WO2013118844A1

WO2013118844A1 - Method and kit for detecting and quantifying detection subject

Info

Publication number: WO2013118844A1
Application number: PCT/JP2013/052949
Authority: WO
Inventors: 悟杉田
Original assignee: オーソ・クリニカル・ダイアグノスティックス株式会社
Priority date: 2012-02-07
Filing date: 2013-02-07
Publication date: 2013-08-15
Also published as: JP5844825B2; JPWO2013118844A1

Abstract

The purpose of the present invention is to provide a method and kit for detecting and quantifying a detection subject in a sample, capable of separating non-specific reactants from the sample in a short period of time. A method for detecting and quantifying a detection subject in a sample has steps for: mixing with the sample a conjugate combining a cohesive substance containing a stimuli-responsive substance and a particulate magnetic substance, and a substance with affinity for a substance other than the detection subject; separating the aggregated conjugate (A) by adding magnetic force to this mixture under conditions in which the stimuli-responsive substance aggregates; and detecting and quantifying the detection subject in the posterior mixture.

Description

Method and kit for detection and quantification of detection target

The present invention relates to a method and kit for detecting and quantifying a detection target in a specimen.

In the field of clinical examination and the like, a trace component in a specimen is detected using an antigen-antibody reaction or the like. Examples of the specimen include specimens obtained from a living body such as various body fluids such as serum, plasma, urine, and lymph.

Such specimens generally contain a large number of substances such as proteins, antibodies, and sugars, and the non-specific reaction between these substances and the carrier for detection of the detection target is the detection result. It is known to have an adverse effect. That is, as shown in FIG. 8, regardless of the presence or absence of the detection target T in the sample, the non-specific reaction substance in the sample binds to the carrier C to form an aggregate. It is difficult to detect T.

Therefore, conventionally, in order to suppress non-specific reactions, techniques for removing the causative components of non-specific reactions in the specimen have been developed. In Patent Document 1 (Japanese Patent Laid-Open No. 8-327629), magnetic particles carrying an antibody that binds to a non-specific reactive substance are added to a specimen, and the non-specific reactive substance is separated from the specimen together with the magnetic particles with a magnet. Patent Document 1 describes that a detection result in which an adverse effect due to a non-specific reaction is suppressed can be obtained by detecting a detection target using a subsequent specimen.

Japanese Patent Laid-Open No. 8-327629

However, in order to increase the efficiency of the binding between the antibody and the non-specific reactant, it is necessary to use a magnetic particle having a large specific surface area, that is, a small particle size. It takes time. Due to this dilemma, in the technique disclosed in Patent Document 1, it takes a long time to separate the non-specific reactant from the specimen.

The present invention has been made in view of the above circumstances, and provides a method and kit for detection and quantification of a detection target in a specimen, which can separate a nonspecific reaction substance from the specimen in a short time. The purpose is to provide.

The present inventors have found that by using the agglutination action, the binding efficiency with the non-specific reactant and the separation efficiency of the non-specific reactant can be compatible, and the present invention has been completed. Specifically, the present invention provides the following.

(1) A method for detecting a detection target in a sample,
A binding substance in which an aggregating substance containing a stimulus-responsive substance and a particulate magnetic substance and an affinity substance for a substance other than the detection target are combined with the specimen are mixed, and the stimulus-responsive substance is mixed with the mixture. A step of separating the aggregated aggregates by applying a magnetic force under a condition in which the aggregates are aggregated, and then detecting the detection target in the mixture.

(2) The method according to (1), wherein the detection of the detection target is performed in a state where the aggregated aggregate is solid-liquid separated in a container in which the mixture is accommodated.

(3) The detection of the detection target includes a step of mixing a carrier carrying an affinity substance for the detection target with the mixture and discriminating whether the carrier is aggregated (1) or (2) the method of.

(4) The method according to any one of (1) to (3), wherein the detection of the detection target is performed in a state where a sensitizer that increases detection sensitivity is added.

(5) A method for quantifying a detection target in a sample,
A binding substance in which an aggregating substance containing a stimulus-responsive substance and a particulate magnetic substance and an affinity substance for a substance other than the detection target are combined with the specimen are mixed, and the stimulus-responsive substance is mixed with the mixture. The method has a step of separating the agglomerated bound substance by applying a magnetic force under a condition where the agglomerated substance is aggregated, and then quantifying the amount of the detection target in the mixture.

