WO2014136974A1 - Composition that recognizes antibodies stemming from immune-mediated peripheral neuropathy and use for said composition - Google Patents

Abstract

Sugar-chain-immobilized fluorescent nanoparticles that allow quick and convenient detection of ganglioside-binding autoantibodies in blood serum from immune-mediated peripheral neuropathy patients are provided, as is a use for said sugar-chain-immobilized fluorescent nanoparticles. These sugar-chain-immobilized fluorescent nanoparticles contain the following: sugar-chain ligand complexes comprising sugar chains, including ganglioside-derived sugar chains, and a prescribed linker compound that has a hydrocarbon chain or the like on the main chain thereof; and fluorescent nanoparticles that have a prescribed core-shell structure. These sugar-chain-immobilized fluorescent nanoparticles are obtained by immobilizing the abovementioned sugar chains.

Description

Composition for recognizing antibody derived from immune peripheral neuropathy and use thereof

The present invention relates to fluorescent nanoparticles in which the sugar chain portion of ganglioside is immobilized and use thereof.

Guillain-Barre syndrome (GBS), which is an immune peripheral neuropathy, is a peripheral neuropathy mainly characterized by acute limb weakness and loss of deep tendon reflexes. It is the most common neurological / muscular disease that acutely reduces limb muscle strength. Besides, various related diseases or subtypes such as Fisher syndrome (FS) and Bickerstaff type brainstem encephalitis (BBE) are known for GBS (Non-patent Document 1).

Ganglioside is an acidic glycolipid with sialic acid and is abundant in nerve cell membrane. Ganglioside is composed of ceramide having fatty acid and hydrophobicity, and hydrophilic oligosaccharide, and it is said that it is a part of oligosaccharide existing mainly in the form exposed on the cell surface that has physiological activity.

An antibody against ganglioside has attracted attention as a pathogen of autoimmune peripheral nerve disease, mainly GBS and FS. In GBS and FS, anti-ganglioside antibodies are detected in blood. The antibody titer of the anti-ganglioside antibody is highest immediately after the onset, and decreases and disappears over time (Non-patent Document 2).

In addition, it has been confirmed that there is molecular homology between gangliosides and pathogens of prior infections (pathogens of infections that often develop prior to immune peripheral neuropathy such as GBS). Has been established, and the onset mechanism due to the molecular homology between the pathogen and the nerve tissue has been proved (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5).

The search for anti-ganglioside antibodies has already been widely used clinically. In GBS, detection of IgG antibodies against GM1, GM1b, GD1a, and GalNAc-GD1a is useful as an auxiliary diagnostic technique in detecting IgG anti-GQ1b antibodies in Fisher syndrome, Bickerstaff type brainstem encephalitis, and acute extraocular muscle paralysis (Non-Patent Document 6).

There is an ELISA method as a method for measuring an anti-ganglioside antibody. The ELISA method is an abbreviation for Enzyme-Linked Immuno-Sorbent Assay, and is a method used when detecting or quantifying the concentration of an antibody or an antigen contained in a sample. When a specific protein is present in a trace amount in a biological sample, it has high specificity (how accurately it can be distinguished from contaminants) and good quantitativeness (it can be detected even in trace amounts, or low concentration) Good reproducibility). ELISA uses a highly specific antigen-antibody reaction and clears the above conditions by using color development / luminescence based on enzyme reaction as a signal.

Therefore, the relationship between GBS-related diseases and anti-ganglioside antibodies has been elucidated at the research level, and the ELISA method (Non-patent Documents 5 and 6) has been put to practical use as a method for measuring anti-glycolipid antibodies that can be used in clinical settings. . A complex of GM1 ganglioside sugar chain and protein has also been synthesized (Non-patent Document 9).

In addition, plasma exchange therapy and massive intravenous injection of immunoglobulin have been established as effective treatments for GBS (Non-patent Document 1).

On the other hand, the inventors have developed sugar chain-immobilized fluorescent nanoparticles that have a uniform particle size, high water dispersibility, and luminescence, and that can easily immobilize sugar chains. (Patent Document 1, Non-Patent Document 7).

Furthermore, the inventors have independently developed a fluorescent linker compound that can be used for the preparation of the sugar chain-immobilized fluorescent nanoparticles (Non-patent Document 8).

