silica particles having a thiol group are also referred to as “thiol organic silica particles”. The content of the thiol group used here is arbitrary, and may be, for example, 21% to 31% (% by weight), but is not limited thereto. Such a numerical value can be estimated from the atomic weight. Theoretically, the upper limit is considered to be 31% by weight. It is conceivable that the lower limit is lower than 21% by containing an organic dye or the like.

  Latex particles such as polystyrene particles are mainly composed of organic components such as polyvinyl alcohol. Therefore, when storing for a long period of time, it is necessary to contain a disinfectant. In fact, Bio-rad's Bio-Plex COOH beads contain thimerosal (mercury agent), which is a pharmaceutical poison. On the other hand, since the organic silica particles that may be used in the present invention have a structure in which an organic component is contained in a silica structure that is an inorganic substance, there is no need for an antibacterial agent even during long-term storage.

  In one embodiment, the method used in the method for creating the organic silica particles used in the present invention can include a bottom-up method and a top-down method. The bottom-up method is a method of increasing the scale of an atom or molecule while interacting and reacting with each other by a physical or chemical method. Control in atomic and molecular order is possible. Examples of the bottom-up method include a laser irradiation method (thin film growth method), a self-assembly method, a chemical vapor deposition method, a sol-gel method, an aggregation precipitation method, and a combinatorial chemistry method. The top-down method is a method in which a bulk material is broken down or miniaturized by processing, and examples thereof include a lithography method and an etching method. In nanoparticle synthesis, a sol-gel method, a gas phase method, a spray method, and the like are performed. Among them, the sol-gel method is a method of synthesizing a solid by gelling a sol-like liquid (Stover, W .; Fink, A .; Bohn, EJ Colloid Interface Sci. 1968, 26, 62-69). ). In order to synthesize the particles of the present invention, a sol-gel method (Stober method) is used. In this sol-gel method, the particles are produced at room temperature in the conventional technique. Alternatively, high temperature conditions disclosed in Patent Document 3 (typically, reaction conditions in the temperature range of 70 to 100 ° C., preferably 80 ° C. to 100 ° C., more preferably 90 ° C. to 100 ° C. But not limited to these) and / or high ammonia conditions (typically, the concentration of the prepared ammonia aqueous solution refers to a final concentration of 20% or more, preferably 20% to 30%, preferably 25% to 30%) %, 26% to 28%, and a more preferable concentration condition is 27%, but is not limited thereto. The particles of the present invention can be produced with a good size control by a one-step reaction. We have applied to fluorescent imaging and multimodal imaging for nano-sized particles. It has been successfully applied to various imaging such as in vitro molecular imaging and in vivo detection of labeled cells (J. Material Chem. 2011, 21, 4689-4695). It has also been found that detection by flow cytometry is possible up to a concentration of the order of pg / ml by a short-time, one-step adsorption reaction between particles and green fluorescent protein (GFP) or fluorescent dye-labeled protein. In the present invention, application research to bead arrays of micro-sized organic silica particles can be developed by utilizing the surface characteristics of the particles. Conduct multi-step binding reactions on the particle surface and study biomolecule measurement methods. The measurement sensitivity and measurement range of bead arrays using organosilica particles are evaluated. Furthermore, the conditions can be examined, and research can be carried out with the goal of realizing performance equal to or better than commercial bead arrays.

In one embodiment, the particles of the present invention are advantageously “non-porous” silica particles or “pore free” silica particles. In one embodiment, this “non-porous” or “no pore” feature is, for example, the absence of “macropores”, the absence of pores (pores) of 20 μm or more, or For example, 100 μm or more, 10 μm or more, 1 μm or more, 0.5 μm or more, 0.2 μm or more, 0.1 μm or more, 0.05 μm or more (http://www.jsac.or.jp/bunseki/pdf/bunseki2009/200907kaisetsu .pdf), which means that there are no pores. In another embodiment, for example, the silica particles of the present invention have a specific surface area of 4.816 (m ² / g) and a pore volume of particles having an average particle diameter of about 900 nm. a 0.0159 ^(cm 3 / g), or, a pore volume of 1.0 cm ³ / g or less, 0.5 cm ³ / g or less, 0.1 cm ³ / g or less, 0.05 cm ³ / g or less , 0.02 cm ³ / g or less, 0.01 cm ³ / g or less, 0.008 cm ³ / g or less, 0.006 cm ³ / g or less, may be less 0.004 cm ³ / g. The “pore” is a term indicating a porous structure, and the pore is roughly classified into a micropore (micropore), a mesopore (mesopore), and a macropore (macropore). “Micropore” or “micropore” refers to a pore having a diameter of 2 nanometers (nm) or less. “Mesopore” or “mesopore” refers to pores having a diameter of 2 to 50 nanometers (nm). “Macropore” or “macropore” refers to a pore having a diameter of 50 nanometers (nm).

  In one embodiment, the particles of the present invention are substantially spherical, and this “substantially spherical” refers to particles that are spherical without taking an irregular structure due to the absence of pores. Say.

  As used herein, “size distribution” or “particle size distribution” is used interchangeably, and this “size distribution” is the mode of distribution of “particle size” in the particle group to be measured. Indicates. “Size distribution” is measured by various techniques, and it is possible to evaluate the size distribution of particles using flow cytometry or the like.

  In this specification, the “particle size distribution width” is an index indicating the degree of dispersibility of the “particle size” in the “size distribution”, and indicates the width in which the particle size of the target particle exists. In the present invention, in one embodiment, the particle size distribution width is a narrow region distribution property in which the particle size distribution width in the target particle group is within ± 25% of the average particle size. Features.

As used herein, in a size control evaluation method in an electron microscope, “size yield” or “size control rate” is used interchangeably and is defined by the following equation: That is,
“Size yield” = {(maximum particle size) − (minimum particle size)} / {2 × (average particle size)} × 100 (%)
Here, “average particle diameter” = {(maximum particle diameter) + (minimum particle diameter)} / 2.
These values are measured, for example, by a transmission electron microscope or calculated based on the measured values.

  It will be appreciated that the particles used in the present invention may preferably have a preferred size yield as a group of particles.

  “Flow cytometry” is a technique in which fine particles (for example, single cells in a sheath fluid) are dispersed in a fluid, and the fluid is flowed finely to optically analyze individual particles. is there. A light beam of a certain wavelength (usually a laser beam) is applied to the fluid, and forward scatter in the direction along the light beam (Forward Scatter = FSC) and side scatter SSC (Side Scatter) in a direction perpendicular to the light beam are detected. . In general, the forward scattered light is used as an index indicating a large description of the fine particle because its intensity is proportional to the surface area of the particle to be measured, and the side scattered light is generated by refraction or scattering of the measurement object. So it can be used as an indicator of the complexity of the internal structure. For example, when it is found that the particle size of the used particle system and the side scattered light have a positive correlation, the side scattered light can be used. In the present invention, by diluting the reaction solution before measurement by flow cytometry (20 to 50 times, or more or less), not only can the volume required for the measurement required by the apparatus be secured, but also dilution Can also be expected to reduce the background.

  The flow cytometer can acquire detection data for each fluorescent silica particle capturing a target biomolecule, and can distinguish each carrier of the present invention. Can do. The carrier of the present invention may be a fluorescent material capable of barcode labeling. In this specification, “barcode labeling” refers to labeling silica particles with various fluorescent properties.

  In this specification, “multiplexing” refers to simultaneous analysis (detection, quantification, etc.) of a plurality of types of targets (biomolecules, cells, etc.) by one measurement operation. The multiplexing kit for flow cytometry used in the present invention is used for flow cytometry. From the viewpoint of immediately applying to flow cytometry, each carrier used in the present invention recognizes a separate target (biomolecule, cell, etc.). It is preferable to provide a combination of such modifications.

  In the present specification, the “acceptor group” refers to a functional group introduced onto a carrier. When the support is silica particles, the relationship between the silica compound used to form the particles and the acceptor group terminal functional group to be introduced has a corresponding relationship described in the following table, for example. The organic silica particles have a propyl group between the terminal functional group and the silicon atom. In the carrier of the present invention, the acceptor group can serve as a linker-imparting agent that binds the capture agent or a binding site with the linker.

As used herein, “fluorescent substance” refers to a substance that emits fluorescence when excited by an external stimulus such as electromagnetic waves (for example, ultraviolet rays, X-rays, electron beams, etc.). Examples of the “fluorescent substance” include rhodamine, fluorescein, hexanoic acid-6- (tetramethylrhodamine-5-carboxamide), hexanoic acid-5- (tetramethylrhodamine-5-carboxamide), Alexa Fluor (R ^{) 647, DY635 TM, DY485 TM} , DY495 TM, DY505 TM, tris dichlororuthenium (II) hexahydrate (tris dichlororuthenium) (II) hexahydrate ) and their although derivatives include, but are not limited to. This fluorescent substance is present in the silica particles in the following modes (1) to (4), but is not limited to these modes. That is, (1) contained alone; (2) a substance combined with a fluorescent substance and a compound selected from N-hydroxysuccinimide (NHS) and isothiocyanate (ITC) and 3- (aminopropyl) triethoxy Reaction product with silane [3- (aminopropyl) triethoxysilane] is contained inside or is present on the surface in the form of binding to silica network; (3) Reaction of mercaptopropyltrimethoxysilane with those bound with maleimide The product is contained inside or on the surface in a form that binds to the silica network; (4) or the fluorescent material and maleimide bound to the surface by reaction with silica particles containing silica compounds with thiols To do.

  For example, 5 (6) -carboxy prepared by the method described later for the preparation of the fluorescent silica particles used in the present invention so that 5 (6) -carboxyfluorescein and DY630 are contained as two types of fluorescent dye compounds. Fluorescein and DY630-containing fluorescent silica particles, 5 (6) -carboxyfluorescein and PerCP (pludinine chlorophyll protein) -containing fluorescent silica particles prepared by the same method, 5 (6) -carboxyfluorescein, PI (propidium iodide) ) And 3 types of fluorescent silica particles containing DY635, 4 (6) -carboxyfluorescein, PE (phycoerythrin), 4 types containing fluorescent silica particles of PerCP and DY635, and the like.

In the formula, each of R ¹ and R ² is a group that can be covalently bonded to the particles of the present invention (for example, silica particles). Examples of such a group capable of covalent bonding include an active ester group such as NHS (N-hydroxysuccinimide) ester group, a maleimide group, an isothiocyanate group, an isocyanate group, a cyano group, and an aldehyde group.

  In one embodiment, the fluorescent dye compound that can be used in the present invention preferably has an excitation wavelength by a laser used for flow cytometry.

Examples of the fluorescent dye compound having an excitation wavelength of 488 nm by an argon laser include 5 (6) -carboxyfluorescein, PE (phycoerythrin, Invitrogen), PE-Cy5 ^™ , PE-Cy7 ^™ (GE), DY495 ^™ , DY505. ^TM (Dyomics GmbH), BODIPY-TAMRA-X (Invitrogen), etc. are mentioned.

Here, R ³ and R ⁴ are groups capable of covalently bonding to the carrier or particle of the present invention, respectively.

Examples of fluorescent dye compounds having an excitation wavelength of 532 nm by a YAG laser include 5-TAMRA, Cy3 ^™ (GE), DY550 ^™ , DY555 ^™ (Domics GmbH), Alexa Fluor (R) 546, Alexa Fluor (R) 555 (Invitrogen). Etc.

The fluorescent dye compound having an excitation wavelength 635nm according to the semiconductor red ^{laser, DY630 TM, DY631 TM, DY633} TM, DY635 TM, DY636 TM, DY650 TM, DY651 TM (Dyomics GmbH), Cy5 TM (GE), BODIPY TM 630 / 650, Alexa Fluor (R) 633, Alexa Fluor (R) 647, APC ^™ (Allophycocyanin) (Invitrogen), Oyster643 ^™ , Oyster656 ^™ (Denovo Biolabels), and the like.

In the formula, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ² , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ can each be covalently bonded to the particles of the present invention, for example, organic silica particles. It is a group.

  As a method for preparing fluorescent silica particles, active groups such as N-hydroxysuccinimide (NHS) ester group, maleimide group, isocyanate group, isothiocyanate group, aldehyde group, paranitrophenyl group, diethoxymethyl group, epoxy group and cyano group A product obtained by reacting and covalently bonding the fluorescent dye compound having a silane coupling agent having a substituent (for example, an amino group, a hydroxyl group, and a thiol group) that reacts with the active group. Can also be used.

  As a technique relating to such modification of silica particles, Japanese Patent Application Laid-Open No. 2009-14729 is known, and this can be referred to.

Since the preparation method of fluorescent silica particles is already known (for example, Table 2006/070582), these can be referred to. Specifically, the fluorescent silica particles can be prepared by performing the following steps (a) and (b).
(A) A fluorescent dye compound (1) having an active ester group such as N-hydroxysuccinimide (NHS) ester and a silane coupling agent (2) having an amino group are reacted to form a dye silane coupling agent composite compound ( 3) and (b) the dye silane coupling agent composite compound (3) obtained in (a) above is polymerized with the silica compound (4) under basic conditions to produce the fluorescent silica particles ( 5) forming. A process of esterifying a carboxylic acid compound and N-hydroxysuccinimide is already known (for example, Table 2006/070582). The fluorescent dye compound (1) having an NHS ester group used in the step (a) can be prepared by carrying out according to the esterification reaction step. However, it is also possible to obtain a commercially available product. The silane coupling agent (2) having an amino group is not particularly limited. For example, γ-aminopropyltriethoxysilane (APS), 3- [2- (2-aminoethylamino) ethylamino] propyl-tri Mention may be made of ethoxysilane, N-2 (aminoethyl) 3-aminopropylmethyldimethoxysilane, and 3-aminopropyltrimethoxysilane.

  The reaction between the fluorescent dye compound (1) having an NHS ester group and the silane coupling agent (2) having an amino group is dissolved in a solvent such as DMSO or water and then stirred at room temperature (for example, 25 ° C.). While reacting. The ratio of the fluorescent dye compound (1) having an NHS ester group used in the reaction and the silane coupling agent (2) is not particularly limited. In this way, the carbonyl group of the fluorescent dye compound (1) and the amino group of the silane coupling agent (2) having an amino group form an amide bond (—NHCO—), and the dye silane coupling agent composite compound (3) is obtained. That is, in the dye silane coupling agent composite compound (3), the fluorescent dye compound and the silica component are bonded via an amide bond.

  In step (b), the dye silane coupling agent composite compound (3) is reacted with the silica compound (4). The silica compound (4) is not particularly limited, but γ-mercaptopropyltrimethoxysilane (MPS), γ-mercaptopropyltriethoxysilane, γ-aminopropyltriethoxysilane (APS), 3-thiocyanatopropyl. Mention may be made of triethoxysilane, 3-glycidyloxypropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane, and 3- [2- (2-aminoethylamino) ethylamino] propyl-triethoxysilane.

  Usually, fluorescent organic silica particles (5) can be prepared by reaction for several hours to several days.

(Exemplary particle production example)
As an example of the present invention, the production or production of mercaptopropyltrimethoxysilane (MPS) particles will be described. It is understood that other particles of the present invention can be appropriately implemented by those skilled in the art based on the following production examples.

