Method for detecting target substance, reagent for detecting target substance, and kit for detecting target substance

Abstract

The present invention aims to improve the inspection accuracy of a target substance detection system using a lectin of Wisteria floribunda. The present invention solves the above-mentioned problem by a method for detecting a target substance, the method comprising: a step of forming a complex containing WFA and the target substance on the carrier by mixing a lectin of Wisteria floribunda (WFA) immobilized on a carrier and a target substance containing a sugar chain bound to WFA in the presence of an alcohol having 1 to 7 carbon atoms composed only of carbon atoms, hydrogen atoms, and oxygen atoms, and a step of detecting the target substance by detecting the complex.

Description

[Technical field]

[0001] The present invention relates to a method for detecting a target substance, a reagent for detecting a target substance, and a kit for detecting a target substance. [Background technology]

[0002] Patent Document 1 discloses a sandwich assay for quantifying glycoprotein as a detection target substance in a test subject using a labeled lectin, wherein the sandwich assay method suppresses the influence of impurities by introducing a simple process.

[0003] [Prior art literature]

[0004] [Patent Literature]

[0005] [Patent Document 1] U.S. Patent Application Publication No. 2017 / 0122940

[0006] [Summary of the Invention]

[0007] [Problems to be solved by the invention]

[0008] Patent Document 1 describes a method for inhibiting nonspecific binding of labeled lectins using a reducing agent such as thioglycerol. However, the inventors have found that the method described in Patent Document 1 does not sufficiently improve detection accuracy as shown in the Examples described below.

[0009] The present invention aims to improve the detection accuracy of a target substance using a lectin from Wisteria floribunda.

[0010]

Methods for solving the problem

[0011] Some embodiments of the present invention relate to a method for detecting a target substance, comprising: a step of forming a complex containing WFA and the target substance on the carrier by mixing a lectin (WFA) of Wisteria floribunda immobilized on a carrier and a target substance containing a sugar chain bound to WFA in the presence of an alcohol having 1 to 7 carbon atoms composed only of carbon atoms, hydrogen atoms, and oxygen atoms; and a step of detecting the target substance by detecting the complex.

[0012] Some embodiments of the present invention relate to a method for detecting a target substance, comprising: a step of forming a complex containing WFA and the target substance on the carrier by mixing a lectin (WFA) of Wisteria floribunda immobilized on a carrier and a target substance containing a sugar chain bound to WFA in the presence of an alcohol having 1 to 7 carbon atoms (but excluding thioglycerol); and a step of detecting the target substance by detecting the complex.

[0013] Some embodiments of the present invention relate to a method for detecting a target substance, comprising: a step of forming a complex containing WFA and the target substance on the carrier by mixing a lectin (WFA) of Wisteria floribunda immobilized on a carrier and a target substance containing a sugar chain bound to WFA in the presence of an alcohol having 1 to 7 carbon atoms (but excluding alcohols containing a thiol group); and a step of detecting the target substance by detecting the complex.

[0014] One embodiment of the present invention relates to a reagent for detecting a target substance, the reagent comprising an alcohol having 1 to 7 carbon atoms consisting only of carbon atoms, hydrogen atoms, and oxygen atoms, wherein the target substance comprises a sugar chain that binds to a lectin (WFA) of Wisteria floribunda.

[0015] One embodiment of the present invention relates to a reagent for detecting a target substance, the reagent comprising: an alcohol having 1 to 7 carbon atoms (but excluding thioglycerol), wherein the target substance comprises a sugar chain that binds to a lectin (WFA) of Wisteria floribunda.

[0016] One embodiment of the present invention relates to a reagent for detecting a target substance, the reagent comprising: an alcohol having 1 to 7 carbon atoms (but excluding an alcohol containing a thiol group). The target substance comprises a sugar chain that binds to a lectin (WFA) of Wisteria floribunda.

[0017] One embodiment of the present invention relates to a kit for detecting a target substance, the kit comprising: a detection substance for the target substance; a lectin of Wisteria floribunda (WFA); and an alcohol having 1 to 7 carbon atoms consisting only of carbon atoms, hydrogen atoms, and oxygen atoms, wherein the target substance contains a sugar chain bound to WFA.

[0018] One embodiment of the present invention relates to a kit for detecting a target substance, the kit comprising: a detection substance for the target substance; a lectin of Wisteria floribunda (WFA); and an alcohol having 1 to 7 carbon atoms (but excluding thioglycerol), wherein the target substance contains a sugar chain bound to WFA.

[0019] One embodiment of the present invention relates to a kit for detecting a target substance, the kit comprising: a detection substance for the target substance; a lectin of Wisteria floribunda (WFA); and an alcohol having 1 to 7 carbon atoms (but excluding thioglycerol), wherein the target substance contains a sugar chain bound to WFA.