Japanese Patent Publication “Japanese Patent Laid-Open No. 2011-209282 (published on October 20, 2011)”

N. Yuki, et al., N. Engl. J. Med., 366 ： 2294-2304 (2012) Odaka M, et al., J. Neurol. Sci., 210: 99-103 (2003) Yuki N, et al., J. Exp. Med., 178: 177-1775 (1993) N. Yuki, et al., Proc. Natl. Acad. Sci. U.S.A, 101: 11404-11409 (2004) N. Yuki, et al., Ann. Neurol., 49: 712-720 (2001) Masataka Kotaka et al., Modern Media 54 (3): 82-86 (2008) H. Shinchi, et al., Chem. Asian J., 7: 2678-2682 (2012) Sato M, et al., J. Biochem., 146: 33-41 (2009) T. Yoshikawa, et al., Glycoconjugate J., 25: 545-553 (2008)

However, the above-mentioned ELISA method currently used as a method for measuring an anti-ganglioside antibody has a problem that it is a time consuming and laborious method. In other words, it is usually necessary to send the patient's serum to the testing company and wait for a week or more for the result to arrive. Therefore, there is a problem that a quick diagnosis cannot be performed and treatment is inevitably delayed.

Under such circumstances, a new method capable of rapidly detecting an anti-ganglioside antibody contained in the serum of a patient and a kit capable of rapidly detecting the antibody are required. An object of the present invention is to develop a rapid and simple diagnostic method using nanoparticles having ganglioside sugar chains immobilized thereon.

Based on this background, we started research, targeted the “glycans” of gangliosides, and made use of the latest nanotechnology to make rapid and simple diagnostic methods (methods for detecting the above antibodies quickly and easily). Has been developing. In the present invention, based on the sugar chain-immobilized fluorescent nanoparticle technology that has been developed so far (Patent Document 1, Non-Patent Document 7), a fluorescent nanoparticle having a ganglioside sugar chain moiety immobilized, such as ganglioside GM1. Particles were newly prepared and used to successfully detect antibodies closely related to GBS in patient sera.

The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide sugar chain-immobilized fluorescent nanoparticles in which a sugar chain part of ganglioside is immobilized and use thereof.

INDUSTRIAL APPLICABILITY The present invention can provide, for example, a simple diagnostic test tool that has specific reactivity to an antibody against ganglioside present in GBS patient serum and enables detection within 3 hours. .

The sugar chain-immobilized fluorescent nanoparticles according to the present invention include one or more sugar chains containing a ganglioside-derived sugar chain, and one or more sugar chains having a hydrocarbon chain or a hydrocarbon-derived chain in the main chain. A sugar chain ligand comprising two or more types of linker compounds, wherein the main chain of the linker compound has an amino group bonded to the sugar chain at one end and a hydrocarbon structure containing a sulfur atom at the other end A composite and a fluorescent nanoparticle in which a particle core composed of first and second metal components is coated with a layer composed of first and third metal components, and the hydrocarbon structure is formed in the layer It is characterized by being fixed.

The sugar chain-immobilized fluorescent nanoparticles (also referred to as ganglioside sugar chain-immobilized fluorescent nanoparticles) according to the present invention specifically bind to an anti-ganglioside antibody such as an anti-GM1 antibody present in the serum of GBS patients. Have an action of forming an aggregate.

GBS has similar clinical symptoms to cerebral thrombosis, and a rapid examination method is required. If the sugar chain-immobilized fluorescent nanoparticles of the present invention are used, the serum of the patient can be used directly, and the test result can be confirmed visually within a few hours. Therefore, the possibility of practical use is extremely high.