(6) The method according to (5), wherein the quantitative determination of the detection target is performed in a state in which the aggregated aggregate is solid-liquid separated in a container in which the mixture is accommodated.

(7) The determination of the detection target includes a step of mixing a carrier carrying an affinity substance for the detection target with the mixture and detecting the degree of aggregation of the carrier (5) or (6) the method of.

(8) The method according to any one of (5) to (7), wherein the quantification of the detection target is performed in a state where a sensitizer that increases detection sensitivity is added.

(9) A kit for detecting and / or quantifying a detection target in a sample,
A combined product in which an aggregating substance containing a stimulus-responsive substance and a fine-particle magnetic substance and an affinity substance for a substance other than the detection target are bound;
A kit comprising an affinity substance for the detection target.

(10) The kit according to (9), wherein the affinity substance is supported on a carrier.

(11) The kit according to (9) or (10), further comprising a sensitizer for increasing detection sensitivity.

According to the present invention, after binding a non-specific reactive substance to an affinity substance, a magnetic force is applied under conditions where the stimulus-responsive substance aggregates. The bound substance is excellent in binding efficiency because it is not aggregated or only slightly aggregated when the non-specific reactant is bound to the affinity substance. Further, when separating the bound matter, the bound matter aggregates, so that the moving speed by magnetic force increases. Thereby, the nonspecific reaction substance can be separated from the specimen in a short time.

It is a schematic block diagram of the combination thing used for the method which concerns on one Embodiment of this invention. FIG. 3 is a flow diagram of a method according to an embodiment of the invention. FIG. 6 shows a mechanism of a method according to an embodiment of the present invention. It is a photograph which shows the state which isolate | separated the cohesive substance which can be used with the method which concerns on one Embodiment of this invention with magnetic force. It is a photograph which shows the state which isolate | separated the particulate matter which concerns on a prior art example with magnetic force. It is a photograph which shows the low redispersibility of the cohesive substance used in FIG. It is a photograph which shows the high redispersibility of the particulate material used in FIG. It is a schematic block diagram which shows aggregation of the nonspecific reaction substance in the method which concerns on a prior art example.

Hereinafter, embodiments of the present invention will be described, but the present invention is not limited thereto.

In the detection method of the present invention, first, a bound substance and a specimen are mixed, and a magnetic force is applied to the mixture under conditions where the stimulus-responsive substance aggregates. First, the combination used here will be described in detail.

[Binder]
The binding substance is a combination of an aggregating substance containing a stimulus-responsive substance and a particulate magnetic substance and an affinity substance for a substance other than the detection target (that is, a non-specific reactive substance to be removed).

(Aggregating substance)
A stimulus-responsive substance is a substance that undergoes a structural change in response to an external stimulus and can adjust aggregation and dispersion. Examples of the stimulus include, but are not limited to, temperature change, light irradiation, acid or base addition (pH change), electric field change, and the like.

In particular, in the present invention, as the stimulus-responsive substance, a temperature-responsive polymer that can be aggregated and dispersed by temperature change can be used. Examples of the temperature-responsive polymer include a polymer having a lower critical solution temperature (hereinafter also referred to as LCST) and a polymer having an upper critical solution temperature (hereinafter also referred to as UCST). For example, a polymer having a lower critical solution temperature with an LCST of 37 ° C. is completely dispersed in an aqueous solution having a temperature lower than the LCST, and can be agglomerated immediately when the water temperature is raised above the LCST. In addition, a polymer having an upper critical solution temperature with a UCST of 5 ° C. is completely dispersed in an aqueous solution having a temperature exceeding the UCST, and can be immediately aggregated when the water temperature is lowered below the UCST.