[0020] By mixing WFA and a target substance in the presence of an alcohol having 1 to 7 carbon atoms consisting only of carbon atoms, hydrogen atoms, and oxygen atoms, an alcohol having 1 to 7 carbon atoms other than thioglycerol, or an alcohol having 1 to 7 carbon atoms other than a mercapto group-containing alcohol, the detection accuracy of the target substance can be improved.

[0021] [Effects of the Invention]

[0022] The detection accuracy of target substances can be improved.

[0023] [Brief description of the attached drawings]

[0024]

Figure 1

[0025]

Figure 2

[0026]

Figure 3

[0027]

Figure 4

[0028]

Figure 5

[0029]

Figure 6

[0030]

Figure 7

[0031]

Figure 8

[0032]

Fig. 9

[0033] 【1. Detection method】

[0034] An example of an embodiment of the detection method disclosed in this specification is shown. Some embodiments of the present disclosure relate to a method for detecting a target substance, the method comprising: a step of forming a complex containing WFA and the target substance on the carrier by mixing a lectin and a target substance containing a sugar chain that binds to the lectin in the presence of an alcohol (forming step), and a step of detecting the target substance by detecting the complex (detecting step). Preferably, the detection can be quantitative.

[0035] The target substance is a substance to be detected. It is preferably a glycoprotein, and more preferably has a sugar chain that binds to Wisteria floribunda agglutinin (WFA). As the target substance, Mac-2-binding protein (M2BP), cell surface-related Mucin 1 (MUC1), α1 acid glycoprotein (AGP), neural cell attachment molecule L1 (L1CAM), KL-6 antigen, etc. can be preferably mentioned.

[0036] The target substance is preferably a substance contained in a sample collected from a living body. Urine or blood can be used as the sample. The blood can be peripheral blood, or plasma or serum prepared from the peripheral blood. Among these, plasma or serum is also preferred. In the present embodiment, the sample can also be diluted with a suitable aqueous medium as needed. Such an aqueous medium is not particularly limited as long as it does not hinder the detection of the target substance, and for example, water, physiological saline, buffer solution, etc. can be cited. The buffer solution is not particularly limited, and a buffer solution with a pH near neutrality (for example, a pH of 6 or more and 8 or less) can be used. For example, Good buffers such as HEPES, MES, Tris, PIPES, and phosphate buffered saline (PBS) can be cited.

[0037] Lectins preferably include WFA. As WFA, for example, naturally occurring WFA or recombinant WFA can be mentioned. Naturally occurring WFA is a 4-mer protein composed of 4 subunits. WFA is known as a lectin contained in the seeds of Wisteria floribunda. From this 4-mer WFA, a monomer or 2-mer WFA can be obtained by a specified treatment using a reducing agent or the like. In this specification, unless otherwise specified, the expression "WFA" refers to both monomeric WFA and multimeric WFA. In addition, in this specification, when indicating a WFA composed of a specified number of subunits, the number of subunits is explicitly expressed, such as "monomeric WFA", "2-mer WFA", and "4-mer WFA".

[0038] In the present embodiment, WFA may be a tetramer WFA, a monomer WFA, or a dimer WFA. Among them, dimer WFA is also preferred from the viewpoint of high reactivity.

[0039] As alcohol, for example, alcohols having 1 to 7 carbon atoms, preferably 2 to 7 carbon atoms, can be cited. The lower limit of the number of carbon atoms of the alcohol can be selected from 1, 2 or 3. In one embodiment, an alcohol consisting only of carbon atoms, hydrogen atoms and oxygen atoms is used. Preferably, thioglycerol is not contained in the above-mentioned alcohols having 1 to 7 carbon atoms. More preferably, thiol-containing alcohols are not included in the above-mentioned alcohols having 1 to 7 carbon atoms. Among them, "thioglycerol is not contained in the alcohols having 1 to 7 carbon atoms" means that in the detection system, reagents, and kits described later, at least alcohols having 1 to 7 carbon atoms other than thioglycerol are contained, and the detection system, reagents, and kits to which thioglycerol is added are not completely excluded. Even if the expression "there is no thiol-containing alcohol in the alcohols having 1 to 7 carbon atoms" means that in the detection system, reagents, and kits described later, at least alcohols having 1 to 7 carbon atoms other than thiol-containing alcohols are included, the detection system, reagents, and kits to which thiol-containing alcohols are added are not completely excluded. That is, thioglycerol may be contained to such an extent that detection of the target substance is not hindered.

[0040] The alcohol may be monovalent or polyvalent. The valence refers to the number of hydroxyl groups. Examples of polyols include divalent, trivalent, and tetravalent alcohols.

[0041] The carbon chain contained in the alcohol may be a straight chain or a branched chain, but is preferably a straight chain.