(Production of organic silica particles with MPS)
(A) Preparation of MPS particles using MPS and ammonia:
As an example, after mixing MPS and 20 wt% or more, for example, 28 wt% ammonia aqueous solution, at a temperature of 80-100 ° C, preferably at 95 ± 5 ° C, 1-12 hours, preferably 7 ± 5 hours, Stir and keep warm and react to produce MPS particles. The produced MPS particles are collected in the form of pellets by high-speed centrifugation, and washed with a 70% aqueous ethanol solution and distilled water alternately by a total of 4 to 8 times by centrifugation. The collected pellets (MPS particles) are dispersed by a high-speed homogenizer, ultrasonic treatment or the like and then used. The particle size of MPS particles can be adjusted and adjusted from nano size to micron size by changing the MPS concentration used for generation. For example, the amount of MPS added to a 28 wt% ammonia aqueous solution (a constant amount) can be adjusted as appropriate so as to obtain a desired particle size. The three relationships between MPS concentration, average particle size, and size yield (%) for 675 μl (constant amount) of 28 wt% aqueous ammonia are illustrated in the table below.

(B) Generation of MPS particles with MPS, isopropanol, and ammonia:
As an example, after mixing MPS, isopropanol, and 28 wt% aqueous ammonia, the temperature is 80 to 100 ° C., preferably 95 ± 5 ° C., and is stirred and kept warm for 1 to 12 hours, preferably 7 ± 5 hours. By reacting, MPS particles can be generated. The collection, washing, and dispersion of the generated PMS particles are as described in (a) above. The particle size of the MPS particles can be adjusted and adjusted by changing the MPS concentration used for production and changing the I / N volume ratio [volume of isopropanol solution (I): volume of 28 wt% aqueous ammonia solution (N)]. The MPS concentration and the I / N ratio with respect to the 28 wt% aqueous ammonia solution (constant amount) can be adjusted as appropriate so as to obtain a desired particle size. The interrelationship between the MPS concentration, I / N ratio, and average particle size for 337.5 μl (constant amount) of 28 wt% aqueous ammonia is illustrated in the following table.

(C) Generation of MPS particles containing a labeled molecule:
As an example, an MPS-labeled molecule conjugate is prepared in advance by reacting a substance that reacts with a thiol group, such as a maleimide compound, with a conjugate of a labeled molecule, such as rhodamine, and a thiol group of MPS. Next, after mixing the conjugate, MPS and aqueous ammonia solution, or after mixing the conjugate, MPS, aqueous ammonia solution and isopropanol, the mixture is stirred and kept warm in the same manner as in (a) above, thereby containing the labeled molecule. MPS particles are generated. One or more labeling molecules can be contained. Further, not only MPS but also other types of silica compounds can be used to prepare other types of silica compounds-labeled molecular conjugates. The particle size can be adjusted by the MPS concentration as described above. The produced MPS particles are ready for use after collection, washing, and dispersion. Specific examples include the following.

(Example of surface layer functionalization by fluorescent dye labeling of MPS particles)
(1) Preparation Example of MPS Particles As an example, 405 μl of isopropanol solution and 290 μl of 28 wt% aqueous ammonia solution are added to and mixed with 10 μl of MPS and reacted at 95 ° C. for 2 hours. Next, the reaction end solution is subjected to a high-speed centrifuge (10,000 × g, 5 minutes), and the pellet is collected. The pellets are washed using 70% ethanol and distilled water alternately. Washing is repeated a total of 6 times by centrifugation. The collected pellets (silica particles) were stirred and dispersed with an ultrasonic crusher, then sampled and observed with a fluorescence microscope. For example, the average particle size was 690 nm, the size control rate was about 15%, and good size control was achieved. The production of the particles below is confirmed.

(2) Functionalization of rhodamine (labeled molecule) by surface labeling As an example maleimide compound, rhodamine Red ^™ C2 maleimide (5 mg) was dissolved in 73.5 μl of DMSO solution, and then MPS prepared in (1) above. 3 μl of the particle solution is added, and the mixture is stirred for about 2 hours under light shielding using a tube mixer. Next, the reaction end solution is subjected to a high-speed centrifuge (10,000 × g, 5 minutes), and the pellet is collected. The pellets were washed using 70% ethanol and distilled water alternately. Washing is repeated a total of 6 times by centrifugation. The collected pellets (silica particles) are stirred and dispersed with an ultrasonic crusher, then sampled and observed with a fluorescence microscope to confirm that the particles emit rhodamine fluorescence. In this way, surface layer functionalized MPS particles can be prepared by rhodamine (labeled molecule) surface layer labeling.

  The carrier of the present invention can be produced using MPS particles and has useful properties. However, the silica particles having the characteristics of the present invention are not limited to MPS particles, and mercaptopropyltriethoxysilane. (MPES), mercaptopropylmethyldimethoxysilane (MPDMS), trimethoxy [2- (7-oxabicyclo [4.1.0] -hept-3-yl) ethyl] silane (EpoPS), thiocycyanatopropyltriethoxysilane It is understood that it can also be made from one or several silica compounds selected from the group consisting of (TCPS) and acryloxypropyltrimethoxysilane (AcPS), which is also described in US Pat. .

  As used herein, “surface layer functionalization” refers to disposing and stabilizing a functional material on the surface layer of the silica particles of the present invention. Moreover, the property of the target particle which can perform this surface layer functionalization is expressed as surface layer functionalization ability. In this specification, “stabilization” means, for example, physical and chemical stability necessary for reproducibly realizing the desired function of the functional substance in the use environment in the silica particles. Is to provide a state. As used herein, “internal functionalization” refers to the inclusion and stabilization of a functional substance within the particles of the present invention. Moreover, the property of the target particle that can perform this internal functionalization is expressed as internal functionalization ability. The particles used in the carrier of the present invention preferably have surface layer functionalization, but are not limited thereto. Both the internal function and the surface layer function can include a label (for example, a fluorescent substance).

  When the particles used in the present invention are silica particles, a modification method for binding a substance that recognizes the target biomolecule to the surface thereof is described below as “silica particle surface modification”. Silica is generally known to be chemically inert and easy to modify.

  Regarding the surface modification of the particles used in the present invention, specifically, a group of fluorescent silica particles having an acceptor group on the surface capable of binding to a desired substance depending on the type of the silica compound (4) used in the step (b). It can be. Examples of the acceptor group include an amino group, a hydroxyl group, a thiol group, a carboxyl group, a maleimide group, and a succinimidyl ester group.

  The relationship between the silica compound (4) used in the reaction when silica is used as the particles in the present invention and the acceptor groups formed on the surface of the silica particle group obtained thereby is shown in the following table.

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

  Examples of the substance that recognizes the target biomolecule that modifies the silica particles include antibodies, antigens, peptides, DNA, RNA, sugar chains, receptors, and the like.

  That is, the substance that recognizes the target biomolecule modified with the silica particles serves as a receptor site itself, for example, antigen-antibody reaction, biotin-avidin reaction, hybridization using base sequence complementarity, etc. The specific molecular recognition of can be used to bind specifically to the target molecule.

  As used herein, “adsorption” refers to integration by van der Waals force, hydrophobic interaction, or electrostatic interaction.

Here, molecular recognition means (1) hybridization between DNA molecules or between DNA-RNA molecules or between RNA molecules, (2) antigen-antibody reaction, (3) between enzyme (receptor) and substrate (ligand). A specific interaction between molecules (which may be a biomolecule such as a physiologically active substance) such as a reaction is referred to, and a portion where the specific interaction is performed is called a recognition site.

  The particles used in the present invention are substances that recognize a desired target molecule according to the type of acceptor group on the surface [for example, antigen, antibody, DNA, RNA, sugar, sugar chain, ligand, receptor, peptide, chemical The substance can be modified by binding or adsorbing to the surface.

Although there is no particular limitation on the method for binding and modifying a substance that recognizes a target molecule on the surface of the particle used in the present invention, the following examples (i) to (iii) are exemplified.
(I) A group of silica particles having a thiol group prepared on the surface using mercaptopropyltrimethoxysilane or the like is bonded to the target molecule via a disulfide bond, a thioester bond, or a bond via a thiol substitution reaction. It can be modified with a molecule recognition substance.
(Ii) In particular, when the substance that recognizes the target molecule as a molecule has an amino group, the thiol group that the particle group has and the amino group that the substance that recognizes the molecule as a molecule has succinimidyl-trans-4- It can also couple | bond using cross-linking agents, such as (N-maleimidylmethyl) cyclohexane-1-carboxylate (SMCC) and N- (6-maleimidocaproyloxy) succinimide (EMCS).
(Iii) The particles having an amino group on the surface prepared by using APS or the like have the same amino group and a thiol group possessed by a substance that recognizes the target molecule as described above. Can be combined. Moreover, this amino group and the amino group which the substance which recognizes the said target molecule molecule | numerator has can also be couple | bonded with crosslinking agents, such as glutaraldehyde. Furthermore, the surface can be modified with a substance that recognizes the target molecule through an amide bond or a thiourea bond.

  Further, particles having a carboxyl group on the surface react with S-NHS (N-hydroxysulfosuccinimide) after adding EDAC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride), for example. By doing so, the binding with the amino group of the scavenger becomes possible. In addition, the coupling | bonding of the capture | acquisition agent using click chemistry by an alkyne and an azide compound etc. is mentioned.

  In one embodiment, the particle can include a label. Such a label may be any entity (eg, substance, energy, electromagnetic wave, etc.) for distinguishing the target molecule or substance from others. It is understood that any substance used as a “medicament” is utilized. In one embodiment, examples of the label added to the particle include a radioactive label (RI (radioisotope)), a fluorescent dye, biotin, a chemiluminescent substance, and the like. The label that can be included in the particles of the invention is preferably distinguishable from the label used for the labeling agent used in the assay, and can be of different label categories (eg, fluorescence and RI) or the same There can be different types in categories (eg, different types of labels).

(Washless assay)
As an assay for practicing the present invention, any assay using particles can be envisaged.

  For example, the target (test substance) targeted by the assay can be any target, such as, but not limited to, biomolecules, antigens, proteins, peptides, nucleic acids, saccharides, variants thereof, and conjugates thereof. Not. Examples include, but are not limited to, assays for qualitative or quantitative methods of biomolecules. An exemplary method for such an assay includes: (A) providing a capture agent-bound carrier having the capture agent bound to the carrier of the present invention; (B) in the presence of a blocking agent, the capture agent is specific. Contacting a sample containing or suspected of containing a test substance that binds to the capture agent-binding carrier; (C) if the test substance is not labeled, a labeled agent that specifically binds to the test substance Contacting with a binding agent that specifically binds to the test substance after contacting or further contacting with a labeled agent that specifically binds to the binding agent (this step is skipped if there are two steps) (Including three steps and four steps) (alternatively, a binding agent may be further intervened multiple times); and (D) directly or indirectly on the capture agent-binding carrier. The bound label Out, comprising the step, of determining the amount of said test substance in said sample the detected label. Such a method can be said to be a two-step or more washless assay. Provision of a capture agent-bound carrier in which a capture agent is bound to the carrier of the present invention may be performed by purchasing a carrier with a capture agent bound in advance, or providing a carrier with no capture agent bound thereto, The carrier may be prepared by reacting and binding the capture agent to the carrier.

  In another embodiment, the particle of the present invention captures one or more target biomolecules (including a physiologically active substance, the same applies hereinafter) to which a quantitative fluorescent label is bound, and the label is attached. The test substance can be quantified by measuring the fluorescence intensity of the bound target biomolecule.

  In the present specification, the “test substance” refers to a substance to be detected or diagnosed in an assay. The test substance may be a biomolecule (including a physiologically active substance), and examples of the test substance include antigens, antibodies, DNA, RNA, sugars, sugar chains, ligands, receptors, peptides, biotin, and fluorescent dyes. Examples include, but are not limited to, chemical substances, fragments thereof, and complexes composed of two or more of these substances.

  In this specification, the “sample” (also referred to as a sample) refers to any substance obtained from a subject or the like. Examples include, but are not limited to, liquid, tears, and the like. Those skilled in the art can appropriately select a preferable sample based on the description of the present specification.

  The labeling with fluorescence for quantifying the test substance may be performed with any fluorescent labeling probe, or a nucleic acid such as DNA, RNA, or the like into which a fluorescent dye compound such as Cy3 or Cy5 has been introduced by a PCR reaction, and a fluorescent protein It may be a label as a pre-labeled target biomolecule such as a fused protein. The fluorescently labeled probe is not particularly limited as long as it specifically binds or adsorbs to a target biomolecule, and examples thereof include an antibody bound with an arbitrary fluorescent dye compound.

In this specification, the “label” refers to a presence (for example, a substance, energy, electromagnetic wave, etc.) for distinguishing a target molecule or substance from others. Examples of such a labeling method include RI (radioisotope) method, fluorescence method, biotin method, chemiluminescence method and the like. In the case of a two-step assay, the label is processed so as to be directly contained in the test substance, and may be already labeled at the time of the assay. In the case of a three-stage assay, the binding agent for the test substance is directly labeled, and also serves as a labeling agent. In the case of the four-step assay, since the binding agent that further binds to the binding agent to the test substance is labeled, the first binding agent and the labeled agent are different substances. When a plurality of test substances targeted by the present invention or factors or means for capturing them are labeled by the fluorescence method, the labeling is performed with fluorescent substances having different fluorescence emission maximum wavelengths. The difference in the maximum fluorescence emission wavelength is preferably 10 nm or more. Any ligand can be used as long as it does not affect the function. As fluorescent substances, allophycocyanin (APC), Alexa Fluor (R) 488, FITC, phycoerythrin (PE), PerCP, Alexa Fluor® 700 is preferred. Also, MCA, AlexaFluor 350, AlexaFluor 488, Qdot (R) 605, FITC, PE / RD1, ECD / PE-TexasRed, PC5 / SPRD / PE-Cy5, PC5.5 / PE-Cy5.5, PEAlexa Fluor (R) 750, PC7 / PE-Cy7, TRITC, Cy3, TexasRed, Alexa Fluor (R) 647, Alexa Fluor (R) 700, Cy5, Cy5.5, APC, APC7 / APC-Cy7, APC Alexa Fluor (R) 750, Hoechst 33342, DAPI , PI, YOYO-1, CPO, PyroninY, 7-AAD, ethidium homodimer-1, SYTO9, SYBRGreenI, LDS751, TO-PRO-3, etc. Used. Alexa Fluor (R) is a water-soluble fluorescent dye obtained by modifying coumarin, rhodamine, fluorescein, cyanine, etc., and is a series corresponding to a wide range of fluorescent wavelengths, compared to fluorescent dyes of other applicable wavelengths. It is very stable, bright and has low pH sensitivity. Examples of combinations of fluorescent dyes having a fluorescence maximum wavelength of 10 nm or more include a combination of Alexa Fluor (R) 555 and Alexa Fluor (R) 633, a combination of Alexa Fluor (R) 488 and Alexa Fluor (R) 555, and the like. it can. Any nucleic acid can be used as long as it can bind to its base moiety. For example, a cyanine dye (eg, CyDye ^™ series Cy3, Cy5, etc.), rhodamine 6G reagent, N-acetoxy-N2— Acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) or the like is preferably used. Examples of the fluorescent substance having a difference in fluorescence emission maximum wavelength of 10 nm or more include a combination of Cy5 and rhodamine 6G reagent, a combination of Cy3 and fluorescein, a combination of rhodamine 6G reagent and fluorescein, and the like. In the present invention, by using such a label, the target object can be modified so that it can be detected by the detection means used. Such modifications are known in the art, and those skilled in the art can appropriately carry out such methods depending on the label and the target object.

  In this specification, the “labeled drug” should be labeled by directly or indirectly binding to a substance to be labeled (for example, a test substance or a binding drug bound to the test substance). Any drug capable of labeling a substance. For example, label-bound antibody, antigen-binding antibody fragment, antigen, receptor, ligand, hormone, avidin, or streptavidin, peptide, natural protein, recombinant protein, fusion protein, or variants thereof (for example, streptavidin (Nutravidin from which sugar chains are removed from), genes (DNA, RNA), peptide gene complexes, protein gene complexes, biotin, drugs such as low molecular weight compounds, and the like, but are not limited thereto. In the case of a two-step assay, the test substance may be bound before the assay.