Examples of the polymer having a lower critical solution temperature used in the present invention include Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N N substitution such as acryloylmorpholine, Nn-propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Polymer composed of (meth) acrylamide derivative; hydroxypropyl cellulose, polyvinyl alcohol partially acetylated product, polyvinyl methyl ether, (polyoxyethylene-polyoxypro Len) block copolymers, polyoxyethylene alkylamine derivatives such as polyoxyethylene laurylamine; polyoxyethylene sorbitan ester derivatives such as polyoxyethylene sorbitan laurate; (polyoxyethylene nonylphenyl ether) acrylate, (polyoxyethylene octylphenyl) (Polyoxyethylene alkylphenyl ether) (meth) acrylates such as ether) methacrylate; and (polyoxyethylene alkyl ether) (meth) acrylate such as (polyoxyethylene lauryl ether) acrylate and (polyoxyethylene oleyl ether) methacrylate And other polyoxyethylene (meth) acrylic acid ester derivatives. Furthermore, copolymers comprising these polymers and at least two of these monomers can also be used. A copolymer of N-isopropylacrylamide and Nt-butylacrylamide can also be used. When a polymer containing a (meth) acrylamide derivative is used, another copolymerizable monomer may be copolymerized with the polymer within a range having a lower critical solution temperature. In the present invention, among others, Nn-propylacrylamide, N-isopropylacrylamide, N-ethylacrylamide, N, N-dimethylacrylamide, N-acryloylpyrrolidine, N-acryloylpiperidine, N-acryloylmorpholine, Nn- From at least one monomer selected from the group consisting of propylmethacrylamide, N-isopropylmethacrylamide, N-ethylmethacrylamide, N, N-dimethylmethacrylamide, N-methacryloylpyrrolidine, N-methacryloylpiperidine, N-methacryloylmorpholine Or a copolymer of N-isopropylacrylamide and Nt-butylacrylamide can be preferably used. In addition, an elastin-derived polypeptide having a repeating sequence of a pentapolypeptide represented by Val-Pro-Gly-X-Gly (X is an amino acid other than proline) can be used.

As the polymer having the upper critical solution temperature used in the present invention, a polymer comprising at least one monomer selected from the group consisting of acryloylglycinamide, acryloylnipecotamide, acryloylasparagineamide, acryloylglutamineamide, and the like can be used. Moreover, the copolymer which consists of these at least 2 types of monomers may be sufficient. These polymers include other copolymerizable monomers such as acrylamide, acetylacrylamide, biotinol acrylate, N-biotinyl-N′-methacryloyl trimethylene amide, acryloyl sarcosine amide, methacryl sarcosine amide, acryloylmethyl uracil, etc. May be copolymerized within the range having the upper critical solution temperature.

In the present invention, a substance such as a pH-responsive polymer that can be aggregated and dispersed by changing pH can be used as the stimulus-responsive substance. The pH at which the pH-responsive substance undergoes a structural change is not particularly limited, but it can suppress a decrease in detection / quantitative accuracy due to denaturation of the first bound substance, the second bound substance described later, and the specimen at the time of applying the stimulus. In this respect, pH 4 to 10 is preferable, and pH 5 to 9 is more preferable.

Examples of such a pH-responsive polymer include polymers containing groups such as carboxyl, phosphoric acid, sulfonyl and amino as functional groups. More specifically, (meth) acrylic acid, maleic acid, styrene sulfonic acid, 2-acrylamido-2-methylpropane sulfonic acid, phosphorylethyl (meth) acrylate, aminoethyl methacrylate, aminopropyl (meth) acrylamide, dimethylamino A monomer having a dissociating group such as propyl (meth) acrylamide may be polymerized, and the monomer having such a dissociating group and other vinyl monomers such as methyl (meta ) (Meth) acrylates such as acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, vinyl esters such as vinyl acetate and vinyl propionate, vinyl compounds such as styrene, vinyl chloride and N-vinylpyrrolidone, (Meth) acrylic And a de and the like or it may be copolymerized.

The particulate magnetic substance is not particularly limited as long as it can bind an affinity substance, and may be composed of, for example, a polyhydric alcohol and magnetite.

The polyhydric alcohol is not particularly limited as long as it is an alcohol structure having at least two hydroxyl groups as a structural unit and capable of binding to iron ions, and examples thereof include dextran, polyvinyl alcohol, mannitol, sorbitol, and cyclodextrin. . For example, Japanese Patent Application Laid-Open No. 2005-82538 discloses a method for producing a particulate magnetic material using dextran. Moreover, the compound which has an epoxy like a glycidyl methacrylate polymer and forms a polyhydric alcohol structure after ring-opening can also be used.