[0042] As the alcohol, only one kind may be used or two or more kinds may be used.

[0043] As the alcohol, preferably, at least one alcohol selected from methanol, ethanol, glycerol, pentanol, heptanol, diglycerol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, triethylene glycol, and a mixture thereof can be cited. More preferably, at least one alcohol selected from glycerol, diglycerol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, triethylene glycol, and a mixture thereof is cited, and glycerol is most preferred.

[0044] The complex of WFA and the target substance is formed by, for example, mixing a sample containing the target substance (or a diluent obtained by diluting the sample with an aqueous medium) and WFA, and incubating for a certain period of time as required. In this case, WFA is preferably immobilized on a carrier.

[0045] The implementation method of fixing WFA to the carrier is not particularly limited. For example, WFA and the carrier can be directly bound, or WFA and the carrier can be indirectly bound via another substance. As a direct binding, for example, physical adsorption can be cited. As an indirect binding, for example, binding via a combination of biotin and avidin or streptavidin (hereinafter also referred to as "avidins") can be cited. In this case, by pre-modifying WFA with biotin and pre-binding the carrier with avidins, WFA and the carrier can be indirectly bound via the binding of biotin and avidins.

[0046] The raw material of the carrier is not particularly limited, for example, it can be selected from organic polymer compounds, inorganic compounds, biopolymers, etc. As organic polymer compounds, latex, polystyrene, polypropylene, etc. can be mentioned. As inorganic compounds, magnetic bodies (iron oxide, chromium oxide and ferrite, etc.), silicon oxide, aluminum oxide, glass, etc. can be mentioned. As biopolymers, insoluble agarose, insoluble dextran, gelatin, cellulose, etc. can be mentioned. It is also possible to use two or more of these in combination. The shape of the carrier is not particularly limited, for example, particles, membranes, microplates, microtubes, test tubes, etc. can be mentioned. Among them, particles are also preferred, and magnetic particles are particularly preferred. By using magnetic particles, the reactivity of WFA and the target substance can be improved.

[0047] The WFA fixed to the magnetic particles is preferably a dimer WFA. The dimer WFA can be obtained, for example, by dissociating the subunits of the tetramer WFA using a thiol reagent or a reducing agent. In addition, the tetramer WFA can also be dimerized by contacting the tetramer WFA with a crosslinking agent. As such a crosslinking agent, a crosslinking agent that forms crosslinks with the amino group in the tetramer WFA is preferred. For example, as a crosslinking agent having a reactive group for the amino group, there can be cited a crosslinking agent having at least one functional group selected from N-hydroxysuccinimide ester group, isothiocyanate group, chlorosulfone group, chlorocarbonyl group, oxyethylene group, chloroalkyl group having 1 to 4 carbon atoms, aldehyde group and carboxyl group. When such a crosslinking agent is used, the tetramer WFA can be effectively dimerized.

[0048] The molar ratio (WFA / crosslinker) when the tetramer WFA and the crosslinker are mixed is preferably 1 / 10 or less, more preferably 1 / 20 or less. On the one hand, the lower limit of the molar ratio (WFA / crosslinker) can be set to 1 / 100 or more in consideration of the balance between the amount of the crosslinker used and the yield of the generated dimer WFA.

[0049] When WFA is fixed to magnetic particles by binding of biotin and avidin, biotinylated dimeric WFA can also be used. Biotinylated dimeric WFA can be obtained, for example, by dimerizing tetrameric WFA using a cross-linking agent containing biotin. The cross-linking agent containing biotin is obtained, for example, by binding biotin and the above-mentioned reactive group of the cross-linking agent via a spacer. Such a spacer is not particularly limited, and examples thereof include compounds having an aminocaproyl group (aminocaproyl group) and the like.

[0050] When a complex of the target substance and WFA is formed, the alcohol is preferably contained in an amount of 2 to 20 w / w%, preferably 4 to 10 w / w%. In the present disclosure, the unit "w / w%" means a percentage converted to weight / weight (hereinafter the same).

[0051] When a complex of WFA is formed with a target substance in the presence of alcohol, a reagent containing alcohol and a reagent containing WFA immobilized on a carrier can be mixed with a sample (or its dilution) as separate reagents, or a mixed solution of alcohol and WFA immobilized on a carrier can be mixed with the sample. The order in which the reagent and the sample (or its dilution) are mixed is not particularly limited, and these can be mixed substantially simultaneously or sequentially. As an example of a reagent containing alcohol, a reagent in which alcohol is added to HISCL (trademark) R1 reagent manufactured by Sysmex Co., Ltd. can be cited. At this time, as a reagent containing WFA immobilized on a carrier, for example, HISCL (trademark) M2BPGi (trademark) R2 reagent manufactured by Sysmex Co., Ltd. can be cited.