It is a figure which shows the chemical structure of GM1-Glc sugar chain which introduce | transduced Glc into the sugar chain part of ganglioside GM1. It is a figure which shows the synthetic pathway of the intermediate body at the time of synthesize | combining GM1-Glc sugar chain. FIG. 3 is a diagram showing a synthesis route of a disaccharide structure necessary for synthesizing a GM1-Glc sugar chain. It is a figure showing the path | route which shows the glycosylation and deprotection for synthesize | combining GM1-Glc sugar chain. FIG. 2 is a view showing a chemical structure of a sugar chain ligand complex (GM1-Glc-f-mono) containing a GM1 sugar chain prepared from a GM1-Glc sugar chain. 1 is a schematic view showing a method for preparing GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP). FIG. It is a figure which shows the particle size distribution by the DLS measurement of GM1-Glc sugar chain fixed fluorescent nanoparticle (GM1-Glc-FNP). It is a figure which shows the result of the MALDI-TOF / MS analysis by Positive mode of GM1-Glc sugar chain fixed fluorescent nanoparticle (GM1-Glc-FNP). It is a figure which shows the result of the MALDI-TOF / MS analysis by Negative mode of the GM1-Glc sugar chain fixed fluorescent nanoparticle (GM1-Glc-FNP). It is a figure which shows the result of the fluorescence emission of the precipitate after the centrifugation in the aggregation experiment of GM1-Glc sugar chain fixed fluorescent nanoparticle (GM1-Glc-FNP) and sugar chain binding protein. It is a figure which shows the fluorescence spectrum of the supernatant after centrifugation in the aggregation experiment of GM1-Glc sugar chain fixed fluorescent nanoparticle (GM1-Glc-FNP) and sugar chain binding protein. It is a figure which shows the result of the fluorescence emission of the precipitate after centrifugation in the aggregation experiment of the GM1-Glc sugar chain fixed fluorescent nanoparticle (GM1-Glc-FNP) and the serum of a patient with immune peripheral neuropathy . Fluorescence of the precipitate after centrifugation when the incubation time was changed in an agglutination experiment between GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP) and sera of patients with immune peripheral neuropathy It is a figure which shows the result of light emission. It is a figure which shows the result of having confirmed that the antibody exists in the precipitate which emits fluorescence by performing SDS-PAGE of a precipitate under reducing conditions. It is a figure which shows the result of having confirmed that the antibody exists in the precipitate which emits fluorescence by performing SDS-PAGE of a precipitate on non-reducing conditions. It is a figure which shows the result confirmed by performing Western blotting that an antibody exists in the precipitate which emits fluorescence. It is a figure which shows the result of the fluorescence emission of the precipitate after centrifugation in the case where GM1 sugar chain and GM1-Glc sugar chain are simultaneously present in a serum sample. It is a figure which shows the fluorescence spectrum of the supernatant after centrifugation in the case where GM1 sugar chain and GM1-Glc sugar chain are simultaneously present in a serum sample. FIG. 3 is a schematic diagram showing a method for preparing fluorescent nanoparticles having tetraethylene glycol and GM1-Glc sugar chains immobilized thereon. FIG. 3 is a schematic diagram showing the structure of fluorescent nanoparticles (TEG-containing GM1-Glc-FNP) in which a GM1-Glc sugar chain and tetraethylene glycol are immobilized. It is a figure which shows the result of having changed the density | concentration of TEG with respect to GM1-Glc sugar chain, and having performed the aggregation experiment of the serum sample using TEG containing GM1-Glc-FNP. It is a figure which shows the result of having examined the relationship between the antibody concentration and reaction time, and the detection sensitivity of an aggregate using TEG containing GM1-Glc-FNP. It is a figure which shows the detection result of the anti- GM1 IgG antibody by TEG containing GM1-Glc-FNP at the time of using 50 samples of anti-GM1 antibody positive sera by ELISA method. It is a figure which shows the detection result of the anti- GM1IgG antibody by TEG containing GM1-Glc-FNP at the time of using 50 samples of anti-GM1IgG antibody negative sera by ELISA method. Serum that was positive for anti-GM1 antibody by ELISA and was not mixed with TEG-containing GM1-Glc-FNP for 3 hours, and when it was allowed to stand for 3 hours, it was left for 12 hours after mixing. It is a figure which shows the detection result of the anti-GM1 IgG antibody in the case. It is a figure which shows the synthetic pathway of GM1 sugar_chain | carbohydrate fixed sugar_chain | carbohydrate ligand complex. It is a figure which shows the result of the fluorescence emission of the precipitate after centrifugation in the aggregation experiment of GM1 sugar chain fixed fluorescent nanoparticle (GM1-FNP) and sugar chain binding protein. It is a figure which shows the fluorescence spectrum of the supernatant after centrifugation in the aggregation experiment of GM1 sugar chain fixed fluorescent nanoparticle (GM1-FNP) and sugar chain binding protein. It is a figure which shows the result of the fluorescence emission of the precipitate after centrifugation in the aggregation experiment of GM1 sugar_chain | carbohydrate fixed fluorescent nanoparticle (GM1-FNP) and the serum of the patient of immune peripheral neuropathy. It is a figure which shows the fluorescence spectrum of the supernatant after centrifugation in the aggregation experiment of the GM1 sugar chain fixed fluorescent nanoparticle (GM1-FNP) and the serum of the patient of immune peripheral neuropathy. It is a figure which shows the result of having mixed TEG containing GM1-Glc-FNP (GM1-Glc: TEG = 5: 5) with PNA and confirming the aggregation of PNA. It is a figure which shows the result of having mixed TEG containing GM1-FNP (GM1: TEG = 10: 0) with PNA and having confirmed aggregation of PNA. It is a figure which shows the result of having mixed TEG containing GM1-GNP (GM1: TEG = 5: 5) with PNA and confirming the aggregation of PNA. Using TEG-containing GM1-Glc-FNP (GM1-Glc: TEG = 5: 5), GM1-FNP (GM1: TEG = 10: 0), and TEG-containing GM1-FNP (GM1: TEG = 5: 5) It is a figure which shows the result of having performed the agglutination test of serum. It is a figure which shows the detection result of the anti- GM1IgG antibody by GM1-FNP at the time of using 50 samples of anti-GM1IgG antibody positive sera by ELISA method. It is a figure which shows the fluorescence intensity of a supernatant when 50 samples of anti-GM1 antibody positive sera and GM1-FNP are mixed by ELISA method. It is a figure which shows the detection result of the anti- GM1IgG antibody by GM1-FNP at the time of using 50 samples of anti-GM1IgG antibody negative sera by ELISA method. It is a figure which shows the fluorescence intensity of a supernatant when 50 samples of anti-GM1 antibody negative serum and GM1-FNP are mixed by ELISA method. Detection of aggregates in the case of sera positive for anti-GM1 IgG antibody by ELISA and having a negative determination result when mixed with GM1-FNP for 3 hours, and left for 12 hours after mixing It is a figure which shows a result. Detection of aggregates when the serum is anti-GM1 IgG antibody-positive sera by ELISA and left to stand for 3 hours after mixing with GM1-FNP, and the determination result is false-positive for 12 hours after mixing It is a figure which shows a result. FIG. 2 is a diagram showing a synthesis route of a GD1a-immobilized sugar chain ligand complex (GD1a-f-mono). It is a figure which shows the result of the fluorescence emission of the precipitate after the centrifugation in the aggregation experiment of GD1a sugar chain fixed fluorescent nanoparticle (GD1a-FNP) and sugar chain binding protein. It is a figure which shows the fluorescence spectrum of the supernatant after centrifugation in the aggregation experiment of GD1a sugar chain fixed fluorescent nanoparticle (GD1a-FNP) and sugar chain binding protein. It is a figure which shows the result of the fluorescence emission of the deposit after centrifugation in the aggregation experiment with GD1a-FNP and the serum of the patient of immune peripheral neuropathy. It is a figure which shows the result of having measured the fluorescence spectrum of the supernatant after centrifugation in the aggregation experiment with GD1a-FNP and the serum of the patient of immune peripheral neuropathy. It is a figure which shows the result of the aggregation experiment which mixed the anti-GM1IgG antibody positive by ELISA method, and the result of making it react with GM1-FNP in Example 18 negative or false positive, and GD1a-FNP. FIG. 19 is a diagram showing the results of an agglutination experiment in which GD1a-GM1-FNP was mixed with serum that was positive for anti-GM1 IgG antibody by ELISA and negative or false positive as a result of reaction with GM1-FNP in Example 18 . FIG. 3 is a diagram showing an intermediate synthesis route when a GQ1b-Glc sugar chain is synthesized. FIG. 3 is a diagram showing a pathway showing glycosylation and deprotection for synthesizing GQ1b-Glc sugar chain. FIG. 2 is a view showing a synthesis route of a GQ1b-Glc-immobilized sugar chain ligand complex (GQ1b-Glc-f-mono). It is a figure which shows the result of having measured the fluorescence and UV-Vis spectrum of the GQ1b-Glc-FNP solution. It is a figure which shows the particle size distribution by DLS measurement of GQ1b-Glc-FNP. It is a figure which shows the result of the MALDI-TOF / MS analysis by Positive mode of GQ1b-Glc-FNP. It is a figure which shows the result of the MALDI-TOF / MS analysis by Negative mode of GQ1b-Glc-FNP. It is a figure which shows the result of the fluorescence emission of the deposit after centrifugation in the aggregation experiment of GQ1b-Glc-FNP and sugar chain binding protein. It is a figure which shows the fluorescence spectrum of the supernatant after centrifugation in the aggregation experiment of GQ1b-Glc-FNP and sugar chain binding protein. It is a figure which shows the result of the fluorescence emission of the precipitate after centrifugation in the aggregation experiment with GQ1b-Glc-FNP and the serum of the patient of immune peripheral neuropathy. 1 is a schematic diagram showing a method for preparing GM1 sugar chain-immobilized fluorescent nanoparticles (GM1-FNP2) in which GM1 sugar chains are immobilized on ZAIS / ZnS core / shell nanoparticles having ZAIS as a core. FIG. It is a figure which shows the result of the fluorescence emission of the precipitate after centrifugation in the aggregation experiment of GM1-FNP2 and the serum and protein of the patient of immune peripheral neuropathy.