When used in a conventional method, a fine magnetic substance (magnetic fine particle) needs to have a large particle diameter from the viewpoint of securing a moving speed by magnetic force. In the present invention, the moving speed by magnetic force is a stimulus response. In order to ensure by utilizing aggregation of the active substance, it is not necessary to have a large particle size. The specific particle size may be appropriately set according to the magnitude of the magnetic force that can be applied, and is not particularly limited, but the average particle size may be 0.9 nm or more and less than 1000 nm, and may be 2.9 nm or more and less than 200 nm. In particular, it is preferably 150 nm or less, 100 nm or less, 95 nm or less, 90 nm or less, 85 nm or less, or 80 nm or less. The average particle diameter in the present invention is measured by a dynamic light scattering method.

Further, the stimulus-responsive substance is not limited to the above-mentioned stimulus-responsive polymer. For example, Japanese Patent No. 3693979, Japanese Patent No. 3916330, Japanese Patent Application Laid-Open No. 2002-85957, Japanese Patent No. 4071738, Japanese Patent No. 2869684 are disclosed. Hydrogels disclosed in Japanese Patent No. 2927601, Japanese Patent No. 3845249, and the like may be used.

(Affinity substance)
The affinity substance may be a monoclonal antibody or a polyclonal antibody that recognizes the antigenic determinant of the non-specific reactant. The antibody used herein may be any type of immunoglobulin molecule, and may be an immunoglobulin molecule fragment having an antigen binding site such as Fab.

The non-specific reaction substance is a substance that should be removed according to the composition and characteristics of the specimen and is not particularly limited. Examples thereof include IgM, albumin, globulin, fibrin, and rheumatoid factor. Further, the non-specific reactive substance may be one kind or two or more kinds.

[Preparation of combined product]
The bound substance is prepared by binding an aggregating substance and an affinity substance. This binding method is not particularly limited. For example, both of the aggregating substance side (for example, stimulus-responsive substance part) and the affinity substance (for example, antibody) side have substances having affinity for each other (for example, avidin and biotin, Glutathione and glutathione S-transferase) are bound, and the aggregating substance and affinity substance are bound via these substances.

Specifically, as described in International Publication WO01 / 009141, biotin is bound to a stimulus-responsive substance by adding biotin or the like to a polymerizable functional group such as methacryl or acryl to form an addition polymerizable monomer. It can be carried out by copolymerizing with other monomers. The binding of avidin or the like to the affinity substance can be performed according to a conventional method. Next, when the biotin-binding stimulus-responsive substance and the avidin-bonded first affinity substance are mixed, the affinity substance and the stimulus-responsive substance are bonded through the binding between avidin and biotin.

Alternatively, a monomer having a functional group such as carboxyl, amino, or epoxy is copolymerized with another monomer during the production of the polymer or the like, and the antibody affinity substance is passed through this functional group according to a method well known in the art. (For example, a method of binding melon gel, protein A, protein G) to a polymer can be used. By binding an antibody to the antibody affinity substance thus obtained, a conjugate of a substance such as a stimulus-responsive polymer and an antibody against the antigen to be detected is produced.

Alternatively, a monomer having a functional group such as carboxyl, amino, or epoxy may be copolymerized with another monomer during the production of the polymer, and an antibody against the antigen to be detected may be directly bonded to these functional groups according to a conventional method.

Alternatively, an affinity substance and a stimulus responsive substance may be bound to a fine particle magnetic substance.

</ RTI> After the aggregating substance is placed under conditions where the stimuli-responsive substance aggregates, the bound product may be purified by separation by centrifugation. For purification of the bound substance, a magnetic substance in the form of fine particles is bound to the stimulus-responsive substance, and after further binding the affinity substance, the magnetic substance is recovered by applying a magnetic force under conditions where the stimulus-responsive substance aggregates. You may carry out by the method to do.