[0052] The detection of the complex is possible by, for example, making the complex of WFA and the target substance further contain a detection substance and detecting a signal from the detection substance. The complex of WFA, the target substance and the detection substance can be formed by mixing the complex of WFA and the target substance and the detection substance and reacting for a certain time as necessary. Alternatively, the complex of WFA, the target substance and the detection substance can be formed by making the detection substance coexist when forming the complex of WFA and the target substance.

[0053] The detection substance is a substance that specifically binds to the target substance, and there is no limitation as long as it is a substance containing a labeling substance. When the target substance is a glycoprotein, the substance that specifically binds to the target substance preferably binds to the protein portion of the target substance. As a substance that specifically binds to the target substance, for example, an antibody can be cited. It can also be any of a monoclonal antibody, a polyclonal antibody, and their fragments (e.g., Fab, F(ab')2, etc.). In addition, commercially available antibodies can also be used.

[0054] The labeling substance used in the detection substance can be a substance commonly used in the art for immunological determination. For example, it can be a substance that generates a signal by itself (hereinafter also referred to as a "signal generating substance"), or it can be a substance that generates a signal by catalyzing the reaction of other substances. Examples of signal generating substances include fluorescent substances and radioactive isotopes. Examples of substances that generate detectable signals by catalyzing the reaction of other substances include enzymes. Examples of enzymes include alkaline phosphatase, peroxidase, β-galactosidase, and luciferase. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor (trademark), and fluorescent proteins such as GFP. Examples of radioactive isotopes include 125 I. 14 C. 32P, etc. Among them, enzymes are also preferred as labeling substances, and alkaline phosphatase is particularly preferred. As labeling substances, haptens such as dinitrophenyl (DNP) or biotin can also be used. By combining a substance that binds to a hapten and has a signal-generating substance or an enzyme with the detection substance, a signal from the complex can be obtained.

[0055] The labeling substance can be bound to a substance that specifically binds to the target substance by a labeling method known in the art. Alternatively, labeling can be performed using a commercially available labeling kit or the like.

[0056] In the present embodiment, the complex is detected based on the labeling substance of the detection substance contained in the complex. Specifically, by detecting the signal generated by the labeling substance of the detection substance contained in the complex, a measured value reflecting the amount or concentration of the labeling substance contained in the sample can be obtained. Among them, the "detection signal" includes the presence or absence of a qualitative detection signal, quantification of the signal intensity, and the intensity of a semi-quantitative detection signal. Semi-quantitative detection refers to expressing the intensity of the signal in stages such as "no signal", "weak", "medium", "strong", etc. In the present embodiment, quantitative or semi-quantitative detection of the signal intensity is preferred.

[0057] The method of detecting the signal itself is well known in the prior art. In the present embodiment, it is appropriate to select a determination method corresponding to the type of the signal derived from the above-mentioned labeling substance. For example, when the labeling substance is an enzyme, the signal of light, color, etc. generated by reacting the substrate for the enzyme can be carried out by using a known device such as a spectrophotometer.

[0058] The substrate of the enzyme can be appropriately selected from known substrates according to the type of the enzyme. For example, when alkaline phosphatase is used as an enzyme, as a substrate, chemiluminescent substrates such as CDP-Star (trademark) (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, CSPD (trademark) (3-(4-methoxyspiro[1,2-dioxetane-3,2-(5'-chloro)tricyclo[3.3.1.13,7]decane]-4-yl)phenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-6-chloro-indolyl phosphate, p-nitrophenyl phosphate, etc. can be cited.

[0059] When the labeling substance is a radioisotope, the radiation as a signal can be measured using a known device such as a scintillation counter. In addition, when the labeling substance is a fluorescent substance, the fluorescence as a signal can be measured using a known device such as a fluorescent microplate reader. Furthermore, the excitation wavelength and the fluorescence wavelength can be appropriately determined corresponding to the type of fluorescent substance used.

[0060] As an example of a reagent containing a detection substance, when the target substance is M2BP, for example, HISCL (trademark) M2BPGi (trademark) R3 reagent manufactured by Sysmex Co., Ltd. In addition, when detecting other target substances, the reagent can be changed according to the substance that specifically binds the anti-M2BP antibody contained in the HISCL (trademark) M2BPGi (trademark) R3 reagent to the target substance.

[0061] In this embodiment, when the labeling substance is an enzyme, the step of contacting the substrate with the labeling substance and the step of detecting the signal are referred to as the "detection step". In addition, when the labeling substance is a fluorescent substance or a radioactive isotope, the step of detecting the signal is referred to as the "detection step".