[1. (Sugar chain-immobilized fluorescent nanoparticles)
The present invention is described in detail below. Note that all of the non-patent documents and patent documents described in the present specification are incorporated herein by reference.

In the present specification, the “sugar chain-immobilized fluorescent nanoparticle” refers to a sugar chain ligand complex described in detail below and a fluorescent nanoparticle (also referred to as a fluorescent metal nanoparticle). It will be. The fluorescent nanoparticles may be produced as described later, or commercially available ones may be used.

In the present specification, the “metal nanoparticle” is not particularly limited as long as it is a nanoparticle containing an inorganic metal component. Examples of the metal component suitably used in the present invention include Si, Ge, Cd, Zn, Cu. , Ag, Ga, As, In, Te, S, Au and the like, but are not limited thereto.

In the present specification, “nanoparticles” are intended to be dispersed in an aqueous solution to form a colloidal solution. Therefore, the average particle diameter of the nanoparticles is preferably in the range of 0.5 to 400 nm, more preferably in the range of 0.5 nm to 100 nm, and further in the range of 1 nm to 10 nm. preferable. The average particle size may be less than 0.5 nm, but the production of such particles is expensive and impractical, and if it exceeds 400 nm, the dispersion stability of the particles tends to change over time. It is not preferable.

In addition, in this specification, the said average particle diameter says the average particle diameter measured by the dynamic light scattering method (DLS).

(1-1. Sugar chains containing sugar chains derived from gangliosides)
The sugar chain-immobilized fluorescent nanoparticles according to the present invention comprise one or more sugar chains containing a ganglioside-derived sugar chain, a linker compound having a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, A sugar chain ligand complex having a hydrocarbon structure in which the main chain of the linker compound has an amino group bonded to the sugar chain at one end and a sulfur atom at the other end; And a fluorescent nanoparticle in which a particle core made of a second metal component is covered with a layer made of a first and third metal component, and the hydrocarbon structure is immobilized on the layer.

Anti-ganglioside antibodies are often found in the serum in the acute phase of immune peripheral neuropathy such as GBS, FS, and BBE, and these antibodies are considered to be a causative agent of immune peripheral neuropathy . For example, anti-GM1 antibody is involved in the development of GBAN, AMAN (acute motor axon type neuropathy), and anti-GM1 antibody produced by Campylobacter lipooligosaccharide acts on GM1 on peripheral nerve axons as a target. It is thought that axonal disorder is caused by.

Therefore, in view of the object of the present invention to perform rapid and simple detection of an anti-ganglioside antibody, it is preferable to use ganglioside which can be a pathogenic substance of immune peripheral neuropathy as ganglioside. From such a viewpoint, for example, it is preferable to use one or more gangliosides selected from the group consisting of GM1, GM1b, GD1a, GalNAc-GD1a, GM2, GD1a, GD1b, GT1a, and GQ1b.

Of these, the ganglioside is more preferably one or more gangliosides selected from the group consisting of GM1, GD1a, and GQ1b.

In this specification, “sugar chain derived from ganglioside” means a sugar chain contained in ganglioside, and the sugar chain contains one or more sialic acids. In the present specification, “a sugar chain containing a ganglioside-derived sugar chain” includes both a ganglioside-derived sugar chain itself and a sugar chain obtained by binding a saccharide to a ganglioside-derived sugar chain. Hereinafter, the “sugar chain containing a ganglioside-derived sugar chain” is also simply referred to as “ganglioside sugar chain”.