The fine magnetic substance can be bonded to a substance such as a stimulus-responsive polymer through a reactive functional group, or an active hydrogen on a polyhydric alcohol or a polyhydric alcohol in the magnetic substance. It may be carried out by a method known in the art such as a method of introducing a bond and graft polymerization (for example, ADV.Polym.Sci., Vol.4, p111, 1965, J. Polymer Sci., Part-A, 3, p1031, 1965).

Next, the procedure of the detection / quantification method will be described with reference to FIGS. When the above binding substance and specimen are mixed, the non-specific reaction substance in the specimen binds to the affinity substance and is captured. As shown in FIG. 1, the conjugate 10 according to an embodiment includes a stimulus-responsive substance 11, and the stimulus-responsive substance 11 passes through an avidin 15 and biotin 17 to an antibody 13 against a nonspecific reactant 50. Are combined. Further, the bonded material 10 includes a fine magnetic material 19, and the stimulus-responsive material 11 is bonded to the surface of the magnetic material 19. At this time, the specific surface area of the magnetic substance 19 is large, and the nonspecific reaction substance 50 can be efficiently captured by the antibody 13. When two or more kinds of non-specific reactive substances are to be captured, a binding substance having an affinity substance for each of them may be added simultaneously or separately.

The step of capturing is preferably performed from the viewpoint of maximizing capture efficiency under conditions where the stimulus-responsive substance does not aggregate (for example, a temperature lower than the lower critical solution temperature when LCST is used). However, if the conjugate before mixing with the specimen is placed under conditions where the stimulus responsive substance does not aggregate, even if the mixture is aggregated with the stimulus responsive substance (for example, when LCST is used, Even under (temperature), it is possible to capture a significant portion of non-specific reactants before high aggregation begins. This aspect is advantageous in that it is possible to eliminate the trouble (control, movement, time, etc.) of transferring the mixture to a condition where the stimulus-responsive substance aggregates.

Next, this mixture is placed under conditions where the stimulus-responsive substance aggregates, and a magnetic force is applied. In FIG. 2, the magnet M is arranged near the lower part of the container that contains the mixture, whereby the aggregated binding substance A that has captured the non-specific reaction substance moves to the lower part of the container and is separated from the mixture. At this time, since the combined material is aggregated, it is attracted toward the magnet in a short time. The steps so far vary depending on specific conditions, but can usually be completed within 5 to 10 minutes. For this reason, the present invention is also advantageous in that the separation step and the subsequent detection and quantification steps can be performed continuously and with high efficiency. In FIG. 3, from the viewpoint of simplification of illustration, the aggregated bonded material A is drawn so as to be directly adsorbed to the magnet M, but the magnet M is generally arranged outside the container.

Next, the detection target in the subsequent mixture is detected or quantified. This detection and quantification are not particularly limited, and may be performed by various methods. In the present invention, since the bonded material A aggregates and is strongly attracted to the magnetic force, it is difficult to redisperse into the mixture. For this reason, detection and quantification can also be performed in a state where the aggregated bound substance A is solid-liquid separated in a container in which the mixture is accommodated. This embodiment is preferable in that it saves the trouble of moving the mixture to another container or a washed container. This mode is preferable from the viewpoint of solid-liquid separation that is surely performed under conditions in which the stimulus-responsive substance aggregates, but depending on the accuracy required for detection and quantification, the conditions may be such that the stimulus-responsive substance does not aggregate. You may go. Similarly, it is preferable from the viewpoint of solid-liquid separation to continue to apply magnetic force during detection and quantification, but depending on the accuracy required for detection and quantification, at least a part of detection and quantification (especially antibody loading) The mixing of the antibody and the mixture) may be performed under conditions that do not apply a magnetic force (eg, no magnet is placed near the container).

As shown in FIGS. 2 and 3, the detection is performed by mixing carrier C carrying an affinity substance (for example, antibody or fragment thereof) for detection target T with the mixture and determining whether carrier C is aggregated or not. Steps may be included. Similarly, the quantification may include a step of mixing a carrier carrying an affinity substance for the detection target T with the mixture and detecting the degree of aggregation of the carrier C (for example, measuring light transmittance). When the detection target T is present, the carrier C carrying the affinity substance bound to the detection target T aggregates, so that detection and quantification can be performed by the aggregation. This process is a conventionally known indirect agglutination immunization process, and known carriers such as latex and gelatin particles can be used as the carrier. In the present invention, detection and quantification can be performed in a state in which the aggregated conjugate A is solid-liquid separated in a container in which the mixture is contained, and thus the above-described conventionally known indirect aggregation immunization process can be used. This is also advantageous.