  As used herein, “binding agent” refers to any substance that binds to a subject. For example, any substance that binds to the test substance corresponds to this, and may or may not be labeled. If an unlabeled binding agent is used in the assay, it can be further labeled with a labeled agent that binds (preferably specifically binds) to the binding agent. Examples of such binding agents include antigen-binding antibody fragments, antigens, receptors, ligands, hormones, avidin, or streptavidin, peptides, natural proteins, recombinant proteins, fusion proteins, or variants thereof (for example, streptoids). Nutravidin from which a sugar chain is removed from avidin), genes (DNA, RNA), peptide gene complexes, protein gene complexes, biotin, drugs such as low molecular weight compounds, and the like.

  As used herein, “contacting” refers to an object such as the carrier of the present invention, either directly or indirectly, to another substance such as a sample, a binding agent, or a labeling agent. This means that they are physically close to each other. “Contact” places an object in an environment where it can interact with another substance (eg, chemical reaction, binding reaction, etc.). It can be present at various concentrations in samples, binding agents, labeling agents, many buffers, salts, solutions, and the like. Examples of the contact include placing in a test tube, a beaker, a microtiter plate, a centrifuge tube, a cell culture flask, a microarray (for example, a gene chip) or the like containing a sample, a binding agent, a labeling agent and the like.

  As used herein, the term “kit” refers to a unit provided with a portion to be provided (eg, carrier, binding agent, labeling agent, instructions, etc.) usually divided into two or more compartments. This kit form is preferred when it is intended to provide a composition that should not be provided in a mixed form but is preferably used in a mixed state immediately prior to use. Such a kit preferably includes a provided part (eg, instructions or instructions describing how to use the detection agent or how to process the reagent). In the present specification, when the kit is used as a reagent kit, the kit usually includes instructions describing how to use the antibody.

  In the present specification, the “instruction” describes a method for using the present invention for a doctor or other user. This instruction manual includes a word indicating that the detection method of the present invention, how to use a diagnostic agent, or administration of a medicine or the like is given. In addition, the instruction sheet may include a word for instructing administration (for example, by injection) to skeletal muscle as an administration site. This instruction is prepared in accordance with the format prescribed by the national supervisory authority (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States, etc.) It is clearly stated that it has been received. The instruction is a so-called package insert and is usually provided as a paper medium, but is not limited thereto. But it can be provided.

  In certain embodiments, the present invention provides a reagent pack or kit for a washless assay comprising one or more containers filled with one or more components of the reagents, carriers of the present invention. In some cases, associated with such containers, authorization by the government agency for manufacture, use or sale for human administration in a manner prescribed by the government agency that regulates the manufacture, use or sale of biological products. It is also possible to indicate information indicating.

The flow cytometer used in the present invention will be described. A flow cytometer is a method in which a particle suspension is flowed into a narrow tube, a laser beam is applied to floating particles, and each particle excited by the laser emits fluorescence, and the laser scatters by the particles. Is a device for detecting and measuring the flow rate, and flow cytometry is an analysis method thereof.
The flow cytometer used in the present invention is not particularly limited, and a conventionally used flow cytometer can be used as it is. For example, including a laser (eg, He / Ne, Argon) source that emits light of a single wavelength in one or more detection zones. Different detection zones can include laser sources that emit light at different wavelengths. The laser may be focused using a beam shaping lens. Prior to the laser irradiation, a sample liquid containing a sheath liquid (for example, IsoFlow ^TM sheath liquid (Beckman Coulter), Daco Sheath Fluid ^TM (Dako Japan Co., Ltd.)), a target biomolecule, fluorescent silica particles, etc. Introduced into the flow cell, a laminar flow is formed in which the sheath liquid and the sample liquid do not mix. When flowing from the flow cell, the sample liquid flows in a sample core surrounded by a sheath liquid flow, and the laser is irradiated when passing through the laser irradiation area.

The flow cytometer used in the present invention allows the one carrier labeled with the target biomolecule to be present in each detection zone by setting a predetermined flow condition, thereby detecting only one carrier. Data can be acquired. Thereby, the flow cytometry is a so-called multiplexing method in which a plurality of targets can be measured simultaneously by one measurement operation. In the present invention, the flow rate at the time of measurement is not particularly limited, but for the quantification of the target biomolecule, the flow rate can be 10 to 1000 particles / second. For example, when FACSCalibur ^™ (Becton Dickinson) is used as a flow cytometer, the difference between the sheath pressure (the pressure at which the sheath fluid flows) and the sample pressure (the pressure at which the sample fluid flows) is, for example, 1 kpsi to 2 kpsi It can be.

  In each detection zone, fluorescence emission from fluorescent silica particles irradiated by a laser through the detection zone using any one or more fluorescent channels (eg, FL1, FL2, FL3) such as photomultiplier tubes Can be collected. In addition, the light (forward scattered light or side scattered light) refracted by the carrier may be collected using an arbitrary scattered light collecting channel such as a photomultiplier tube.

The flow cytometer used in the present invention is a data integration analysis means (hardware) for recording, storing, and analyzing data obtained by detecting fluorescence emission or scattered light derived from each target biomolecule that has passed through the detection area by a fluorescence channel or the like. Hardware or software).
Commercially available products such as FACSDiVa, EXPO32, Summit, AppSan, LysisII, and CellQuest may be used as the data collection and analysis means. The quantitative system or quantitative method uses a combination of a plurality of types of fluorescent particles that differ only in fluorescence, that is, when the particle sizes are substantially uniform and the particle sizes are substantially uniform (for scattered light). If it is difficult to identify the difference between particles), the particles used can be recognized only by receiving fluorescence. As the flow cytometer used in the present invention, FACSCalibur ^™ (Becton Dickinson), PERFLOWANA ^™ (Furukawa Electric), EPICS ALTRA ^™ (Beckman Coulter), CyAn ^™ (Dako Japan), JSAN ^™ (Bay Bioscience), FISHMAN-R ^TM (on-chip biotechnology), a flow cytometer using a micro-channel chip), EC800 ^TM cell analyzer (Sony), SP6800 ^TM spectrum cell analyzer (Sony) capable of fluorescence spectrum analysis, A commercially available flow cytometer may be used.

(Description of Preferred Embodiment)
In the following, preferred examples of the carrier of the present invention are provided, but it is understood that the present invention is not limited thereto.

(Carrier including particles having a structure for binding a capturing agent)
In one aspect, the present invention includes a carrier for achieving washless in a two or more stage assay comprising particles having a structure for binding a capture agent, wherein the carrier is used to achieve washless. A support having an effective pore volume is provided. This is because the labeling substance not directly or indirectly bound to the test substance enters the pores in the particle and is measured as the fluorescence of the particle in the same manner as the labeling substance bound to the particle. Therefore, it is necessary to wash out the labeling substance that has entered the pores by washing. Without wishing to be bound by theory, a particle with a pore volume below a certain level will achieve a washless because the background will drop to the extent that it will not substantially interfere with the evaluation of the assay results. It is demonstrated in the present invention that this can be done. In addition, since the pore volume correlates with the specific surface area and the swelling constant, those skilled in the art can understand the correlation with reference to the description of the present specification. Using the swelling constant as a guide, particles can be selected for use as a carrier to achieve washless in a two or more stage assay. Based on such a theory, the pore volume that is useful in the present invention, that is, that can achieve washless, is, for example, 1.0 cm ³ / g or less, 0.5 cm ³ / g or less, 0. 1cm ³ / g or less, 0.05cm ³ / g or less, 0.02cm ³ / g or less, 0.01cm ³ / g or less, 0.008cm ³ / g or less, 0.006cm ³ / g or less, 0.004cm ³ / G or less, but is not limited thereto.

  In another aspect, the carrier of the present invention can have a blocking index (BI) effective to achieve washless. Although not wishing to be bound by theory, (1) the block “efficiency” of the blocking agent to the carrier is good, or (2) the pore volume of the carrier is small and the labeling substance does not enter the pores. Both are thought to lead to invention keys (washless, or increased sensitivity). Latex beads have been found in the course of the present invention that (1) the blocking agent “inefficiency” of the blocking agent is poor, and that the pore volume of the carrier is large and the labeling substance easily enters the pores. Blocking indexes can also be useful to achieve washless. It is understood that the pore volume and the blocking index are each independent evaluation parameters, and it is understood that even if either one is satisfied, the washlessness can be achieved, and they may both achieve a certain threshold value. Is understood. 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% for blocking index to be effective to achieve washless Thus, it is preferably 98% or more and 99% or more, and usually 85% or more. Since the skim milk etc. performed in the example also achieves 85%, this numerical value can be a guideline, but is not limited thereto.

  In order to realize such a washless, it is only necessary to have such a specific numerical pore volume and / or blocking index, so any material can be used, not limited to organic silica particles, Particles of other materials such as inorganic silica particles can also be used. It is understood that the organic silica particles that can be used in the present invention can be any of those produced by the methods described in the examples or elsewhere in this specification, examples of which are disclosed in Patent Document 3. Although what is described is illustrated, it is not limited to them.

The structure for binding the capture agent possessed by the carrier of the present invention may vary depending on the capture agent used. For example, when capturing biomolecules such as proteins (antibodies, antigen-binding antibody fragments, etc.), thiol Group, amino group, carboxyl group, maleimide group, N-succinimide ester group, and isocyanate group, mercaptopropyl group, aminopropyl group, thiocyanate propyl group, maleimide group, N-succinimide ester group, isocyanate group, etc. Can do. Methods for imparting such structures are known in the art, such as The Molecular Probes® Handbook 11th Edition, http://www.invitrogen.com/site/us/en/home/References/Molecular -Probes-The-Handbook.html, http://www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Crosslinking-and-Photoactivatable-Reagents.html, http: / /www.invitrogen.com/site/us/en/home/References/Molecular-Probes-The-Handbook/Crosslinking-and-Photoactivatable-Reagents/Chemical-Crosslinking-Reagents.html
And is incorporated herein by reference.

  In a preferred embodiment, the particles used in the carrier of the present invention are silica particles, more preferably organic silica particles, and the structure for binding the scavenger is a thiol group, amino group, carboxyl group, maleimide group. N-succinimide ester group and a group selected from the group consisting of isocyanate group, mercaptopropyl group, aminopropyl group, thiocyanate propyl group, maleimide group, N-succinimide ester group, and isocyanate group Contains groups. As such organic silica particles, it is understood that any of those produced by the methods described in the Examples or elsewhere in this specification can be used, and examples thereof are described in Patent Document 3. However, the present invention is not limited thereto.

  In another aspect, the present invention provides a kit for a two-step or more washless assay comprising the carrier of the present invention and a capture agent. In this kit, it is understood that any of the above embodiments can be used as the carrier of the present invention. As the capture agent that can be used in the two-step or more washless assay kit of the present invention, any one can be used. For example, proteins such as antibodies, nucleic acid molecules such as DNA, lipid molecules, other A small molecule and the like can be exemplified, but not limited thereto. Such a kit is assumed to contain a container containing a carrier and a container containing a capture agent or blocking agent. Such a kit may include other components (for example, a container containing a buffer) in addition to these components. In the kit of the present invention, for example, a carrier and a blocking agent are commercially available as a kit, and a capture agent (such as an antibody) is assumed to be in-house. Therefore, it is considered that a capture agent such as an antibody may be manufactured by the purchaser himself or obtained from another source. Alternatively, the kit may first include a linker-imparting agent and a blocking agent. The scavenger may already be bound to the particles. For example, as a reference kit, http://www.bio-rad.com/evportal/en/JP/LSR/Category/85824182-db2a-4c6e-ac82-4e07dd9ab904/ Bio-Plex-Suspension-Array-System, http://www.bio-rad.com/evportal/en/JP/LSR/Category/LMFT1B15/COOH-%E3%83%93%E3%83%BC%E3 % 82% BA% E3% 81% 8A% E3% 82% 88% E3% 81% B3% E9% 96% A2% E9% 80% A3% E8% A9% A6% E8% 96% AC, http: / /www.bio-rad.com/prd/en/JP/LSR/PDP/LMFT6SE8Z/Bio-Plex-Pro-%E7%A3%81%E6%80%A7-COOH-%E3%83%93%E3 % 83% BC% E3% 82% BA% E3% 81% 8A% E3% 82% 88% E3% 81% B3% E9% 96% A2% E9% 80% A3% E8% A9% A6% E8% 96 % AC, etc., and such a configuration can be taken into consideration as appropriate. The capture agent is often purchased separately by the user, but is not limited thereto. When the kit of the present invention includes a capturing agent, it may be bound to particles, but it need not be. Providing the capture agent separately from the particles in the kit is preferably used, for example, when the binding activity of the capture agent is reduced unless the capture agent is bound to the particles immediately before use.

  In another aspect, the present invention provides a two-step or more washless assay kit comprising the carrier of the present invention and a blocking agent. It will be appreciated that any of the embodiments described herein can be used in this kit as a carrier of the present invention. Such a two-step or more washless assay kit is provided as a kit to which a user binds an arbitrary capture agent. As the blocking agent used in the two-step or more washless assay kit of the present invention, any blocking agent can be used as long as it is for suppressing non-specific binding. Examples of the blocking agent can include, for example, skim milk, albumin, gelatin, serum, surfactant, synthetic blocking agent, and mixtures thereof.

  In another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier of the present invention and a linker-imparting agent. It will be appreciated that any of the embodiments described herein can be used in this kit as a carrier of the present invention. Such a two-step or more washless assay kit is provided as a kit to which a user binds an arbitrary capture agent. As the linker-providing agent used in the two-step or more washless assay kit of the present invention, any linker can be used as long as it can bind the target capture agent. Examples of linker-providing agents in the assay include, for example, maleimide group, N-succinimide ester group, isocyanate group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group on one side. And at least one functional group selected from the group consisting of phenyl azide groups, on the other hand, maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide Ester group, pyridyldithio group A phenylazide group, and optionally protected thiol group, amino group, carboxyl group, at least one functional group selected from the group consisting of at least one functional group selected from Those containing a spacer in between can be used.