[0062] In this embodiment, Bound / Free (B / F) separation to remove unreacted free components that do not form a complex may be performed between the formation and detection steps of the above-mentioned complex. Unreacted free components refer to components that do not constitute a complex. For example, unreacted sample components, WFA, detection substances, etc. are mentioned. The means of B / F separation is not particularly limited. When the carrier is a particle, B / F separation can be performed by recovering only the carrier that captures the complex by centrifugal separation. When the carrier is a container such as a microplate or a microtube, B / F separation can be performed by removing the liquid containing unreacted free components. In addition, when the carrier is a magnetic particle, B / F separation can be performed by sucking out the liquid containing unreacted free components by a nozzle while the magnetic particles are magnetically constrained by a magnet, which is preferred from the viewpoint of automation. After removing the unreacted free components, the carrier that captures the complex can also be washed with a suitable aqueous medium such as PBS.

[0063] The detection result of the signal can be used as the measured value of the target substance. For example, when the intensity of the signal is quantitatively detected, the measured value of the signal intensity itself or a value calculated from the measured value can be used as the measured value of the target substance. As the value calculated from the measured value of the signal intensity, for example, a value obtained by subtracting the measured value of a negative control sample from the measured value, a value obtained by dividing the measured value of the positive control sample by the measured value, and a combination thereof can be cited.

[0064] The target substance can also be detected using a fully automatic immunoassay device HISCL (trademark) series manufactured by Sysmex Corporation.

[0065] 【2. Reagents and kits】

[0066] (1) Reagents

[0067] Some embodiments of the present disclosure relate to a reagent used for detecting a target substance. Preferably, the present embodiment includes a reagent containing an alcohol having 1 to 7 carbon atoms used in a step of a part of the method described in 1 above. Figure 1 (A) shows the appearance of the container 51 of the reagent.

[0068] The reagent preferably contains 3 to 30 w / w%, preferably 4 to 15 w / w%, of an alcohol having 1 to 7 carbon atoms. In addition, the reagent 51 preferably contains, for example, an aqueous solvent. The aqueous solvent is not particularly limited as long as it does not interfere with the detection described in 1 above, and examples thereof include water, physiological saline, and a buffer solution. The buffer solution is not particularly limited as long as it has a buffering effect at a pH near neutral (for example, a pH of 6 to 8). Examples of the buffer solution include Good buffers such as HEPES, MES, Tris, and PIPES, and phosphate-buffered saline (PBS). HEPES is preferred. Examples of such a buffer solution include HISCL (trademark) R1 reagent manufactured by Sysmex Corporation.

[0069] In addition, the reagent containing an alcohol having 1 to 7 carbon atoms may also contain the WFA immobilized on the carrier described in 1 above. In this case, the reagent also preferably contains, for example, an aqueous solvent. The aqueous solvent is not particularly limited as long as it does not interfere with the detection described in 1 above, and examples thereof include water, physiological saline, buffer solution, etc. The buffer solution is not particularly limited as long as it has a buffering effect at a pH near neutral (e.g., a pH of 6 to 8). Examples of the buffer solution include, for example, Good buffers such as HEPES, MES, Tris, and PIPES, and phosphate buffered saline (PBS). HEPES is preferred. Examples of such a buffer solution include, for example, HISCL (trademark) M2BPGi (trademark) R2 reagent manufactured by Sysmex Corporation.

[0070] Furthermore, the reagent may also contain the detection substance described in 1 above.

[0071] The reagent may also contain at least one additive selected from stabilizers such as β-mercaptoethanol and DTT; protective agents such as albumin; surfactants such as polyoxyethylene sorbitan monooleate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, and stabilizers such as sodium azide and bovine serum albumin. The reagent may also contain metal salts (preferably chlorides) of sodium, potassium, calcium, manganese, and the like.

[0072] (2) Test kit

[0073] Some embodiments of the present disclosure relate to a kit for use in detecting a target substance. The kit contains an alcohol having at least 1 to 7 carbon atoms, WFA, and a detection substance. Figure 1 (B) An example showing the outline of the kit. Figure 1As shown in (B), the kit 50 includes an outer box 55, a first container 51 storing a reagent containing an alcohol having 1 to 7 carbon atoms as described in 2. (1), a second container 52 storing a reagent containing WFA immobilized on a carrier as described in 1, a third container 53 storing a reagent containing a detection substance capable of binding to the target substance as described in 1, and an accompanying document 54 of the kit. The accompanying document 54 may record the handling method, storage conditions, expiration date, etc. of the kit.

[0074] When the reagent containing WFA immobilized on a carrier and contained in the second container 52 contains an alcohol having 1 to 7 carbon atoms, the first container 51 contains the aqueous solvent described in 2. (1) above and at least one reagent selected from additives and metal salts as needed.

[0075] When the alcohol having 1 to 7 carbon atoms is contained in the reagent contained in the first container 51, the WFA immobilized on the carrier contained in the second container 52 may be in a dry form. In addition, when the WFA contained in the kit is not immobilized on the carrier during circulation, the reagent for immobilizing the WFA on the carrier before use, the WFA and the carrier may be packaged separately.