However, if the accuracy required for detection and quantification is extremely high, etc., the detection and quantification should be performed after transferring the mixture to another container or a washed container as necessary. May be. In this case as well, detection and quantification may be carried out by various conventionally known methods. In addition to the indirect aggregation immunization step, labeled immunization using a labeling substance (enzyme, radioisotope, chemiluminescent substance, fluorescent substance, etc.) A process etc. are also possible.

Detection and quantification are preferably performed in a state where a sensitizer that increases detection sensitivity is added. In general, the sensitizer increases not only the detection sensitivity of the detection target but also the detection sensitivity of the non-specific reaction substance, but in the present invention, since the non-specific reaction substance is separated, the detection target detection is performed. Only the advantage of increased sensitivity can be enjoyed. Such a sensitizer is appropriately selected depending on the detection and quantification method. For example, in the case of indirect immunoaggregation, water-soluble polymers such as polyethylene glycol and dextran that promote aggregation of the carrier can be used. In addition, the timing of addition of the sensitizer may not be added together with the above-described carrier, and may be added alone or together with the binder.

After the detection and quantification are completed, the container is washed, and the detection target and the bound substance (if present) are discharged. At this time, it is preferable that the container is placed under a condition in which no magnetic force is applied from the viewpoint of easily discharging the bonded product.

As described above, in the present invention, the separation process can be completed in a short time of 5 to 10 minutes. For this reason, as a device for carrying out the method of the present invention, a conventional device for performing detection and quantification by adding a plurality of reagents can be used without major modification simply by replacing the reagent to be used with a conjugate or an antibody-supporting carrier. You can also. Examples of the plurality of reagents added in the conventional apparatus include a combination of a diluent or a sensitizer and an antibody-supporting carrier, a first substance containing a stimulus-responsive substance, and a first affinity substance for the detection target. A combination of a bound first bound substance and a second bound substance in which a charged or hydrophilic second substance and a second affinity substance for a detection target are bound (see JP 2009-168636 A) ) And the like.

The present invention also includes a kit for detecting and / or quantifying a detection target in a specimen. This kit includes a binding substance in which an aggregating substance containing a stimulus-responsive substance and a particulate magnetic substance is bound to an affinity substance for a substance other than the detection target, and an affinity substance for the detection target. Combining the binding substance with the affinity substance for the detection target is based on the use of agglutination, and there is no new idea to achieve both the binding efficiency with the non-specific reactant and the separation efficiency of the non-specific reactant. It ’s hard to imagine. The kit may contain one or more affinity substances for each non-specific reaction substance such as IgM, albumin, globulin, fibrin, rheumatoid factor.

In the kit of the present invention, the affinity substance is preferably in a form supported on a carrier. Moreover, it is preferable that the kit of this invention is further equipped with the sensitizer which increases a detection sensitivity. Since these details are as described above, they are omitted.

<Example 1>
(Preparation of stimuli-responsive magnetic fine particles with affinity substances bound to non-specific reactive substances)
First, an antibody as a ligand for human IgM as a non-specific reaction substance was biotinylated by a well-known sulfo-NHS-Biotin method (Asahi Techno Glass) to prepare a biotin-labeled anti-human IgM antibody.

On the other hand, 250 μL of Thermo-Max LSA Streptavidin (0.2 mass%, average particle diameter 100 nm) manufactured by JNC, which is a magnetic fine particle to which streptavidin and a temperature-responsive polymer are bonded, was placed in a 1.5 mL microtube. The microtube was heated to 42 ° C. to aggregate Thermo-Max LSA Streptavidin, collected with a magnet, and then the supernatant was removed. To the tube after removal, 250 μL of TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) was added, and the aggregate was dispersed by cooling.