Accordingly, the ganglioside sugar chain may be, for example, the GM1 sugar chain itself (hereinafter sometimes referred to as “GM1 sugar chain” or “ganglioside GM1 sugar chain”) represented by the following chemical formula 14, for example: A sugar chain represented by Chemical Formula 1 (hereinafter referred to as “GM1-Glc sugar chain”), which is a sugar chain obtained by binding one glucose as the additional sugar to the reducing end of the sugar chain represented by Chemical Formula 14. 8).

Of course, the present invention is not limited to this. For example, a GD1a sugar chain (hereinafter sometimes referred to as “GD1a sugar chain” or “ganglioside GD1a sugar chain”) represented by the following chemical formula 15; “GQ1b sugar chain” “sometimes referred to as ganglioside GQ1b sugar chain”, sugar chain represented by chemical formula 16 in which one additional glucose is bonded to the reducing end of GQ1b sugar chain (hereinafter referred to as “GQ1b-Glc sugar chain”) The ganglioside sugar chain having a different structure is also included in the applicable range.

The upper limit on the number of additional sugars (eg, glucose in the GM1-Glc sugar chain; hereinafter referred to as “further sugar”) to be bound to the ganglioside-derived sugar chain is that the sugar chain cluster effect on the nanoparticles is Since it is important for bonding, it is preferably 5 or less, more preferably 3 or less, and particularly preferably 1 or less.

The binding position of the further sugar is preferably the reducing end of the sugar chain derived from ganglioside. Moreover, as the kind of the above-mentioned saccharide, any kind can be used as long as it can undergo a reductive amination reaction with an amino group of a linker compound described later. For example, glucose, galactose, mannose, etc. can be used.

As the ganglioside sugar chain, one type or two or more types can be used. When it is desired to detect only an anti-ganglioside antibody against a specific ganglioside, only one type of sugar chain may be used. For example, when only an anti-ganglioside antibody against GM1 is to be detected, one type of GM1 sugar chain itself or the above-described GM1-Glc sugar chain shown in Chemical Formula 14 may be used.

On the other hand, when it is desired to detect two or more anti-ganglioside antibodies, two or more of the above ganglioside sugar chains may be used. For example, since IgG antibodies against GM1, GM1b, GD1a, and GalNAc-GD1a are detected in the serum of GBS patients, two or more anti-ganglioside antibodies are used using two or more of the ganglioside sugar chains. Can also be detected.

(1-2. Linker compound, sugar chain ligand complex)
The sugar chain ligand complex, which is a component of the sugar chain-immobilized fluorescent nanoparticle according to the present invention, is composed of a linker compound capable of binding to any fluorescent nanoparticle and a ganglioside sugar chain.

Therefore, the sugar chain ligand complex is required not to form a non-specific interaction based on hydrophobicity with the anti-ganglioside antibody. Preferably, the sugar chain ligand complex is composed of one or more ganglioside sugar chains and one or more linker compounds each having a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, The main chain of the linker compound has a hydrocarbon structure having an amino group bonded to the ganglioside sugar chain at one end and a sulfur atom at the other end.

The hydrocarbon-derived chain is a hydrocarbon chain composed of carbon and hydrogen, and some of the carbon and hydrogen may be replaced with other atoms and substituents. That is, the hydrocarbon-derived chain has an amino group at the terminal, and a part of the carbon-carbon bond (C—C bond) which is the main chain structure of the hydrocarbon chain is a carbon-nitrogen bond (C—N Bond), carbon-oxygen bond (C—O bond), and amide bond (CO—NH bond).

The hydrocarbon structure containing a sulfur atom means a hydrocarbon structure composed of carbon and hydrogen in which some carbon is replaced with sulfur. The hydrocarbon structure containing a sulfur atom may be a chain (including both a straight chain and a branched chain) or a ring, and may have both a chain structure and a ring structure. May be included.

In the sugar chain-immobilized fluorescent nanoparticles according to the present invention, the hydrocarbon structure containing a sulfur atom may have a hydrocarbon structure containing an S—S bond or an SH group. That is, the hydrocarbon structure containing a sulfur atom may contain a disulfide bond (SS bond) or a thiol group (SH group).

In the sugar chain-immobilized fluorescent nanoparticles according to the present invention, the amino group is preferably an aromatic amino group. At pH 3-4, which is the optimum condition for the reductive amination reaction, it is necessary that the amino group is not protonated. Therefore, an aromatic amino group in which an unshared electron pair exists on a nitrogen atom even at pH 3 to 4 due to conjugation with an aromatic group is preferable.