  In more detailed embodiments, examples of linker imparting agents that can be included in the kits of the present invention include maleimidobutyric acid N-succinimidyl ester, N- (α-maleimidoacetoxy) -succinimide ester, N- (β-maleimide) Propionic acid) hydrazide.TFA, N, N-dichlorocyclohexylcarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ε-maleimidocaproic acid, N- (ε-maleimidocaproic acid) hydrazide N- (ε-maleimidocaproyloxy) succinimide ester, N- (γ-maleimidobutyryloxy) succinimide ester 1,6-hexane-bis-vinylsulfone, N-κ-maleimidoundecanoic acid, N- (κ- Maleimidoundecanoic acid) hydrazide, succi N-imidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amidocaproate), succinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxy Succinimide ester, 4- (4-N-maleimidophenyl) -butyric acid hydrazide.HCl, N-hydroxysuccinimidyl-4-azidosalicylic acid 3- (2-pyridyldithio) propionylhydrazide, N- (p-maleimidophenyl) Isocyanate N-succinimidyl (4′-azidophenyl) 1,3′-dithiopropionate, succinimidyl 4-hydrazinonicotinate acetone hydrazone, N-succinimidyl 6- (4′-azido-2′-nitrophenylamino) hexa Succinimidyl 3- (bromoacetamido) propionate, succinimidyl 4-formylbenzoate, succinimidyl 4-hydrazide terephthalate hydrochloride, N-succinimidyl iodoacetate N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4- (N -Maleimidomethyl) cyclohexane-1-carboxylate, NHS-PEO2-maleimide, NHS-PEO4-maleimide, NHS-PEO8-maleimide, NHS-PEO12-maleimide, succinimidyl 4- (p-maleimido-phenyl) butyrate succinimid Zyl-6- (β-maleimidopropionamido) hexanoate, 4-succinimidyloxycarbonylethyl-α- (2-pyridyldithio ) Toluene succinimidyl- (4-psoralen-8-yloxy) butyrate, N-succinimidyl 3- (2-pyridyldithio) propionate, ethylene glycol bis (sulfo-succinimidyl succinate), N- (ε- Maleimidocaproyloxy) sulfosuccinimide ester, N- (γ-maleimidobutyryloxy) sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N- (κ-maleimidoundecanoyloxy) sulfosuccinimide Ester, sulfosuccinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamide) hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl (4-azido Salicylamido) hexanoate, sulfosuccinimidyl (4-azido-phenyldithio) propionate, sulfosuccinimidyl 2- [7-azido-4-methylcoumarin-3-acetamido] ethyl-1,3′-dithiopro Pionate, sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) ethyl, 1,3′-sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, Sulfosuccinimidyl 2- (p-azido-salicylamido) ethyl 1,3′-dithiopropionate, sulfo-NHS- (2-6- [biotinamide] -2- (p-azidobenzamido) hexanoamide) Ethyl-1,3'-dithiopropionate, sulfosuccinimidyl (perfluoroazidobenzami D) Ethyl 1,3′-dithiopropionate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate, sulfosk Examples include, but are not limited to, cinimidyl 4- (p-maleimidophenyl) butyrate, 1,6-bismaleimidohexane, bis-maleimidoethane, maleimidodiethylene glycol, disuccinimidyl glutarate and the like.

  In yet another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier of the present invention, a capture agent, and a blocking agent. It will be appreciated that any of the embodiments described herein can be used in this kit as a carrier of the present invention. Such a two-step or more washless assay kit is provided as a special-purpose kit in which a capture agent is also determined. As the capturing agent and the blocking agent, any of those described in the present specification can be used. However, when there is a preferable blocking agent for a certain capturing agent, a preferable combination thereof may be used.

  In yet another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier of the present invention, a capture agent, and a linker-imparting agent. It will be appreciated that any of the embodiments described herein can be used in this kit as a carrier of the present invention. Such a two-step or more washless assay kit is provided as a special-purpose kit in which a capture agent is also determined. As the capture agent and linker imparting agent, any of those described in the present specification can be used. However, when there is a preferred linker imparting agent for a certain capture agent, a preferable combination thereof may be used. In such a case, any linker-providing agent capable of binding a capture agent may be used.

  In still another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier of the present invention, a linker-imparting agent, and a blocking agent. It will be appreciated that any of the embodiments described herein can be used in this kit as a carrier of the present invention. Such a two-step or more washless assay kit is provided as a special purpose kit in which the capture agent is not determined, but the linker-imparting agent and blocking agent to be used are determined. As the linker-providing agent and the blocking agent, any of those described in the present specification can be used, but when the use of a certain capturing agent is envisaged, there are preferred linker-providing agents and blocking agents for the capturing agent. In that case, a preferable combination thereof may be used. If there is a preferred blocking agent for a certain linker-imparting agent, that preferred combination may be used. In such a case, if the result of the kit of the present invention is unsatisfactory, the user can devise such as changing the composition of the blocking agent.

  In yet another aspect, the present invention provides a kit for a washless assay having two or more steps, comprising the carrier of the present invention, a capture agent, a linker-imparting agent, and a blocking agent. It will be appreciated that any of the embodiments described herein can be used in this kit as a carrier of the present invention. Such a two-step or more washless assay kit is provided as a special-purpose kit in which a capture agent is also determined. As the capture agent, linker imparting agent and blocking agent, any of those described in the present specification can be used, but when there is a preferred linker imparting agent and blocking agent for a certain capture agent, a preferred combination thereof is used. May be. If there is a preferred blocking agent for a certain linker-imparting agent, that preferred combination may be used.

  In another aspect, the present invention provides a two or more washless assay using the carrier of the present invention. It will be appreciated that any of the embodiments described herein can be used in this assay as a carrier of the present invention.

  In another aspect, the present invention provides a two-step or more washless assay, which comprises the following steps: (A) capture agent binding wherein the capture agent is bound directly or indirectly to the carrier of the present invention. A step of providing a carrier (eg, binding via biotin as illustrated in FIG. 20); (B) a test substance to which the capture agent specifically binds in the presence or absence of a blocking agent; Contacting the sample containing or suspected of containing with the capture agent-binding carrier; (C) if the test substance is not labeled, further contacting with a labeled agent that specifically binds to the test substance; or A step of contacting a binding agent that specifically binds to the test substance, and further contacting a labeled agent that specifically binds to the binding agent (this step is skipped if there are two stages, 3 stages and And (D) detecting the label bound directly or indirectly to the capture agent binding carrier, and (D) Determining the detected label as the amount of the test substance in the sample. In the case of two steps, the one directly bound to the capture agent-bound carrier is measured, and in the case of three or more steps, the one indirectly bound is measured. In this case, the labeled drug directly bound to the particle is considered to be due to non-specific binding, and since it is difficult to determine the amount of the test substance, a drug bound indirectly via a capture agent is considered to be the test substance. It will be judged as the amount of the substance. It will be appreciated that any of the embodiments described herein can be used in this assay as the carrier, capture agent, blocking agent, etc. of the present invention. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed. When performed in the presence of a blocking agent, the assay of the present invention has the characteristic that the effects of blocking are significantly reduced compared to conventional assays. In some embodiments, for example, when using DNA, a washless assay can be achieved without using a blocking agent, and such an assay could not be achieved by conventional assay techniques. In this respect, it can be said that there is a remarkable effect. Thus, in a form made in the absence of a blocking agent, the assay of the present invention can be said to be washless and blockingless.

  In addition, after the carrier is bonded to the capture agent, the portion where the capture agent is not bonded is bonded to the blocking agent. The test substance is brought into contact with particles bound to both the capture agent and the blocking agent. Some blocking agents are present on the particle surface and others are present in the reaction solution itself. It is understood that both in the presence of a blocking agent are included.

  In one embodiment, the labeling agent used in the present invention is a fluorescent label. In this case, the autofluorescence of the particles used in the present invention is significantly lower than the fluorescence of the fluorescent label, and it is easy to use those that do not interfere with detection. Furthermore, it is preferable that such autofluorescence recognizes an effect of facilitating detection of fluorescence of the fluorescent label.

  One of the features of the assay and assay kit of the present invention is that they can be quantitatively measured and / or qualitatively measured. In a preferred embodiment, these features can be used.

(Capture agent binding carrier)
In another aspect, the present invention is a carrier for achieving washless in a two or more stage assay, comprising 1) particles having a structure for binding a capture agent, and 2) a capture agent, the capture agent And the particles are directly associated or bound, or linked via a linker, the carrier provides a carrier having a pore volume effective to achieve a washless. It is understood that the carrier and the capturing agent used here may be any of those described above (a carrier including particles having a structure that binds the capturing agent).

  In one embodiment, the capture agent used in the present invention and the particle can be directly associated or bound. Alternatively, the capture agent and the particle may be bonded via a linker. When a linker is used, a linker-imparting agent to be used may be one that can bind the target object and / or has little influence on the assay. Such a linker-imparting agent is, for example, selected from the group consisting of maleimide group, N-succinimide ester group, isocyanate group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group, and phenyl azide group. At least one functional group selected from the group consisting of maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group, phenylazide group , And optionally protected thiol group, amino group, carboxyl group, at least one functional group selected from the group consisting of at least one functional group selected from, optionally between the functional groups Those containing spacers can be used. Also preferably, the linker-providing agent is capable of binding to the structure of the functional group present in the particle preparation material. These linker imparting agents can be preferably used when the particles are organic silica.

  Examples of the linker-imparting agent used in the capture agent-binding carrier of the present invention include maleimidobutyric acid N-succinimidyl ester, N- (α-maleimidoacetoxy) -succinimide ester, N- (β-maleimidopropionic acid) hydrazide TFA, N, N-dichlorocarbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-ε-maleimidocaproic acid, N- (ε-maleimidocaproic acid) hydrazide, N- ( ε-maleimidocaproyloxy) succinimide ester, N- (γ-maleimidobutyryloxy) succinimide ester 1,6-hexane-bis-vinylsulfone, N-κ-maleimidoundecanoic acid, N- (κ-maleimidoundecanoic acid) Hydrazide, succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxy- (6-amidocaproate), succinimidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, 4- (4-N-maleimidophenyl) -butyric acid hydrazide.HCl, N-hydroxysuccinimidyl-4-azidosalicylic acid 3- (2-pyridyldithio) propionylhydrazide, N- (p-maleimidophenyl) isocyanate N- Succinimidyl (4′-azidophenyl) 1,3′-dithiopropionate, succinimidyl 4-hydrazinonicotinate acetone hydrazone, N-succinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, N-imidyl 3- (bromoacetamido) propionate, succinimidyl 4-formylbenzoate, succinimidyl 4-hydrazide terephthalate hydrochloride, N-succinimidyl iodoacetate N-succinimidyl (4-iodoacetyl) aminobenzoate, succinimidyl 4- (N-maleimide) Methyl) cyclohexane-1-carboxylate, NHS-PEO2-maleimide, NHS-PEO4-maleimide, NHS-PEO8-maleimide, NHS-PEO12-maleimide, succinimidyl 4- (p-maleimido-phenyl) butyrate succinimidyl- 6- (β-maleimidopropionamido) hexanoate, 4-succinimidyloxycarbonylethyl-α- (2-pyridyldithio) toluene Cinimidyl- (4-psoralen-8-yloxy) butyrate, N-succinimidyl 3- (2-pyridyldithio) propionate, ethylene glycol bis (sulfo-succinimidyl succinate), N- (ε-maleimidocaproyloxy) Sulfosuccinimide ester, N- (γ-maleimidobutyryloxy) sulfosuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N- (κ-maleimidoundecanoyloxy) sulfosuccinimide ester, sulfosuccin Imidyl 6- (3 ′-[2-pyridyl-dithio] propionamido) hexanoate, m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, sulfosuccinimidyl (4-azido-salicylami) ) Hexanoate, sulfosuccinimidyl (4-azido-phenyldithio) propionate, sulfosuccinimidyl 2- [7-azido-4-methylcoumarin-3-acetamido] ethyl-1,3'-dithiopropionate Sulfosuccinimidyl-2- (m-azido-o-nitrobenzamido) ethyl, 1,3′-sulfosuccinimidyl 6- (4′-azido-2′-nitrophenylamino) hexanoate, sulfosk Cinimidyl 2- (p-azido-salicylamido) ethyl 1,3′-dithiopropionate, sulfo-NHS- (2-6- [biotinamide] -2- (p-azidobenzamido) hexanoamido) ethyl- 1,3′-dithiopropionate, sulfosuccinimidyl (perfluoroazidobenzamido) ethyl 1, 3′-dithiopropionate, sulfosuccinimidyl (4-iodoacetyl) aminobenzoate, sulfosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate, sulfosuccinimidyl 4- Examples thereof include (p-maleimidophenyl) butyrate, 1,6-bismaleimidohexane, bis-maleimidoethane, maleimidodiethylene glycol, disuccinimidyl glutarate and the like, but are not limited thereto. Also preferably, the linker-providing agent is capable of binding to the structure of the functional group present in the particle preparation material. These linker imparting agents can be preferably used when the particles are organic silica.

  Alternatively, in another embodiment, a maleimide group, an N-succinimide ester group, an isocyanate group, or the like that is bonded to the scavenger and then bonded to the particles may be used. Such examples are also exemplified in the examples, for example, the binding of maleimide activated neutravidin (Maleimide Activated NeutrAvidin; a protein having a maleimide group bound thereto) to particles. In addition, a capture agent in which a protein or a linker is fused in order to directly associate or bind the capture agent with the particle can also be used. Thus, it is understood that when a linker-providing agent is utilized, the linker-providing agent may be first bound to the particle or first bound to the capture agent.

  In one aspect, the present invention provides a two-step or more washless assay kit comprising the capture agent-binding carrier of the present invention and a blocking agent. It will be appreciated that any of the embodiments described herein can be used in this assay as a carrier of the present invention. In addition, as the blocking agent used here, it is understood that any of those exemplified in (a carrier including particles having a structure binding a capture agent) can be used.

  In one aspect, the present invention provides a two or more washless assay using the capture agent binding carrier of the present invention. It will be appreciated that any of the embodiments described herein can be used in this assay as a carrier of the present invention.

  In one aspect, the present invention provides a two or more washless assay using the capture agent binding carrier of the present invention. Wherein the assay comprises (A) providing a capture agent binding carrier of the invention; (B) a test substance to which the capture agent specifically binds in the presence or absence of a blocking agent. (C) when the test substance is not labeled, further contact with a labeled agent that specifically binds to the test substance or specific to the test substance A step of contacting a binding agent that specifically binds, and then a further contact with a labeling agent that specifically binds to the binding agent (this step is skipped if there are two steps, steps 3 and 4 And (D) the label bound directly or indirectly to the capture agent binding carrier is detected and detected. The sign The step of determining the amount of test substance in the medium including,. It will be appreciated that any of the embodiments described herein can be used in this assay as the carrier, capture agent, blocking agent, etc. of the present invention. It will also be appreciated that any of the embodiments illustrated in (a carrier comprising particles having a structure that binds a capture agent) can also be used in this assay. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed. When performed in the presence of a blocking agent, the assay of the present invention has the characteristic that the effects of blocking are significantly reduced compared to conventional assays. In some embodiments, for example, when using DNA, a washless assay can be achieved without using a blocking agent, and such an assay could not be achieved by conventional assay techniques. In this respect, it can be said that there is a remarkable effect. Thus, in a form made in the absence of a blocking agent, the assay of the present invention can be said to be washless and blockingless.

  One of the features of the assay and assay kit of the present invention is that they can be quantitatively measured and / or qualitatively measured. In a preferred embodiment, these features can be used.

(Blocking partial binding carrier)
In another aspect, the present invention provides a carrier for achieving washless in a two-step or more assay, comprising 1) particles having a structure for binding a capture agent, 2) a capture agent, and 3) a blocking moiety. In addition, the capture agent and the particles are directly associated or bound, or are linked via a linker, the carrier providing a carrier having a pore volume effective to achieve a washless. The carriers, capture agents and blocking moieties, linkers or linker-providing agents, binding modes, etc. used here are described in the above (carriers containing particles having a structure for binding the capture agents) and (capture agents-binding carriers). It is understood that any embodiment can be used, where it is understood that the blocking moiety may be similar to the blocking agent used herein. A blocking agent is called a blocking moiety when bound to and integrated with particles.

  In another aspect, the present invention provides a two-step or more washless assay kit comprising the blocking moiety-binding carrier of the present invention. Such a kit can be provided as a ready-made assay kit. The carriers, capture agents and blocking moieties, linkers or linker-providing agents, binding modes, etc. used here are described in the above (carriers containing particles having a structure for binding the capture agents) and (capture agents-binding carriers). It is understood that any embodiment can be used, where it is understood that the blocking moiety may be similar to the blocking agent used herein.

  In another aspect, the present invention provides a two or more washless assay using the blocking moiety binding carrier of the present invention. It will be appreciated that any of the embodiments described herein can be used in this assay as a carrier of the present invention. In addition, the carrier used, the capture agent and the blocking moiety, the linker or linker-providing agent, the binding mode, and the like are described in the above (carrier including particles having a structure for binding the capture agent) and (capture agent-binding carrier). It is understood that any embodiment can be used, where it is understood that the blocking moiety may be similar to the blocking agent used herein.