[0076] As an example of the reagent contained in the third container 53, for example, when the target substance is M2BP, the HISCL (trademark) M2BPGi (trademark) R3 reagent manufactured by Sysmex Co., Ltd. can be cited. In addition, when detecting other target substances, it can be changed according to the substance that makes the anti-M2BP antibody contained in the HISCL (trademark) M2BPGi (trademark) R3 reagent specifically bind to the target substance.

[0077] Furthermore, in the test kit, a container containing a diluent for diluting a sample (e.g., HISCL (trademark) R4 reagent manufactured by Sysmex Co., Ltd.), a container containing a matrix reagent (e.g., HISCL (trademark) R5 reagent manufactured by Sysmex Co., Ltd.), a container containing an aqueous medium for washing, etc. may also be placed in the outer box 55. Furthermore, the description of the terms described in 1 above is applicable to this item.

[0078] [Example]

[0079] [1. Reagents - Test subjects - Measurement]

[0080] (1-1) Buffer for sample dilution (first reagent)

[0081] HISCL (trademark) M2BPGi R1 reagent manufactured by Sysmex Corporation was used.

[0082] (1-2) Magnetic particles immobilizing WFA lectin (second reagent)

[0083] As the target substance, HISCL (trademark) M2BPGi R2 reagent manufactured by Sysmex Corporation was used in the system using M2BP and MUC1.

[0084] (1-2') Magnetic particles immobilizing LTL lectin (second reagent)

[0085] Biotinylation of (1-2'-1)LTL lectin

[0086] LTL [manufactured by VECTOR Laboratories, trade name: Lotus tetragonolobus Lectin] was added to 20 mM phosphate buffer (pH 7.5) so that the concentration of LTL became 2.5 mg / mL to obtain a solution containing LTL.

[0087] To the obtained solution containing LTL, 5-(N-succinimidyloxycarbonyl)pentyl D-biotinamide (manufactured by Dojin Chemical Research Institute, trade name: Biotin-AC5-Osu) as a cross-linking agent containing biotin was added until the LTL / cross-linking agent (molar ratio) became 1 / 100. The obtained solution was incubated at 25° C. for 90 minutes to react the LTL and the cross-linking agent containing biotin to obtain a reaction product.

[0088] Purification of (1-2'-2) biotinylated LTL

[0089] The reaction product obtained in (1-2'-1) was purified by high performance liquid chromatography under the following conditions to obtain purified biotinylated LTL.

[0090] Dissolution solvent: phosphate buffer (pH 6.5)

[0091] Separation column: gel filtration column

[0092] Preparation of a solution containing (1-2'-3) streptavidin-bound particles

[0093] The complex in which streptavidin (STA) was immobilized on the surface of magnetic particles (average particle size 2 μm) (the amount of streptavidin per 1 g of magnetic particles: 2.9 to 3.5 mg, hereinafter also referred to as "STA-bound magnetic particles") was washed three times with 0.01 M HEPES buffer (pH 7.5). The washed STA-bound magnetic particles were added to 0.01 M HEPES buffer (pH 7.5) so that the STA concentration became 18 to 22 μg / mL (the concentration of the STA-bound magnetic particles was 0.48 to 0.52 mg / mL), thereby obtaining a solution containing STA-bound particles.

[0094] Preparation of (1-2'-4)LTL Immobilization Vector

[0095] The biotinylated LTL was added to the obtained solution containing STA-bound particles in such a manner that the concentration of the biotinylated LTL was 20 μg / mL, so that the streptavidin of the STA-bound magnetic particles and the biotin of the biotin LTL were bound. The obtained product was washed three times with 0.1 M MES buffer (pH 6.5) to obtain an LTL-immobilized carrier. The obtained LTL-immobilized carrier was suspended in MES buffer to obtain a solution containing an LTL-immobilized carrier.

[0096] (1-3) Solution containing labeled M2BP antibody (third reagent)

[0097] As the target substance, in the system using M2BP, HISCL (trademark) M2BPGi (trademark) R3 reagent manufactured by Sysmex Corporation was used.

[0098] (1-3') Solution containing labeled MUC1 antibody (reagent No. 3)

[0099] As the MUC1 antibody, MY.1E12 (Journal of Immunological Methods Volume 270, Issue 2, 15 December 2002, Pages 199-209) was used. The MUC1 antibody was dissolved in a phosphate buffer, and the EDTA concentration of the obtained solution was adjusted to 1 mM. An appropriate amount of 2-mercaptoethylamine hydrochloride solution was added to the antibody solution, and the reaction was carried out at 37°C for a specified time. Thereafter, the antibody solution was desalted using a PD-10 column (GE Healthcare Japan) and adjusted to a specified concentration.