To this dispersion, 50 μL (0.75 mg / mL) of the above biotin-labeled anti-human IgM antibody dissolved in PBS buffer (0.01 M phosphate buffer, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4) was added. In addition, it was mixed by inverting at room temperature for 20 minutes. Thereafter, the microtube was heated to 42 ° C. to aggregate Thermo-Max LSA Streptavidin, recovered with a magnet, and then the supernatant was removed to separate excess biotin-labeled anti-human IgM antibody (B / F separation). ). Aggregates were dispersed by adding 250 μL of TBS buffer to the microtube after separation and cooling. Furthermore, the aggregate was added to a PBS buffer (pH 7.4) solution containing 0.5% (w / v) BSA (manufactured by Sigma), 0.5% (w / v) Tween (registered trademark) 20, 10 mM EDTA. Was dispersed to prepare a stimulus-responsive magnetic particle solution to which an affinity substance for human IgM was bound.

(Sample preparation)
Human sera that became negative in “Lumipulse II TP-N” and “Esrline TP” (Fujirebio Inc.) were treated with Treponema antibody kit “Mediace TPLA” (Sekisui Medical) and biochemical automatic analyzer “CA-90”. ”(Furuno Denki Co., Ltd.) of human sera that are determined to be positive (suspected to be false positive due to non-specific reaction),“ Vitros Microchip IgM ”(Ortho Clinical Diagnostics Inc.) Then, the IgM concentration measured to be 300 mg / dL or more was adopted as the specimen. Separately, the measurement results (positive and negative) of “Lumipulse II TP-N” (Fujirebio), “Esrline TP” (Fujirebio) and Treponema antibody kit “Mediace TPLA” (Sekisui Medical) Matched human sera were employed as positive and negative control samples, respectively.

(Magnet addition to biochemical automatic analyzer)
A reaction cuvette prepared by adhering a neodymium magnet (Neomag Co., Ltd.) having dimensions of 1 mm × 1 mm × 1 mm to a surface orthogonal to the photometric surface of a disposable plastic cuvette of the biochemical automatic analyzer “CA-90” was prepared.

(Preparation of TP measurement reagent)
In the “Mediace TPLA” kit, the sample dilution buffer was replaced with the stimulus-responsive magnetic particle solution prepared above and bound with an affinity substance for human IgM. Serum (A): A standard curve was prepared at 5 concentrations.

Samples, positive control samples, and negative control samples were analyzed using the “Mediace TPLA kit” prepared above in a biochemical automatic analyzer “CA-90” equipped with a cuvette with a neodymium magnet. The results are shown in Table 1.

When the stimulus-responsive magnetic particle solution is used to efficiently remove IgM in the specimen, the measurement result by “Mediace TPLA” turns negative and the measurement result by “Lumipulse II TP-N” and “Espline TP” Matched. Thereby, it turned out that the bad influence by the nonspecific reaction of IgM in the latex agglutination method was suppressed. In addition, regarding the analysis results in the positive sample and the negative sample as controls, there was no change due to the use of the stimulus-responsive magnetic particle solution.

<Reference example>
The reagents and equipment used and their abbreviations are shown below.
1. “Therma-Max LSA Streptavidin (30)” manufactured by JNC
: “TM-LSA (30)” (concentration: 2 mg / ml)
2. “Dynabeads MyOne ™ Streptavidin C1” manufactured by Life Technologies Corporation
: “DyMOS-C1” (bead average particle diameter: 1 μm, concentration: 10 mg / ml)
3. 3. Semi-micro cell for spectrophotometer Seiko Industrial Co., Ltd. neodymium permanent magnet (dimensions: 5 x 9 x 2 mm)
5). Eppendorf pipette 100 μL and Eppendorf 100 μL tips

Heat 1 mL of “TM-LSA (30)” to 37 ° C., collect “TM-LSA (30)” particles with a magnet for 5 minutes, discard the supernatant, add 1 mL of sterile water, It stirred at room temperature (21 degreeC) in the state which removed the collection magnet. The “TM-LSA (30)” particles were uniformly dispersed and then diluted twice with sterilized water to prepare a dispersion of “TM-LSA (30)” at 1 mg / mL.