  In yet another aspect, the present invention is a two-step or more washless assay using the blocking moiety-binding carrier of the present invention, comprising (A) providing the blocking moiety-binding carrier of the present invention; (B) said capture Contacting the sample with a sample that contains or appears to contain a test substance to which the agent specifically binds; (C) a labeled agent that specifically binds to the test substance when the test substance is not labeled; Further contacting or contacting with a binding agent that specifically binds to the test substance, and then further contacting with a labeled agent that specifically binds to the binding agent (if this step is in two stages) (Which is skipped and includes steps 3 and 4) (or a binding agent may be further intervened multiple times); and (D) directly on the capture agent-bound carrier. Other detects the label indirectly bound, comprising the steps of determining the amount of said test substance in said sample the detected label, provides a two-stage or more wash-less assay. It will be appreciated that any of the embodiments described herein can be used in this assay as the carrier, capture agent, blocking agent, etc. of the present invention. In addition, the carrier used, the capture agent and the blocking moiety, the linker or linker-providing agent, the binding mode, and the like are described in the above (carrier including particles having a structure for binding the capture agent) and (capture agent-binding carrier). It is understood that any embodiment can be used, where it is understood that the blocking moiety may be similar to the blocking agent used herein. Preferably, the two-step or more washless assay of the present invention does not wash between steps. Alternatively, preferably in the two-step or more washless assay of the present invention, in each step, it may be advantageous to perform the next step directly after the previous step is completed. When performed in the presence of a blocking agent, the assay of the present invention has the characteristic that the effects of blocking are significantly reduced compared to conventional assays.

(Example)
In one illustrative example, micro-sized organosilica particles are made. Next, the sandwich reaction performed on the particle surface is examined (FIG. 1 left). The sample to be measured can be carried out using bovine serum albumin which is a relatively inexpensive protein, the primary antibody is a monoclonal antibody, and the secondary antibody is a polyclonal antibody. Further, lysozyme and glutathione S transferase can be used. Cytokines and the like can also be performed.

  (1) Production of particles: Uniform organic silica particles having a diameter of 1 to 10 μm are produced as beads array particles. A method has already been established, and Patent Document 3 and the like can be referred to. Illustratively, micro-sized thiol organic silica particles are prepared for use as beads array particles. It has been demonstrated that 3-mercaptopropyl-trimethoxysilane can be produced as a raw material in a one-step reaction. Fig. 2 shows an electron microscope image. The present invention can be carried out by producing uniform-sized organic silica particles having an average particle diameter of about 3.2 μm.

(2) Sandwich reaction:
(A) Primary antibody binding method Several kinds of useful methods already known in the art such as an adsorption method and a method using a chemical cross-coupling agent can be used. A person skilled in the art can examine conditions with higher sensitivity and a wider measurement range.

For example, antibody binding to the particle surface was examined using thiol organosilica particles. It is understood that a method using particle adsorption, a method using a coupling agent, and the like can be implemented. An example is shown in FIG. A method using 4-maleimidobutyric acid N-succinimidyl ester is exemplified. Antigen-antibody reactions including sandwich reactions on organosilica particles have been demonstrated. It has also been confirmed that the antigen-antibody reaction on the particle surface is possible in the adsorption method.
(B) Blocking skimming rum, a commercially available blocking reagent, etc. can be examined. Actually, it has been confirmed using skim milk, gelatin, and a commercially available blocking reagent. Illustratively, blocking of 0.25% to 1.0% Block Ace (commercially available product) can be determined as a standard condition from the result of a comparative experiment using the primary evaluation system (FIG. 1 left).
(C) Reaction conditions The optimum size and concentration of particles, the concentration of secondary antibody (detection antibody), reaction time, buffer conditions, etc. can be examined. It is also possible to study the use of a small amount of sample and shorten the measurement time.

(3) Evaluation (a) Primary evaluation of bovine serum albumin (BSA) measurement: selection of antibody Establishing a primary evaluation system (Fig. 1 left), antibody binding method, blocking condition study, antibody affinity to antigen Etc. can be evaluated. In this example, various anti-BSA antibodies can be bound to the particle surface. Various antibodies such as mouse monoclonal antibodies, rabbit and sheep polyclonal antibodies were examined. One hour after addition of various concentrations of FITC-labeled BSA, measurement was performed by flow cytometry. As shown in FIG. 4, the binding activity of BSA to the particles was clearly different depending on the antibody. The mouse monoclonal antibody (black) can only measure from 100 ng / ml to 10 ng / ml, while the rabbit polyclonal antibody (green) can measure a wide range from 100 ng / ml to 2.5 ng / ml. . The primary evaluation made it possible to select an antibody with high binding activity suitable for sandwich measurement. In addition, this evaluation can be applied as a simple evaluation method for various antigen-antibody reactions.
(B) Examination of reaction time The time dependency of the antigen-antibody reaction can be evaluated in a primary evaluation system. After the sheep / anti-BSA antibody was bound to the particle surface, various concentrations of FITC-labeled BSA with fluorescent labeling were added to start the reaction, and the measurement was performed 0.5, 1, 2 hours later. As shown in the measurement results in FIG. 4, an increase in the fluorescence intensity at each concentration was observed in a time-dependent manner. No change in the measurement range was observed, and the possibility of short-time measurement was found.
(C) Examination of reaction temperature The temperature dependence of the antigen-antibody reaction can be evaluated in a primary evaluation system. After the sheep anti-BSA antibody was bound to the particle surface, various concentrations of FITC-labeled BSA with fluorescent labeling were added, and the reaction was carried out at 25 ° C. or 37 ° C. for 1 hour, followed by measurement. The reaction at 37 ° C. at each concentration showed high fluorescence intensity and good concentration dependency.
(D) Evaluation of Washing Process Flow cytometry can be measured using at least two parameters (particle scattering light and fluorescence). Therefore, since it could be distinguished from the background fluorescence of the reaction solution by the scattered light, it was verified that it could be measured without a washing operation after the reaction. As a result, there was no significant change in the measurement range with and without washing. Based on these results, the following measurements were made with “no wash” for the purpose of differentiation from existing products. These conditions can be modified or combined as appropriate.

4) Measurement results (a) BSA measurement (sandwich measurement)
A sandwich measurement (right in FIG. 1) using a rabbit polyclonal antibody and a sheep polyclonal antibody with high binding activity was examined. After binding the rabbit anti-BSA antibody to the particle surface, BSA solutions of various concentrations were added and reacted. Next, FITC-labeled sheep / anti-BSA antibody was added and reacted, followed by measurement. FIG. 8 and Table 1 (see Example 7) show typical results and measured values. Quantitative measurement was possible in the range of 50 ng / ml to 5 pg / ml. Moreover, the coefficient of variation (% CV) of the measured value (n = 3) of each concentration was 1.5 to 5.5%. We succeeded in sandwich measurement of antigen protein with high sensitivity and wide measurement range. In addition to this result, it was confirmed that quantitative measurement was possible in a BSA concentration range of 200 ng / ml to 2 fg / ml (for example, FIG. 10). In addition, there was a result that a correlation between the concentration and the fluorescence was recognized even when the BSA concentration was in the fg / ml range, but it was confirmed that it could be judged that further examination was necessary from the viewpoint of reproducibility. In addition, precautions at the time of measurement have been confirmed, such as differences in fluorescence intensity even at the same concentration due to differences in stirring devices and incubator devices during the measurement process.
(B) Measurement of anti-BSA antibody (sandwich measurement)
A system for measuring antibodies was examined. After binding BSA to the particle surface, various concentrations of rabbit anti-BSA antibody solutions were added and reacted. Next, PE-labeled anti-rabbit antibody can be added and reacted, and then measured. It has been confirmed that a typical result is shown in FIG. Quantitative measurement was possible in the range of 200 ng / ml to 5 pg / ml. It has been confirmed that we have succeeded in establishing a sandwich assay with high sensitivity and a wide measurement range for antibody proteins.
(C) Biotinylated protein measurement (sandwich measurement)
A system for measuring biotinylated protein was established. After binding the rabbit / anti-BSA antibody to the particle surface, various concentrations of biotin-labeled anti-rabbit antibody solutions were added and reacted. Next, PE-labeled avidin was added and reacted, and then measured. Quantitative measurement was possible in the range of 20 ng / ml to 2 pg / ml. We have succeeded in establishing a highly sensitive and wide measuring system using both the antigen-antibody reaction of biotinylated protein and avidin biotin binding. All the above measurement results were obtained without “cleaning”. Compared with cleaning, the measurement time and labor can be greatly reduced. It will be an assay system that can be automated in the future.

  In a preferred embodiment, the present invention can perform A) high sensitivity, B) expansion of the measurement range, and the like.

A) High sensitivity:
1) Antibody binding method: The appropriate binding method varies depending on the antibody. The study can proceed based on the already established method. The measurement sample can be fluorescently labeled to evaluate the binding and antibody activity of the primary antibody and the effect of blocking (primary evaluation (two-step assay), FIG. 1 left). Research can be conducted on those capable of detecting pg / ml or less.
2) Antibody affinity: depends on the characteristics of the antibody to be purchased. Efficient study is possible by performing the above primary evaluation.
3) Blocking method: It is important for suppressing non-specific binding and reducing the background during measurement. Retrieval research of reagents can be conducted. It is possible to proceed efficiently with the primary evaluation.

B) Expansion of measurement area:
1) Particle size: In the present invention, particles of about 5 μm are exemplified, but other particles can also be used. The optimum size of the organic silica particles in the bead array can be studied.
2) Reaction conditions: Conditions such as reaction time, temperature, and stirring can be examined.
3) Others: Examination of antibody concentration and the like is considered to be the same as that of ordinary ELISA, and those skilled in the art can appropriately design. If the reaction system on the plate and the reaction system on the beads in the liquid show different reaction rates, they can be adjusted appropriately.

  In the present invention, detection in the order of pg / ml is obtained in one-step detection. It is highly possible that the goal can be achieved by examining the optimum conditions at the above points. Based on the research results, a bead array of biologically important molecules such as cytokines and CRP can be produced.

  In the present invention, it is demonstrated that quantitative measurement in the range of several pg / ml to several hundred ng / ml can be performed, which is equivalent to a bead array produced using organic silica particles. In addition to the measurement of BSA antigen protein, it has been demonstrated that in addition to this, measurement of antibodies and biotinylated proteins can be performed with high sensitivity and a wide range. In the present invention, it has been confirmed that the performance of a bead assay using organic silica particles is equivalent to that of commercially available beads. In the present invention, as exemplified in the examples, the investigation can be carried out in a reaction system centered on BSA, but is not limited thereto.

  In the present invention, the obtained research results show that sandwich measurement by antigen-antibody reaction is possible on organic silica particles, and development as beads assay particles has been ensured. The present invention can also perform multiplex fluorescence of particles for multiplex assays. Furthermore, it can be applied to sandwich measurement even with particles subjected to multiple fluorescence.

(References)
The following is a list of documents that can be considered in the implementation of the present invention.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used in the present specification are well known and commonly used in the art. For example, Current protocols in immunology (John Wiley & Sons, Inc) , Current protocols in Cytometry (John Wiley & Sons, Inc), Current protocols in Protein science (John Wiley & Sons, Inc), Current protocols in Molecular biology (John Wiley & Sons, Inc), Current protocols in nucleic acid chemistry (John Wiley & Sons, Inc., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press), etc., which are incorporated herein by reference in their entirety, which may be relevant.

  References such as scientific literature, patents and patent applications cited herein are hereby incorporated by reference in their entirety to the same extent as if each was specifically described.

  The present invention has been described with reference to the preferred embodiments for easy understanding. In the following, the present invention will be described based on examples, but the above description and the following examples are provided only for the purpose of illustration, not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described in the present specification, but is limited only by the scope of the claims.

  Examples are provided below. This example is provided for illustrative purposes only, and is not intended to limit the invention.

(Example 1: Example of production of particles)
(Production Example A)
To 100 μl of 95% mercaptopropyltrimethoxysilane (MPS), 700 μl of isopropanol solution and 300 μl of 28 wt% aqueous ammonia solution are added and mixed, and reacted at 100 ° C. for 3 hours. Next, the reaction end solution is subjected to a high-speed centrifuge (10,000 × g, 5 minutes), and the pellet is collected. The pellets are washed using 70% ethanol and distilled water alternately. Washing is repeated a total of 6 times by centrifugation. The collected pellets (silica particles) were stirred and dispersed with an ultrasonic crusher, then sampled and observed with a fluorescence microscope. For example, the average particle size was about 3500 nm, the size control rate was about 17%, and the size was good. Production of particles under control is confirmed.

(Production Example B)
To 20 μl of 80% mercaptopropyltrimethethoxysilane (MPES), 490 μl of isopropanol solution and 490 μl of 28 wt% aqueous ammonia solution are added and mixed, and reacted at 99 ° C. for 3 hours. Next, the reaction end solution is subjected to a high-speed centrifuge (10,000 × g, 5 minutes), and the pellet is collected. The pellets are washed using 70% ethanol and distilled water alternately. Washing is repeated a total of 6 times by centrifugation. The collected pellets (silica particles) were stirred and dispersed with an ultrasonic crusher, then sampled and observed with a fluorescence microscope. For example, the average particle size was about 900 nm, the size control rate was about 23%, and the good size Production of particles under control is confirmed.

(Example 2: Measurement example of pore volume of particles)
The particles produced in Production Example B of Example 1 were measured by a gas adsorption method using a Beckman Coulter specific surface area pore distribution measuring device SA3100. The specific surface area was 4.816 m ² / cm ² and the total pore volume was 0.0159 cm ³ / g.

(Example 3: Binding of capture agent to particle surface)
(Example 3-1)
20 μl of 0.5M 4-maleimidobutyric acid N-succinimidyl ester was added to 2 mg of the thiol organic silica particles produced in Example 1 and reacted at 25 ° C. for 2 hours. Washing by centrifugation was performed to remove unreacted 0.5M 4-maleimidobutyric acid N-succinimidyl ester. The particles became particles having N-succinimidyl ester on the surface. The mixture was reacted with 100 μg of rabbit anti-bovine serum albumin (BSA) antibody (B ethyl) at 25 ° C. for 3 hours. The unreacted antibody was removed by washing by centrifugation. In this operation, it is considered that the pore volume does not substantially vary depending on the operation.

(Example 3-2)
20 μl of 0.5M 4-maleimidobutyric acid N-succinimidyl ester was added to 2 mg of the thiol organic silica particles produced in Example 1 and reacted at 25 ° C. for 2 hours. Washing by centrifugation was performed to remove unreacted 0.5M 4-maleimidobutyric acid N-succinimidyl ester. Reaction was performed with 100 μg of sheep anti-bovine serum albumin (BSA) antibody (Bethyl) at 25 ° C. for 3 hours. The unreacted antibody was removed by washing by centrifugation. In this operation, the pore volume does not substantially vary depending on the operation.

(Example 3-3)
20 μl of 0.5M 4-maleimidobutyric acid N-succinimidyl ester was added to 2 mg of the thiol organic silica particles produced in Example 1 and reacted at 25 ° C. for 2 hours. Washing by centrifugation was performed to remove unreacted 0.5M 4-maleimidobutyric acid N-succinimidyl ester. Reaction was performed with 100 μg of bovine serum albumin (BSA) (Sigma) at 25 ° C. for 4 hours. Washing by centrifugation was performed to remove unreacted BSA. In this operation, the pore volume does not substantially vary depending on the operation.