[0100] A maleimide crosslinker as an ALP labeling agent was dissolved in N,N-dimethylformamide (DMF) to prepare a maleimide crosslinker solution. An appropriate amount of the maleimide crosslinker solution was added to the ALP solution and placed at 37°C for a specified time to introduce maleimide groups. Thereafter, the solution was desalted using a PD-10 column (GE Healthcare Japan) and adjusted to a specified concentration.

[0101] The antibody solution and the ALP solution were mixed at a specified ratio and placed in a refrigerator to allow coupling reaction to occur. Thereafter, 2-mercaptoethylamine hydrochloride solution was added to stop the reaction. The ALP antibody solution was subjected to gel filtration to recover the desired fraction.

[0102] The obtained desired fraction was dissolved in a BSA-containing buffer to a predetermined concentration and used as the third reagent for labeling the MUC1 antibody.

[0103] (1-3") Solution containing labeled haptoglobin antibody (reagent No. 3)

[0104] A labeled haptoglobin antibody was purchased from ABCAM (manufacturer's product number: AB13429), labeled in the same manner as 1-3', and used as the third reagent for the labeled haptoglobin antibody.

[0105] (1-4) Buffer for measurement (the fourth reagent)

[0106] HISCL (trademark) R4 reagent manufactured by Sysmex Corporation was used.

[0107] (1-5) Matrix (Fifth Reagent)

[0108] As the fifth reagent, HISCL (trademark) R5 reagent (manufactured by Sysmex Corporation) using CDP-Star (trademark) (Applied Biosystems) as a chemiluminescent substrate for alkaline phosphatase was used.

[0109] (1-6) Subjects

[0110] As the measurement subjects, a stock solution of human serum and serum prepared by diluting the human serum 2-fold, 4-fold, and 8-fold using HISCL (trademark) subject diluent were used.

[0111] [2. Example 1]

[0112] The effect of the addition of glycerol on the dilution linearity was investigated. Using the above-mentioned reagents 1 to 5, M2BP was measured by the fully automatic immunoassay device HISCL (trademark) 5000 (manufactured by Sysmex Corporation). During the measurement, glycerol was added to the R1 reagent of HISCL (trademark) M2BPGi (trademark) in such a way that it became 15 w / w%. 100 μl of R1 reagent, 30 μl of R2 reagent and 10 μl of the test subject were mixed, and after incubation according to the settings of the device, B / F separation was performed, and the R3 reagent was added to obtain the measurement value (luminescence intensity).

[0113] Based on the measurement results, the correlation coefficient (R 2 value).

[0114] like Figure 2 As shown in Figure 2, compared with the reference (no addition), the test system with glycerol added also showed good R 2 The value showed a significant improvement in the linearity of the test subject dilution.

[0115] [3. Example 2]

[0116] The effect of the addition of glycerol and other substances on the dilution linearity was investigated. Tween 80, NaCl, bovine serum albumin (BSA) or glycerol was added to the R1 reagent of HISCL (trademark) M2BPGi (trademark) to give 15 w / w %, and the dilution linearity of the M2BP determination was investigated in the same manner as in Example 1. Figure 3 The results show that, compared with the reference (no addition), glycerol alone showed good R 2 The linearity of the dilution of the test subjects was significantly improved.

[0117] [4. Example 3]

[0118] Next, methanol (C1), ethanol (C2), glycerol (C3), pentanol (C5), heptanol (C7), and octanol (C8) with different carbon numbers were added to the R1 reagent of HISCL (trademark) M2BPGi (trademark) to obtain 15 w / w % to confirm the effect of M2BP measurement on the linearity of the dilution of the test sample. The measurement was carried out in the same manner as in Example 1. Figure 4 As shown, the linearity of the dilution of the test substance is improved in alcohols having 1 to 7 carbon atoms, especially 2 to 7 carbon atoms. On the other hand, even when octanol having 8 carbon atoms is added, the linearity of the dilution of the test substance is not improved.

[0119] [5. Example 4]

[0120] The dilution linearity of the test object was confirmed by changing the lectin and the target substance. As the lectin, WFA or Lotus tetragonolobus Lectin (LTL) was used. As the target substance, M2BP, MUC1 or haptoglobin was used. In the system using LTL, as the second reagent, the magnetic particles immobilizing the LTL lectin described in (1-2') were used. When measuring MUC1, as the third reagent, the solution containing the labeled MUC1 antibody described in (1-3') was used. When measuring haptoglobin, as the third reagent, the labeled haptoglobin antibody described in (1-3") was used. In all detection systems, as the first reagent, the R1 reagent of HISCL (trademark) M2BPGi (trademark) was used, and glycerol was added to the first reagent in the same way as in Example 1. The measurement was performed in the same way as in Example 1.