Collect 1 mL of “DyMOS-C1”, collect the “DyMOS-C1” particles with a magnet for 5 minutes, discard the supernatant, add 1 mL of sterilized water, and remove the recovery magnet at room temperature ( (21 ° C.). The “DyMOS-C1” particles were uniformly dispersed and then diluted 10-fold with sterilized water to prepare a dispersion of “DyMOA-C1” 1 mg / mL.

1 mL each of the prepared dispersions of “TM-LSA (30)” and “DyMOS-C1” were heated at 37 ° C. for 5 minutes, and added to separate semi-microcells equipped with neodymium magnets. This semi-micro cell was allowed to stand at room temperature (21 ° C.) for 3 minutes. The state of the semi-micro cell at this point is shown in FIG. 4 (“TM-LSA (30)”) and FIG. 6 (“DyMOS-C1”). “TM-LSA (30)” was integrated in the magnet and no dispersion was observed, while “DyMOS-C1” was mostly integrated in the magnet, but some dispersion was observed.

Then, using a 100 μL pipette, pipetting of the entire amount of 100 μL was performed three times so that the tip did not touch the magnet and the particle group accumulated in the magnet, and the flow of the discharged liquid did not directly collide. The supernatant was gently collected and transferred to another container so that the tip did not touch the magnet and the particles collected on the magnet, and the amount of particles was confirmed visually. The state of the semi-micro cell at this point is shown in FIG. 5 (“TM-LSA (30)”) and FIG. 7 (“DyMOS-C1”). The supernatant of “TM-LSA (30)” was colorless and transparent, and the presence of particles could not be confirmed, whereas the supernatant of “DyMOS-C1” showed clear turbidity due to a large amount of particles. It was. As a result, it was found that the bonded product of the present invention aggregated and was strongly attracted by the magnetic force, so that it was difficult to re-disperse.

10 Bound Substance 11 Stimulus Responsive Substance 13 Antibody (Affinity Substance)
19 Magnetic substance 50 Non-specific reaction substance A Aggregated bound substance C Carrier M Magnet T Target to be detected

Claims

A method for detecting a detection target in a sample,
A binding substance in which an aggregating substance containing a stimulus-responsive substance and a particulate magnetic substance and an affinity substance for a substance other than the detection target are combined with the specimen are mixed, and the stimulus-responsive substance is mixed with the mixture. A step of separating the aggregated aggregates by applying a magnetic force under a condition in which the aggregates are aggregated, and then detecting the detection target in the mixture.
The method according to claim 1, wherein the detection target is detected in a state where the aggregated aggregate is solid-liquid separated in a container containing the mixture.
The method according to claim 1 or 2, wherein the detection of the detection target includes a step of mixing a carrier carrying an affinity substance for the detection target with the mixture and discriminating according to the presence or absence of aggregation of the carrier.
The method according to any one of claims 1 to 3, wherein the detection target is detected in a state in which a sensitizer that increases detection sensitivity is added.
A method for quantifying a detection target in a sample,
A binding substance in which an aggregating substance containing a stimulus-responsive substance and a particulate magnetic substance and an affinity substance for a substance other than the detection target are combined with the specimen are mixed, and the stimulus-responsive substance is mixed with the mixture. A method of separating the aggregated aggregates by applying a magnetic force under a condition in which the aggregates are aggregated, and then quantifying the amount of the detection target in the mixture.
The method according to claim 5, wherein the quantitative determination of the detection target is performed in a state where the aggregated aggregate is solid-liquid separated in a container in which the mixture is accommodated.
The method according to claim 5 or 6, wherein the quantification of the detection target includes a step of mixing a carrier carrying an affinity substance for the detection target with the mixture and detecting the degree of aggregation of the carrier.
The method according to any one of claims 5 to 7, wherein the detection target is quantified in a state in which a sensitizer for increasing detection sensitivity is added.
A kit for detecting and / or quantifying a detection target in a sample,
A combined product in which an aggregating substance containing a stimulus-responsive substance and a fine-particle magnetic substance and an affinity substance for a substance other than the detection target are bound;
A kit comprising an affinity substance for the detection target.
10. The kit according to claim 9, wherein the affinity substance is supported on a carrier.
The kit according to claim 9 or 10, further comprising a sensitizer for increasing detection sensitivity.