(Example 3-4)
2 mg of thiol organosilica particles prepared with mercaptopropyltriethoxysilane (MPES), 2 mg of Netavidin, 2.5 mg / ml of maleimide activated neutravidin (Maleimide Activated NeutrAvidin; Pierce) and 25 ° C. for 2 hours Reaction was performed. Centrifugation washing was carried out to remove unreacted Maleimide Activated NeutrAvidin. In this operation, the pore volume does not substantially vary depending on the operation.

(Example 3-5: Experimental example for binding a capture agent to the particle surface)
The following was performed as an example of an experimental method for binding the capture agent to the particle surface.

(1) The dry weight of the organic silica particles (average particle size 3,500 nm) is measured. It was 41.91 mg. The organic silica particles have a specific gravity of 2.3 g / ml, and there are 8.12 × 10 ⁸ particles. Dispersed in distilled water.

  (2) After (1), a linker-imparting agent (maleimidobutyric acid N-succinimidyl ester) was then bound.

(3) Next, a capture agent (antibody, anti-CD20 antibody (Rixan)) was bound, the particles were washed, dried and weighed. When the weight difference between (3) and (1) is the binding amount of the capture agent (antibody), and this was measured, there was an increase of 0.23 mg. When the molecular weight of the antibody is calculated as 150 kDa, 7.43 × 10 ¹² antibody molecules per 1 mg of particle and 3.83 × 10 ⁵ antibody molecules per particle are bound. Binding of 0.010 antibodies per square nm (one antibody per approximately 100 square nm) was observed.

  (4) Next, a blocking agent was bound, dried and weighed. When the weight difference from (3) was the binding amount of the blocking agent, there was an increase of 2.7 mg (0.064 mg of binding per 1 mg of particles). When the washing operation was performed, the weight difference increased by 0.12 mg, and only 0.003 mg of binding per 1 mg of particles was observed. The blocking agent is loosely bound to the particles and antibody protein, and this gentle binding is considered to work for no washing and blocking effect.

In addition, when the amount of protein bound to the particles by adsorption without using a linker-imparting agent was measured, there was an increase of 0.35 mg, and with anti-CD20 antibody (Rixan), 2.58 × 10 ¹³ antibody molecules, 1 Binding of 1.33 × 10 ⁶ antibody molecules per particle, 0.035 antibodies per square nm particle surface area (one antibody per approximately 28.6 square nm) was observed.

When the amount of protein binding to the particles by adsorption is measured without using a linker with BSA (molecular weight 66 kDa), 3.77 × 10 ¹⁴ protein molecules per 1 mg particle, 1.95 × 10 ⁷ BSA molecules per particle Bonding of 0.506 BSA per square nm of particle surface area (one BSA per approximately 2 square nm) was observed.

  A large amount of scavenger could be attached to the particle surface with or without a linker. It is known that the binding activity of the capture agent varies depending on characteristics such as the surface potential of the capture agent, surface functional groups, secondary structure, and tertiary structure, and the method of binding to the particles. From the results of this example, it is understood that the binding between the organic silica particles and the scavenger can be changed as necessary, such as adsorption or binding by a linker.

(Example 4: Examination of blocking reagent and blocking index (A):
In Example 4, the blocking reagent was examined and the blocking index was measured.

  In this example, examination was performed using skim milk, gelatin, and a commercially available blocking reagent. A comparative experiment using the primary evaluation system (left in FIG. 1) was conducted.

  Various blocking agents (4.0% Block Ace, 5% skim milk / phosphate buffer (PBS), 2.5% gelatin) prepared by various dilutions in 5 μl of 2 mg / ml thiol organosilica particles prepared in Example 1・ Phosphate buffer solution) Add 5 μl, stir well, react with FITC-labeled BSA for 30 minutes, add 300 μl of PBS, stir, measure by flow cytometry, and measure the fluorescence of FITC-labeled BSA bound to the particles. It measured and the blocking index (Blocking Index: BI) was computed with the following method.

Blocking index (BI) =
((Particle fluorescence without blocking treatment)-(Particle fluorescence with blocking treatment))
/ (Particle fluorescence without blocking treatment) × 100 (%).

(result)
The following table shows the results of fluorescence intensity and blocking index.

  As described in the above table, the BI is 99% or more in a phosphate buffer containing 0.125% to 2.0% block ace and 0.313% to 2.5% skim milk. The BI was 95% or more in a phosphate buffer containing 15% to 1.25% gelatin.

(Comparative example 1: Examination of blocking reagent and blocking index (B): (comparative example))
Various blocking agents (0.16% Block Ace / Phosphate Buffer, 0.2% Skim Milk / Phosphate Buffer) 5 μl were added to 5 μl of 2 mg / ml thiol organic silica particles produced in Example 1 and stirred well. Thereafter, 300 μl of PBS was added for dilution. After reacting with 50 μg / ml FITC-labeled sheep anti-bovine serum albumin (BSA) antibody (Bethyl) and 30 μl for 30 minutes, the FITC fluorescence of the particles was measured by flow cytometry, and the blocking index (Blocking Index: BI) was calculated. .

(result)
The following table shows the results of fluorescence intensity and blocking index.

  Similarly, a rabbit anti-bovine serum albumin (BSA) antibody (Bethyl) was bound to the surface of Bio-rad Bio-Plex COOH beads. The method was performed using Bio-Plex amine coupling kit and ProteOn amine coupling kit according to the protocol of Bio-rad.

Add 1 μl of 4% Block Ace to 5 μl of Bio-Plex COOH beads (approx. 1.25 × 10 ⁷ ) to which the surface of a rabbit anti-bovine serum albumin (BSA) antibody is bound, and after stirring well, add 300 μl of PBS. Dilution was performed. After reacting with 50 μg / ml FITC-labeled sheep anti-bovine serum albumin (BSA) antibody (Bethyl), 30 μl for 30 minutes, the FITC fluorescence of the particles was measured by flow cytometry, and the blocking index (Blocking Index: BI) was calculated. .

(result)
The following table shows the results of fluorescence intensity and blocking index.

Further, the fluorescence intensity after centrifugal washing three times was 833, and BI was 50.3%.

  As shown in the results of the above table, the thiol organosilica particles have a high blocking efficiency, and a large difference in BI was observed compared to the Bio-Plex COOH beads.

(Example 5: (a) Primary evaluation of bovine serum albumin (BSA) measurement (two-step assay): comparison of antibody binding activity)
In this example, primary evaluation (two-step assay) of bovine serum albumin (BSA) measurement was performed, and antibody binding activities were compared.

The following anti-BSA antibodies were bound to the surface of the thiol organosilica particles produced in Example 1.
Polyclonal antibody / rabbit (Bethyl, A10-127A)
・ Sheep (Bethyl, A10-113A)
・ Rabbit (INTER-CELL AG7140)
Mouse monoclonal antibody / 25G7 antibody (Abbiotec ^™ 250417)
The coupling method is as follows.

  20 μl of 0.5M 4-maleimidobutyric acid N-succinimidyl ester was added to 2 mg of the thiol organic silica particles produced in Example 1 and reacted at 25 ° C. for 2 hours. Washing by centrifugation was performed to remove unreacted 0.5M 4-maleimidobutyric acid N-succinimidyl ester. The antibody was reacted with 100 μg of the above at 25 ° C. for 3 hours. The unreacted antibody was removed by washing by centrifugation.

  After adding 5 μl of 0.8% BlockAce PBS solution to 5 μl of anti-BSA antibody-bound thiol organic silica particles in which anti-BSA antibody was bound to the thiol organic silica particles produced in Example 1, various concentrations were added. 50 μl of FITC-labeled BSA (2.6 ng / ml to 5000 ng / ml) was added, and measurement was performed by flow cytometry after 1 hour.

  The results are shown in FIG.

As shown in FIG. 4, the binding activity between the antibody and BSA could be evaluated. The mouse monoclonal antibody (25G7 antibody (Abbiotec ^™ )) (marked with ●) can only measure from 100 ng / ml to 10 ng / ml, whereas the rabbit polyclonal antibody (Bethyld) (marked with x) has a capacity of 100 ng / ml to 2. A wide range of quantitative measurements was possible up to 5 pg / ml. The primary evaluation made it possible to select an antibody with high binding activity suitable for sandwich measurement. In addition, this evaluation can be applied as a simple evaluation method for various antigen-antibody reactions. A schematic diagram of the particles to which BSA is bound is shown in FIG.

(Example 6: (b) Primary evaluation of bovine serum albumin (BSA) measurement: examination of reaction time)
In this example, primary evaluation of bovine serum albumin (BSA) measurement was performed. In this example, the reaction time was examined.

  After 5 μl of a 0.8% BlockAce PBS solution was added to 5 μl of sheep anti-BSA antibody-bound thiol organic silica particles in which sheep anti-BSA antibody was bound to the thiol organic silica particles prepared in Example 1, After addition of 50 μl of FITC-labeled BSA at a concentration of 2.6 ng / ml to 5000 ng / ml, 0.5 μl, 1 and 2 hours later, 200 μl of PBS was added and stirred, followed by flow cytometry.

  As shown in FIG. 6, the increase in fluorescence intensity at each concentration was recognized in a time-dependent manner. No change in the measurement range was observed, and the possibility of short-time measurement was found.

  In addition, by diluting the reaction solution (20 to 50 times, or more or less) before measurement by flow cytometry, not only can the volume required for the measurement required by the instrument be secured, but also the back-up by dilution. The effect of lowering the ground can also be expected.

(Example 6: (c) Primary evaluation of bovine serum albumin (BSA) measurement: examination of reaction temperature)
In this example, primary evaluation of bovine serum albumin (BSA) measurement was performed. In this example, the reaction temperature was examined.

  After adding 5 μl of a 0.8% BlockAce PBS solution to 5 μl of sheep anti-BSA antibody-bound thiol organic silica particles in which sheep anti-BSA antibody was bound to the thiol organic silica particles prepared in Example 1, Of FITC-labeled BSA at a concentration of 2.6 ng / ml to 5000 ng / ml was added, and after 1 hour of reaction at 25 ° C. or 37 ° C., measurement was performed by flow cytometry.

  The results are shown in FIG.

  As shown in FIG. 7, the reaction at 37 ° C. at each concentration generally showed higher fluorescence intensity and better concentration dependency.

(Example 7: a) Measurement of BSA (sandwich measurement))
In this example, measurement was performed by sandwich assay.

  After adding 5 μl of a 0.8% BlockAce PBS solution to 5 μl of rabbit anti-BSA antibody-bound thiol organic silica particles in which a rabbit anti-BSA antibody was bound to the thiol organic silica particles produced in Example 1, 300 μl of BSA / PBS solution having a concentration (5 pg / ml to 50 ng / ml) was added and allowed to react for 1 hour. Thereafter, 50 μg / ml FITC-labeled sheep anti-BSA antibody / PBS solution (50 μl) was added without performing the washing operation, and the mixture was reacted for 1 hour, followed by measurement by flow cytometry without washing.

  The results are shown in FIG. 8 and Table 1.

  As shown in FIG. 8 and Table 1, quantitative measurement was possible in the range of 50 ng / ml to 5 pg / ml. Moreover, the coefficient of variation (% CV) of the measured value (n = 3) of each concentration was 1.5 to 5.5%. We succeeded in sandwich measurement of antigen protein with high sensitivity and wide measurement range. FIG. 9 shows a schematic diagram of the sandwich assay (three-stage assay) of this example.

(Example 8: b) Measurement of anti-BSA antibody (sandwich measurement))
In this example, another example of measurement in a sandwich assay was performed.

  5 μl of 0.8% BlockAce in PBS was added to 5 μl of BSA-bound thiol organosilica particles obtained by binding BSA to the thiol organosilica particles produced in Example 1, and after stirring well, various concentrations (from 2 pg / ml to 200 ng / ml) of rabbit anti-BSA antibody / PBS solution (300 μl) was added and allowed to react for 1 hour. Thereafter, 30 μl of 4 μg / ml R-phycoerythrin (PE) -labeled anti-rabbit IgG antibody / PBS solution was added and reacted for 1 hour without washing, and then measurement was performed by flow cytometry without washing.

  The results are shown in FIG.

  As shown in FIG. 10, quantitative measurement was possible in the range of 200 ng / ml to 2 pg / ml. It was possible to measure antibody protein with high sensitivity and wide measurement range.

(Example 9: c) Measurement of biotinylated protein (sandwich measurement))
In this example, another example of measurement in a sandwich assay was performed.

  After adding 5 μl of a 0.8% BlockAce PBS solution to 5 μl of rabbit anti-BSA antibody-bound thiol organic silica particles in which a rabbit anti-BSA antibody was bound to the thiol organic silica particles produced in Example 1, 300 μl of a biotin-labeled rabbit IgG antibody / PBS solution at a concentration (2 pg / ml to 20 ng / ml) was added and allowed to react for 1 hour. Thereafter, 30 μl of 10 μg / ml R-phycoerythrin (PE) -labeled streptavidin was added and allowed to react for 1 hour without washing, and then measurement was performed by flow cytometry without washing.

  The results are shown in FIG.

  As shown in FIG. 11, quantitative measurement was possible in the range of 20 ng / ml to 2 pg / ml. FIG. 12 shows a schematic diagram of the sandwich assay of this example.

FIG. 12A is a measurement finding on a BD FACSCalibur ^™ flow cytometer. The measurement conditions are as follows.

  The type of cytometer used is FACSCalibur. Detailed parameters of the detector / amplifier and the like are as shown in the following table (numerical values and the like are numerical values displayed in FACSCalibur).

The event was set to 5000. Measurements were performed under the same conditions in other measurements. The voltage of fluorescence (FL1, FL2, etc.) was normally set to the maximum (999) and adjusted as necessary.
(In addition, each detection wavelength is as follows.
SSC: 488 ± 10 nm
FL: 530 / ± 30 nm
FL2: 585 ± 42 nm
FL3: 670 nm LP
FL4: 661 ± 16 nm
)
The results are shown in FIG. 12A. The SSC and FL2 profiles of the bead assay are shown in (A) and (C), respectively. In (A), the region of the particle scattered light is designated by R8. SSC and FL2 gated in region R8 are shown in FIGS. (The X-axis of each profile represents the intensity of scattered light (SSC-H) and fluorescence (FL2-H), and the Y-axis represents the particle counts (counts) at each intensity. (D) In the profile The geometric mean (GeoMean) of the particle fluorescence was 1938.97, and this value was defined as the value of the particle fluorescence to which the streptavidin labeled with PE was bound.

  The gate can be set using SSC, FSC, particle fluorescence, fluorescence lifetime, particle volume, cell volume, or a combination thereof. The range of the gate can also be adjusted, and it is possible to analyze a particle having a specific size or fluorescence characteristic by specifying a gate in a specific narrow range. They can be used properly in the properties of the drugs and particles used and the device characteristics.

  As the definition of the fluorescence value, numerical values such as mean fluorescence intensity (MFI), median, median, peak, peak channel, fluorescence lifetime, and the like can be used, and they can be properly used in terms of measurement items, particle properties, and device characteristics.

(Example 10: Quantification of biotin-labeled antibody binding to Neutravidin)
In this example, the biotin-labeled antibody binding to Neutravidin was quantified.

  Add 5 μl of 5% BSA / PBS solution to 5 μl of Neutra-avidin-bound thiol-organic silica particles in which Neutra-avidin (Pierce) is bound to thiol-organic silica particles produced with mercaptopropyltriethoxysilane (MPES), and stir well. Then, 50 μl of biotin-labeled anti-rabbit IgG antibody / PBS solution at various concentrations (1 pg / ml to 100 ng / ml) was added and reacted for 1 hour. Thereafter, 0.5 μg / ml FITC-labeled anti-goat IgG antibody / PBS solution (50 μl) was added and reacted for 3.5 hours without washing, and after washing, PBS (290 μl) was added and stirred, followed by flow cytometry. The measurement was performed.