[0121] The results are shown in Figures 5 to 7 As a lectin, the detection system of WFA was used ( Figure 5 and Figure 6 ), regardless of the target substance (M2BP and MUC1), the linearity of the dilution of the test substance was improved by the addition of glycerol. On the other hand, the detection system using LTL-haptoglobin as a lectin ( Figure 7) With the addition of glycerol, the dilution of the test object decreased linearly.

[0122] This indicates that the addition of alcohol, although not a target substance, is useful for the detection system using WFA.

[0123] [6. Example 5]

[0124] In order to confirm the effect of other alcohols, diglycerol, ethylene glycol, diethylene glycol, dipropylene glycol, and PEG6000 were added in addition to glycerol, and the target substance was M2BP to explore the dilution linearity of the test subject. In addition, a reducing agent (thioglycerol) used in the treatment of inhibiting nonspecific binding of labeled lectins in Patent Document 1 was added to compare the performance with glycerol. Each additive was added to the first reagent, and the measurement was performed in the same manner as in Example 1. Figure 8 As shown in the figure, the linearity of the dilution of the test object is improved by diglycerol, ethylene glycol, diethylene glycol, and dipropylene glycol. On the other hand, when PEG6000, which is a high molecular weight glycerol, is added, the linearity of the dilution of the test object is significantly reduced. In addition, when thioglycerol is added, the performance of the linearity of the dilution of the test object is also significantly reduced.

[0125] From the above results, it is clear that even when other glycerol compounds are added as alcohols having 3 to 6 carbon atoms, improvement in the linearity of the dilution of the test sample is confirmed.

[0126] [7. Example 6]

[0127] Next, the concentration of glycerol added to the R1 reagent was changed to 0 w / w%, 5.0 w / w%, 12.5 w / w%, 15.0 w / w%, and 20.0 w / w%, and the linearity of the test dilution when the target substance was M2BP was investigated. Each additive was added to the first reagent, and the measurement was performed in the same manner as in Example 1. Fig. 9 As shown, at any of the concentrations of 5.0 w / w%, 12.5 w / w%, 15.0 w / w%, and 20.0 w / w%, the dilution of the test substance increased linearly.

[0128] From the above results, it was confirmed that the addition of an alcohol having 1 to 7 carbon atoms to the detection system using WFA improves the dilution linearity of the test subject. In addition, it is thought that the improvement of the dilution linearity of the test subject can also be expected to improve the test accuracy.

Claims

1. A method for detecting a target substance, comprising: A step of forming a complex containing WFA and the target substance on the carrier by mixing a Wisteria floribunda agglutinin (WFA) immobilized on the carrier and the target substance containing a sugar chain bound to WFA in the presence of at least one alcohol selected from the group consisting of methanol, ethanol, glycerol, pentanol, heptanol, diglycerol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, triethylene glycol and a mixture thereof, and A step of detecting a target substance by detecting the complex.

2. The detection method according to claim 1, wherein In the forming step, the WFA, the target substance, and a detection substance that binds to the target substance and has a labeling substance are mixed in the presence of the alcohol to form a complex containing the WFA, the target substance, and the detection substance. In the detection step, the complex is detected based on the labeling substance of the complex. 3 . The detection method according to claim 1 , wherein in the forming step, the complex is formed in the presence of an alcohol at a concentration of 2 to 20 w / w %.

4. The detection method according to claim 1 or 2, wherein the target substance is glycoprotein.

5. The detection method of claim 4, wherein the target substance is: Mac-2-binding protein (M2BP), cell surface-associated Mucin 1 (MUC1), α1 acid glycoprotein (AGP), neural cell attachment molecule L1 (L1CAM) or KL-6 antigen. The detection method according to claim 2 , wherein the detection substance is an antibody that specifically binds to the target substance.

7. The detection method according to claim 2, wherein the labeling substance is at least one selected from the group consisting of a hapten, an enzyme, and a fluorescent substance.

8. The detection method according to claim 1 or 2, wherein the carrier is a particle.

9. The detection method according to claim 1 or 2, wherein in the detection step, the target substance is quantified.

10. Use of at least one alcohol selected from the following in the manufacture of a reagent for detecting a target substance: methanol, ethanol, glycerol, pentanol, heptanol, diglycerol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, triethylene glycol, and a mixture thereof, The target substance contains a sugar chain that binds to Wisteria floribunda lectin (WFA).

11. Use of the following substances in the manufacture of a kit for detecting a target substance: Detection substances of target substances; Wisteria floribunda agglutinin (WFA); and At least one alcohol selected from the group consisting of methanol, ethanol, glycerol, pentanol, heptanol, diglycerol, ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, triethylene glycol, and mixtures thereof, The target substance contains sugar chains bound to WFA.

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