  The results are shown in FIG.

  As shown in FIG. 13, the quantitative measurement was possible in the range of 100 ng / ml to 1 pg / ml.

(Example 11: Quantification of the amount of adsorbed binding of anti-GST antibody)
In this example, the amount of adsorbed binding of the anti-GST antibody was quantified.

  After adding 173 μl of 5% BSA / PBS solution to 5 μl of thiol organosilica particles to which sheep / anti-GST antibody of various concentrations (3 pg / ml to 10 ng / ml) were bound by adsorption reaction on the particle surface and stirring well 2 μl of 1 μg / ml FITC-labeled anti-goat IgG antibody / PBS solution was added and reacted for 1 hour. Thereafter, 200 μl of PBS was added without washing, and the mixture was stirred and measured by flow cytometry.

  The results are shown in FIG.

  As shown in FIG. 14, quantitative measurement was possible in the range of 10 ng / ml to 3 pg / ml.

(Example 12: Comparison with and without washing)
In this example, a comparison of assays with and without washing (with and without wash) was performed.

  (A) Washless: 5 μl of 0.8% BlockAce in PBS was added to 5 μl of rabbit anti-BSA antibody-bound thiol organic silica particles obtained by binding rabbit anti-BSA antibody to the thiol organic silica particles produced in Example 1, and well After stirring, 300 μl of BSA / PBS solution having various concentrations (5 pg / ml to 50 ng / ml) was added and reacted at 37 ° C. for 1 hour. Thereafter, 30 μl of a 50 μg / ml FITC-labeled sheep anti-BSA antibody / PBS solution was added without washing operation, and the mixture was reacted at 37 ° C. for 1 hour, and then measured by flow cytometry without washing.

  (B) After adding 5 μl of 0.8% BlockAce in PBS to 5 μl of thiol organic silica particles to which Washl (+) Rabbit anti-BSA antibody is bound and stirring well, various concentrations (5 pg / ml to 50 ng / ml) 300 μl of BSA / PBS solution was added and reacted at 37 ° C. for 1 hour. Thereafter, centrifugation (500 × g, 10 minutes) was performed, the supernatant was removed, PBS was added to disperse, and then centrifugation (500 × g, 10 minutes) was performed again to remove the supernatant. After dispersing the particles in 300 μl of PBS, 50 μg / ml FITC-labeled sheep anti-BSA antibody / PBS solution (30 μl) was added and reacted at 37 ° C. for 1 hour. Thereafter, centrifugation (500 × g, 10 minutes) was performed, the supernatant was removed, PBS was added to disperse, and then centrifugation (500 × g, 10 minutes) was performed again to remove the supernatant. The particles were dispersed in 300 μl of PBS and then measured by flow cytometry.

  The results are shown in FIG.

  As shown in FIG. 15, high sensitivity and wide-range measurement were possible even without washing as with washing.

(Example 13: Necessity of washing in assay using Bio-Plex)
(A) Washless: Add 5 μl of a 0.8% BlockAce PBS solution to 5 μl of Bio-Plex COOH beads (approximately 1.25 × 10 ⁷ ) to which a rabbit anti-bovine serum albumin (BSA) antibody is bound, and mix well. Then, 300 μl of BSA / PBS solution having various concentrations (5 pg / ml to 50 ng / ml) was added and reacted at 37 ° C. for 1 hour. Thereafter, 30 μl of a 50 μg / ml FITC-labeled sheep anti-BSA antibody / PBS solution was added without washing operation, and the mixture was reacted at 37 ° C. for 1 hour, and then measured by flow cytometry without washing.

(B) Washl (+): 5 μl of 0.8% BlockAce in PBS was added to 5 μl of Bio-Plex COOH beads (approximately 1.25 × 10 ⁷ cells) bound with a rabbit anti-bovine serum albumin (BSA) antibody on the surface. In addition, after stirring well, 300 μl of BSA / PBS solution having various concentrations (5 pg / ml to 50 ng / ml) was added and reacted at 37 ° C. for 1 hour. Thereafter, centrifugation (500 × g, 10 minutes) was performed, the supernatant was removed, PBS was added to disperse, and then centrifugation (500 × g, 10 minutes) was performed again to remove the supernatant. After dispersing the particles in 300 μl of PBS, 50 μg / ml FITC-labeled sheep anti-BSA antibody / PBS solution (30 μl) was added and reacted at 37 ° C. for 1 hour. Thereafter, centrifugation (500 × g, 10 minutes) was performed, the supernatant was removed, PBS was added to disperse, and then centrifugation (500 × g, 10 minutes) was performed again to remove the supernatant. The particles were dispersed in 300 μl of PBS and then measured by flow cytometry.

  The results are shown in FIG.

  As shown in FIG. 16, the concentration dependency of BSA was not observed without washing, but quantitative measurement was possible in the range of 500 pg / ml to 500 ng / ml with washing.

(Example 14: About four-step reaction)
In this example, as a four-step reaction, measurement using an antigen-antibody reaction and avidin biotin binding was performed.

  5 μl of 0.8% BlockAce PBS solution was added to 5 μl of BSA-bound thiol organosilica particles obtained by binding BSA to the thiol organosilica particles produced in Example 1, and after stirring well, various concentrations (0.09 ng / ml-200 ng / ml) Sheep anti-BSA antibody (50 μl) was added and allowed to react for 1 hour. Thereafter, 50 μl of 1.0 μg / ml biotin-labeled anti-Goat IgG antibody / PBS solution was added and allowed to react for 1 hour without washing operation. Then, 2.0 μg / ml FITC-labeled anti-Avidin / PBS solution (50 μl) was added without washing, and the mixture was allowed to react for 1 hour. After washing, 300 μl of PBS was added, stirred, and measured by flow cytometry. Went.

  The results are shown in FIG.

  As shown in FIG. 17, quantitative measurement was possible in the range of 200 ng / ml to 274 pg / ml.

(Example 15: Measurement using multistage antigen-antibody reaction)
In this example, measurement using multistage antigen-antibody reaction was performed.

  Add 5 μl of 0.8% BlockAce in PBS to 5 μl of thiol organosilica particles to which sheep anti-BSA antibody is bound, and after stirring well, BSA / PBS solutions of various concentrations (20 pg / ml to 200 ng / ml) 300 μl was added and allowed to react for 1 hour. Thereafter, 30 μl of 1 μg / ml rabbit anti-BSA antibody / PBS solution was added and the mixture was allowed to react for 1 hour without washing. Thereafter, 30 μl of 4 μg / ml R-phycoerythrin (PE) -labeled anti-rabbit IgG antibody was added and reacted for 1 hour without washing, and then measurement was performed by flow cytometry without washing.

  The results are shown in FIG.

  As shown in FIG. 18, quantitative measurement was possible in the range of 20 ng / ml to 2 pg / ml.

(Example 16: Antigen-antibody reaction, measurement using avidin biotin binding)
In this example, measurements were made using antigen-antibody reaction and avidin-biotin binding.

  5 μl of 0.8% BlockAce in PBS was added to 5 μl of BSA-bound thiol organic silica particles obtained by binding BSA to the thiol organosilica particles produced in Example 1, and after stirring well, various concentrations (from 1 ng / ml to (1000 ng / ml) 300 μl of Sheep anti-BSA antibody was added and allowed to react for 1 hour. Thereafter, 30 μl of 10 μg / ml biotin-labeled anti-goat IgG antibody / PBS solution was added and reacted for 1 hour without washing operation. Thereafter, 30 μl of 50 μg / ml R-phycoerythrin (PE) -labeled anti-avidin / PBS solution was added and reacted for 1 hour without washing. After washing, 200 μl of PBS was added and stirred, followed by flow cytometry. Measurements were made.

  The results are shown in FIG.

  As shown in FIG. 19, quantitative measurement was possible in the range of 100 ng / ml to 1 ng / ml.

(Example 17: Examination of relationship between pore volume of particles and blocking index and washless)
Organic silica particles: pore volume (actual value) and blocking index (actual value), commercially available product: pore volume (document value), blocking index (actual value) and others: Sephadex beads (for example, 0.7-0) And a size of 9 cm ³ / g pore size that can be used for flow cytometry).

(Example 18: Determination of the weight of a capture agent (for example, an antibody) that binds to one particle) and determination of the weight of a blocking agent that binds to one particle bound to an antibody)
Link the antibody (Rixan) to the particles synthesized from MPS (diameter 3,500 nm, weight 41.91 mg (calculated that 1.94 × 10 ⁷ particles in 1 mg are present)) synthesized in Example 1 etc. After bonding, the sample was dried and weighed 42.14 mg, confirming an increase of 0.23 mg. When the calculation was performed with the molecular weight of the antibody being 150 kDa, it was found that 2.8 × 10 ⁻¹⁰ mg, 3.83 × 10 to 5 antibody molecules were bound per particle. Next, the mixture was mixed with a blocking agent (0.8% Block Ace), centrifuged, the supernatant was discarded, and the weight was measured and an increase of 2.70 mg was observed. It was found that 3.3 × 10 ⁻⁹ mg of blocking agent was bound per particle.

(Example 19: Comparison of washless effect using various blocking agents)
The washless effect can be compared using skim milk, albumin, serum, surfactant, and various synthetic blocking agents.

(Example 20: Binding of capture agent to particle surface: Example of binding with various linkers)
In the present example, binding of a capturing agent to the particle surface: an example in which various types of linkers are used for binding.

  For example, maleimide butyric acid N-succinimidyl ester, a surface of an organic silica particle having a thiol group on the surface thereof, which is further converted back to an amino group or a carboxy group and then bonded with a linker can be used.

(Example 21: Measurement and analysis using autofluorescence of particles)
In this embodiment, measurement and analysis are performed using the autofluorescence of particles. Demonstrate the possibility that autofluorescence can have a positive effect in the assay. Here, it is possible to show the possibility of improving the assay with particles that contain a fluorescent dye in advance to enhance autofluorescence.

(Example 22: Production of neutravidin-binding particles)
Here, as synthesized in Example 1, 760 μl of 2.5 mg / ml maleimide-activated neutravidin (Pierce) was added to 2 mg of thiol organic silica particles (synthesized from MPES) and reacted at 25 ° C. for 2 hours. went. Centrifugal washing was performed to remove unreacted maleimide-activated neutravidin.

  In this example, all the thiol organic silica particles to which nutravidin is bound are particles synthesized with mercaptopropyltriethoxysilane (MPES). The other particles used in the examples use mercaptopropyltrimethoxysilane (MPS) unless otherwise specified, but it is understood that other particles may also be utilized.

Example 23: Multi-stage assay where the binding of particles and capture agent is adsorption
(Three steps)
After adding 173 μl of a 5% BSA / PBS solution to 5 μl of thiol organosilica particles to which sheep / anti-GST antibodies of various concentrations (78 ng / ml to 5000 ng / ml) were bound by adsorption reaction on the particle surface and stirring well Then, 5 μl of 20 μg / ml biotin-labeled anti-Goat IgG antibody / PBS solution was added and allowed to react for 1 hour. Thereafter, 5 μl of a 100 μg / ml R-phycoerythrin (PE) -labeled anti-avidin / PBS solution was added and reacted for 1 hour, and 500 μl of distilled water was added, followed by flow cytometry without washing. The measurement was performed.

  The results are shown in FIG.

  As shown in FIG. 21, quantitative measurement was possible in the range of 5000 ng / ml to 78 pg / ml.

(Example 24: Washless one-step measurement)
In this example, one-step measurement of washless was performed.

Bio-rad Bio-Plex COOH beads (0.8 × 10 ⁷ / ml) not subjected to blocking treatment, etc. 10 μl (FIG. 22, A) (marked with ■), or thiol organic silica particles 5 μl (FIG. 23, B) On the other hand, after adding 10 μl of FITC-labeled bovine serum albumin (BSA) at various concentrations (8 ng / ml to 5000 ng / ml) and reacting at room temperature for about 3 minutes, add 300 μl of PBS and agitate well. Measured by a meter.

  The results are shown in FIGS.

  In a one-step assay, washless quantitative evaluation was possible with both Bio-rad Bio-Plex COOH beads and thiol organosilica particles. Bio-rad Bio-Plex COOH beads allowed quantitative measurement in the range of 5000 ng / ml to 40 ng / ml, and thiol organosilica particles in the range of 5000 ng / ml to 8 ng / ml. At the same concentration, the fluorescence intensity of thiol organosilica particles was higher than that of Bio-Plex COOH beads.

  The reason why a washless assay is possible for both particles is that, in a one-step assay, a labeling substance (FITC-labeled bovine serum albumin) bound to the particle and a substance penetrating into the particle are measured simultaneously. . Since the labeling substance that has penetrated into the particles depends on the solution concentration, the particle fluorescence becomes concentration-dependent. Therefore, the one-step washless assay evaluates the binding of the measurement object to the particles and the influence other than the binding (such as penetration).

(Example 25: Blocking index: Sephadex G-50)
The blocking index was calculated for 5 μl of Sephadex G-50 (Sigma) (1 mg / ml) (from which particles of 40 μm or more were removed using a filter) or 5 μl of thiol organic silica particles. The same amount of 0.8% BlockAce was added to the particles and then diluted with 300 μl of PBS. Subsequently, 50 μg / ml FITC-labeled sheep anti-bovine serum albumin (BSA) antibody (Bethyl) and 30 μl were reacted for 60 minutes, and then the FITC fluorescence of the particles was measured by flow cytometry to calculate the blocking index (BI).

(result)
The result is
1) Sephadex G-50 = 37%
2) Thiol organic silica particles = 99%
Met.

(Example 26: Assay using Sephadex G-50)
(One-step assay)
For Sephadex G-50 (Sigma) (1 mg / ml) 10 μl (■ mark) (particles of 40 μm or more removed by using a filter) or 5 μl (● mark) of thiol organic silica particles not subjected to blocking treatment, etc. After adding 10 μl of FITC-labeled bovine serum albumin (BSA) at various concentrations (8 ng / ml to 5000 ng / ml) and reacting at room temperature for about 3 minutes, add 300 μl of PBS, stir well, and then perform flow cytometry without washing. Measured with

  The results are shown in FIG.

  As shown in FIG. 24, washless quantitative evaluation was possible for both Sephadex G-50 and thiol organosilica particles in a one-step assay. FITC-labeled bovine serum albumin was at a low concentration, and Sephadex G-50 showed high fluorescence.

  The reason why a washless assay is possible for both particles is that, in a one-step assay, a labeling substance (FITC-labeled bovine serum albumin) bound to the particle and a substance penetrating into the particle are measured simultaneously. . Since the labeling substance that has penetrated into the particles depends on the solution concentration, the particle fluorescence becomes concentration-dependent. Therefore, the one-step washless assay evaluates the binding of the measurement object to the particles and the influence other than the binding (such as penetration).

(Two-step assay)
Sephadex G-50 (Sigma) (1 mg / ml) 10 μl (marked with ■) (particles larger than 40 μm removed using a filter) (10 ng / ml) sheep / anti-GST antibody (500 μl) was stirred at 37 ° C. for 1 hour, reacted, and bound by adsorption. Thereafter, 5 μl of 5% BSA / PBS solution was added without washing, and the mixture was stirred for 30 minutes for blocking treatment. Thereafter, 50 μl of 1 μg / ml FITC-labeled anti-Goat IgG antibody / PBS solution was added and allowed to react for 1 hour without washing operation. Thereafter, measurement was performed by flow cytometry without washing.

  The results are shown in FIG.

  As shown in FIG. 25, the thiol organosilica particles could be quantitatively measured in the range of 10 ng / ml to 10 pg / ml. However, Sephadex G-50 could not be quantitatively measured. Sephadex G-50 has a high swelling constant of 9 to 11 ml / g (which is considered to correlate with the pore volume) and a low blocking index of 37%, which suggests that the labeled protein penetrates well into the particles. As a result, it is conceivable that the fluorescence due to permeation or the like was higher than the increase in fluorescence of the particles due to the antigen-antibody reaction.

  In the washless assay of two or more stages, it was shown that the labeled protein penetrates into the particles and nonspecific adsorption greatly affects the measurement of the antigen-antibody reaction.

(Example 27: Silica gel)
An experiment similar to the above is performed on silica gel (pore volume 0.7-0.9 ml / g). Sephadex G-50 is assumed to have a pore volume of about 10 ml / g, thus demonstrating the effectiveness of the present invention. The polyorgano silica particles are 0.016 ml / g or less. The same experiment as in Example 26 is performed on silica gel (pore volume 0.7-0.9 ml / g), and the same evaluation as Sephadex G-50 is performed. The results of the washless assay indicate that it can be used with a pore volume of 1 ml / g or less.

<Measurement of blocking index: Silica Gel (236756 SIGMA-ALDRICH)>
The blocking index was calculated for 5 μl of silica gel (236756 SIGMA-ALDRICH) (2 mg / ml). The same amount of 0.8% BlockAce was added to the particles and then diluted with 300 μl of PBS. Thereafter, 50 μg / ml FITC-labeled sheep anti-bovine serum albumin (BSA) antibody (Bethyl) was reacted with 30 μl for 60 minutes, and then FITC fluorescence of the particles was measured by flow cytometry to calculate a blocking index.
Silica gel (236756 SIGMA-ALDRICH) = 50%.

<One-step assay: silica gel (236756 SIGMA-ALDRICH)>
10 μl of FITC-labeled bovine serum albumin (BSA) at various concentrations (8 ng / ml to 5000 ng / ml) was added to 10 μl of silica gel (236756 SIGMA-ALDRICH) (2 mg / ml) not subjected to blocking treatment, etc. at room temperature. After reaction for about 3 minutes, 300 μl of PBS was added and stirred well, followed by measurement by flow cytometry without washing.

  The results are shown in FIG.

  In a one-step assay, a washless quantitative evaluation was possible with silica gel (236756 SIGMA-ALDRICH).

<Two-step assay: silica gel (236756 SIGMA-ALDRICH)>
Silica gel (236756 SIGMA-ALDRICH) (2 mg / ml) 5 μl (500 μl of sheep anti-GST antibody at various concentrations (10 pg / ml to 10 ng / ml) was stirred at 37 ° C. for 1 hour, reacted and bound by adsorption. Then, 5 μl of 5% BSA / PBS solution was added without washing, and the mixture was stirred for 30 minutes for blocking treatment, and then 1 μg / ml FITC-labeled anti-goat IgG antibody / PBS solution 50 μl without washing. After that, it was measured by flow cytometry without washing, and quantitative measurement was possible in the range of 10 ng / ml to 1 ng / ml, but below 0.3 ng / ml Concentration dependence was not recognized.

  The results are shown in FIG.

  Silica gel (236756 SIGMA-ALDRICH) has a particle size of 10.0 to 14.0 μm and a pore volume of 0.7 to 0.9 ml / g (from Sigma Web). The blocking index was as low as 50% and quantitative measurements in the two-step assay were limited. From this result, it is considered that a washless assay is possible even if the pore volume is large and the blocking index is low, but the measurement concentration range is limited as compared with the case where the pore volume is small and the blocking index is high. In addition, it is considered that particles having a large pore volume and a low blocking index require particle size distribution and uniformity of pore volume of individual particles. Based on these results, particles having a small pore volume are desirable as a support for a washless assay.

(Example 28: Fluorescent dye-containing thiol organic silica particles)
<Preparation of dye-containing thiol organic silica particles>
Rhodamine red-C2-maleimide (Invitrogen, R6029) 5 mg was dissolved in 50 μl DMSO. Further, 14.1 μl of mercaptopropyltrimethoxysilane (95%, Aldrich) and 9.4 μl of DMSO were added and reacted at room temperature for 2 hours to prepare a dye organic silica conjugate. 10 μl of mercaptopropyltriethoxysilane (MPES), 245 μl of 2-propanol, and 245 μl of 28 wt% aqueous ammonia solution were added to 1 μl of the dye-organic silica conjugate diluted 10-fold with DMSO, and mixed at 100 ° C. Let react for hours. Next, the reaction end solution is subjected to a high-speed centrifuge (10,000 × g, 5 minutes), and the pellet is collected. The pellets are washed using 70% ethanol and distilled water alternately. Washing is repeated a total of 6 times by centrifugation. The collected pellets (silica particles) were stirred and dispersed with an ultrasonic crusher, then sampled and observed with a fluorescence microscope. For example, the average particle size was about 810 nm, the size control rate was about 16%, and the good size Production of particles under control is confirmed.

<Two-step assay: Dye-containing thiol organosilica particles>
Rhodamine red-containing thiol organosilica particles (1.2 mg / ml) 5 μl Various concentrations (0.1 ng / ml to 1,000 ng / ml) of sheep / anti-GST antibody 500 μl were stirred and reacted at 37 ° C. for 1 hour for adsorption And combined. Thereafter, 50 μl of 0.8% BlockAce solution was added without performing a washing operation, and the mixture was stirred for 30 minutes for blocking treatment. Thereafter, 50 μl of 1 μg / ml FITC-labeled anti-goat IgG antibody / PBS solution was added and reacted for 1 hour without washing operation. Thereafter, measurement was performed by flow cytometry without washing.

  The results are shown in FIG. As shown in FIG. 28, the pigment-containing thiol organosilica particles could be quantitatively measured in the range of 0.1 ng / ml to 1,000 ng / ml.

(Example 29: Epoxy organic silica particles)
<Preparation of epoxy organic silica particles>
Trimethoxy [2- (7-oxabicyclo [4.1.0] -hept-3-yl) ethyl] silane (2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane) (98% Aldrich) 50.2 μl To the mixture, 877.8 μl of distilled water and 72 μl of 28 wt% aqueous ammonia solution are added and mixed, and reacted at 25 ° C. for 18 hours. Next, the reaction end solution is subjected to a high-speed centrifuge (10,000 × g, 5 minutes), and the pellet is collected. The pellets are washed using 70% ethanol and distilled water alternately. Washing is repeated a total of 6 times by centrifugation. The collected pellets (silica particles) were stirred and dispersed with an ultrasonic crusher, then sampled and observed with a fluorescence microscope. For example, the average particle size was about 4,150 nm and the size control rate was about 27%.

<One-step assay: Epoxy organosilica particles>
3 μl of FITC-labeled bovine serum albumin (BSA) at various concentrations (8 ng / ml to 5000 ng / ml) is added to 5 μl of epoxy organosilica particles (45 mg / ml) which are not subjected to blocking treatment, and reacted at room temperature for about 3 minutes. Thereafter, 300 μl of PBS was added and well stirred, and then measured by flow cytometry without washing.

  The results are shown in FIG.

<Two-step assay: Epoxy organosilica particles>
Epoxy organosilica particles (45 mg / ml) 5 μl 500 μl of sheep anti-GST antibody at various concentrations (3 ng / ml to 1,000 ng / ml) were stirred at 37 ° C. for 1 hour, reacted and bound by adsorption. Thereafter, 50 μl of 0.8% BlockAce solution was added without performing a washing operation, and the mixture was stirred for 30 minutes for blocking treatment. Thereafter, 50 μl of 1 μg / ml FITC-labeled anti-goat IgG antibody / PBS solution was added and reacted for 1 hour without washing operation. Thereafter, measurement was performed by flow cytometry without washing.

  The results are shown in FIG. As shown in FIG. 30, the epoxy organic silica particles could be quantitatively measured in the range of 3 ng / ml to 1,000 ng / ml.

(Example 30: DNA assay: DNA washless bead assay)
Unlike antibodies, the two-stage assay experiment was conducted because the hybridized substance was bound to the particle rather than hybridized on the particle.

  3 μg / ml PE-conjugated streptavidin (eBioscience) / PBS was added to 100 μl of the thiol organosilica particles prepared in Example 1 and reacted at room temperature for 2 hours, then washed, dispersed in 100 μl of PBS, and PE conjugate. Streptavidin bonded organosilica particles were prepared.

The following oligonucleotides were used.
(A) Flu: Fluorescein bound to the 5 terminal: Fluorescein-GGCAGATCGCAGTCAGTCAC-3 ′ (SEQ ID NO: 1)
(B) Bio: Biotin bonded to 5 terminals: Biotin-GTGAACTGACTGACGATCTCCCC-3 ′ (SEQ ID NO: 2)
(C) Amine: Amino group bonded to the 5 terminal: Amino-GTGACTGACTGACGATCTGCCC-3 ′ (SEQ ID NO: 3)
Equal amounts of each 20 μM aqueous solution were mixed in 50 μl to prepare mixed solutions (A) + (B) and (A) + (C). Annealing was done. (After heating at 100 ° C, gradually cool to room temperature)
5 μl of the mixed solution (A) + (B) or (A) + (C) was added to 5 μl of PE-conjugated streptavidin-bonded organic silica particles, and stirred well. Then, after reacting at room temperature for 5 minutes, 300 μl of PBS was added, and then washing was performed, and measurement was performed by flow cytometry.

  The fluorescence (GeoMean) of (A) + (B) was 341.85, and (A) + (C) was 173.37.

  From this result, the oligonucleotide (A) was annealed and bound to (B), and was bound to streptavidin on the particle surface by biotin (B), and an increase in the fluorescence of the particle was observed. In (A) + (C), it was predicted that annealing was performed in the same manner, but since biotin was not present, it could not bind to the particles, and no increase in fluorescence as in (A) + (B) was observed. .

(Example 31: DNA washless bead assay II (hybridization on particles))
20 μl of 1M 4-maleimidobutyric acid N-succinimidyl ester was added to 100 μl of the thiol organic silica particles produced in Example 1 and reacted at 25 ° C. for 2 hours. Centrifugal washing was performed to remove unreacted 4-maleimidobutyric acid N-succinimidyl ester. Next, an oligonucleotide (amino-GTGACTGACTGACGGATCTGCC-3 ′ (SEQ ID NO: 3)), 20 μM, 50 μl added with an amino group at the 5 end was added and reacted at 25 ° C. for 1.5 hours. The oligonucleotide was removed. A schematic diagram is shown in FIG. Dispersed in 100 μl of distilled water to prepare an oligonucleotide surface modified particle dispersion. Add 5 μl of oligonucleotide (Flu-GGCAGATCGCAGTCAGTCAC-3 ′ (SEQ ID NO: 2)) in which 20 μM of fluorescein is bound to 15 μl of oligonucleotide surface-modified particle dispersion, heat at 100 ° C., and slowly cool to room temperature. Annealing was performed. Thereafter, 300 μl of distilled water was added to perform the washing operation, and then measurement was performed by flow cytometry. As a control experiment, the same experiment was also performed on unmodified thiol organic silica particles.

  The fluorescence (GeoMean) of the oligonucleotide surface modified particles was 1706.36, and the surface unmodified thiol organosilica particles were 404.23.

  From this result, it was confirmed that the fluorescein-bound oligonucleotide and the complementary oligonucleotide on the particle surface were annealed and the fluorescence of the particle was increased. In the control experiment, since there was no oligonucleotide having a complementary sequence on the particle surface, it could not bind to the particle, and no increase in fluorescence as in the oligonucleotide surface-modified particle was observed.

  A schematic diagram of the assay in this example is shown in FIG.

  As mentioned above, although this invention was illustrated using the preferable embodiment and Example of this invention, it is understood that the scope of this invention should be interpreted only by a claim. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

The present invention realizes an assay that does not require a washing step, particularly an assay by flow cytometry, which could not be achieved conventionally. Prior art products of beads assay particles and the range of hundreds ng / ml from a few pg / ml equivalent in, the quantitative measurements spanning the concentration range of ^106, can be performed in wash less. The object to be measured can be diluted or concentrated as necessary to measure a wide range of concentrations. Therefore, the present invention is expected to be applicable in the biotechnology field. In addition to commercialization as an assay system for research, it can also be used for research and development as a clinical laboratory system.

SEQ ID NO: 1 is an oligonucleotide having fluorescein linked to the 5 termini used in Examples 30 and 31.
SEQ ID NO: 2 is an oligonucleotide in which biotin is bound to the 5 terminal used in Example 30.
SEQ ID NO: 3 is an oligonucleotide having an amino group bonded to the 5 end used in Examples 30 and 31.

Claims (15)

A carrier for achieving washless in a two or more stage assay,
Including particles having a structure for binding a scavenger;
The carrier is a carrier having a pore volume effective for realizing washlessness. The carrier according to claim 1, wherein the pore volume is 1.0 cm ³ / g or less. The carrier according to claim 1, wherein the carrier has a blocking index (BI) effective to achieve washless. The carrier according to claim 1, wherein the particles are silica particles. The carrier according to claim 1, wherein the particles are organic silica particles. The structure for binding the scavenger is thiol group, amino group, carboxyl group, maleimide group, N-succinimide ester group, and isocyanate group, mercaptopropyl group, aminopropyl group, thiocyanate propyl group, maleimide group, N-succinimide ester. The carrier according to any one of claims 1 to 5, comprising a group selected from the group consisting of a group and an isocyanate group. The carrier according to any one of claims 1 to 6, wherein the particles include a label. A kit for a two-step or more washless assay, comprising the carrier according to claim 1 and at least one of a linker-imparting agent, a capturing agent, and a blocking agent. 9. The kit of claim 8, wherein the blocking agent is selected from the group consisting of skim milk, albumin, serum, surfactant, synthetic blocking agent, and mixtures thereof. The particles are organic silica particles, and the linker-imparting agent is composed of maleimide group, N-succinimide ester group, isocyanate group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group, and phenylazide group. At least one functional group selected from the group consisting of maleimide group, N-succinimide ester group or isocyanate group, hydrazide group, carboxy group, iodoacetyl group, sulfo-N-succinimide ester group, pyridyldithio group , Phenyl azide group, and optionally protected thiol group, amino group, carboxyl group, at least one functional group selected from the group consisting of at least one functional group selected from Claims comprising spacers between groups The kit according to. A two-step or more washless assay using the carrier of claim 1. (A) Providing a capture agent-bound carrier in which a capture agent is bound directly or indirectly to the carrier according to claim 1;
(B) contacting a sample that contains or appears to contain a test substance to which the capture agent specifically binds in the presence or absence of a blocking agent with the capture agent-binding carrier;
(C) When the test substance is not labeled, after further contacting with a labeled drug that specifically binds to the test substance, or after contacting with a binding drug that specifically binds to the test substance, Further contacting a labeling agent that specifically binds to the binding agent; and (D) detecting the label directly or indirectly bound to the capture agent binding carrier and detecting the detected label in the sample. The process of determining the amount of a substance,
A two or more washless assay. A carrier for achieving washless in a two or more stage assay,
1) particles having a structure for binding the capture agent; and 2) a capture agent,
The capture agent and the particle are directly associated or bound, or bound via a linker;
The carrier has a pore volume effective to achieve washless,
Carrier. The carrier according to claim 12, wherein the capture agent is selected from the group consisting of a protein, a peptide, avidin, streptavidin, a nucleic acid, a saccharide, a variant thereof, and a conjugate thereof. A carrier for achieving washless in a two or more stage assay,
1) particles having a structure for binding the capture agent, 2) the capture agent, and 3) a blocking moiety,
The capture agent and the particle are directly associated or bound, or bound via a linker;
The carrier has a pore volume effective to achieve washless,
Carrier.