As a suitable ligand complex used in the present invention, a compound shown in WO2005 / 0797965 or a compound shown in Non-Patent Document 8 can be mentioned. Specifically, the general formula (17)

(Wherein p and q are each independently an integer of 0 or more and 6 or less), and the X has an aromatic amino group at the terminal and a carbon-nitrogen bond in the main chain. It is preferable to provide a hydrocarbon-derived chain having a structure including one chain, two chains, or three chains, and Y as a hydrocarbon structure including a sulfur atom or a sulfur atom. Further, as Z, a structure in which a linker compound having a linear structure having a carbon-carbon bond or a carbon-oxygen bond and a sugar having a reducing end are bonded via the aromatic amino group. It is preferable to have.

X is the general formula (18), general formula (19), general formula (20) or general formula (21).

(Wherein m ¹ to m ⁵ are each independently an integer of 0 or more and 6 or less, R ′ is hydrogen (H) or R), and R is a sugar chain-derived compound (for example, reduced More preferably, the Z is represented by the formula (22) or the formula (23).

(Wherein n ¹ and n ² are each an integer of 1 or more and 6 or less). More preferably, the ligand conjugate usable in the present invention is, for example, the general formula (24), the general formula (25), the general formula (26), the general formula (27), the general formula (28), or the general formula. (29)

(Wherein m ¹ to m ⁵ are each independently an integer of 0 to 6; n ¹ and n ² are each independently an integer of 1 to 6; q is an integer of 0 to 6) Examples thereof include those having a structure in which a sugar having a reducing end is introduced into an aromatic amino group of a linker compound having a structure as described above. The linker compound represented by the general formula (29) is a linker compound that has been developed by the inventors and has itself fluorescence (Non-patent Document 8).

The linker compound that can be used in the present invention, for example, performs a condensation reaction between thioctic acid and an amine compound whose aromatic amino group end is protected by a protecting group to deprotect the protecting group at the end of the aromatic amino group. Manufactured by.

In addition, the linker compound represented by the general formula (28) is, for example, a condensation reaction between a dimer of γ-mercaptobutyric acid and an amine compound in which two molecular amino terminal ends are protected by a protecting group. And deprotecting the protecting group at the terminal of the aromatic amino group.

The above thioctic acid is

The amine compound is not particularly limited as long as it has an aromatic amino group end protected by a protecting group.

In one embodiment of the present invention, a linker compound in which n ¹ is 4 in the structure represented by the general formula (29) (the following formula (30))

In addition, a ligand complex into which a sugar having a reducing end (ganglioside sugar chain) is introduced is used. A method for producing the ligand complex will be described in (1-6. Method for producing sugar chain-immobilized fluorescent nanoparticles). The linker compound represented by the general formula (29) is a linker compound that exhibits fluorescence as described above (Non-patent Document 8, hereinafter referred to as “fluorescent linker compound”), and the fluorescence is used as an index. Since the sugar chain ligand complex can be purified, it is particularly preferably used.

In contrast, since the linker compounds represented by the general formulas (24) to (28) are not fluorescent linker compounds, when these linker compounds are used, the sugar chain ligand complex is obtained as a white solid, Since the sugar chain ligand complex obtained by using the linker compounds represented by the general formulas (24) to (28) can also be immobilized on the fluorescent nanoparticles described later, it is preferably used in the present invention. Can do.

A synthesis procedure of a sugar chain ligand complex in which a sugar having a reducing end is introduced into a linker compound in which n ¹ and q are 0 in the structure represented by the general formula (26) is disclosed in WO2005 / 07965. The disclosed sugar chain ligand conjugate in which a saccharide having a reducing end is introduced into the linker compound represented by the general formula (24), (25), (27), (28) is also synthesized by the same procedure. can do.

Thus, the linker compound that can be used in the present invention has a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, and has an amino group at one end of the main chain. Furthermore, since the linker compound has an amino group at the terminal, a sugar chain molecule can be easily introduced. The amino group may be a modified amino group (for example, an amino group modified with an acetyl group, a methyl group or a formyl group), an aromatic amino group, or an unmodified amino group. There may be.

The linker compound can be used alone or in combination of two or more. That is, in the present invention, one kind of linker compound (for example, a linker compound represented by the formula (30)) is used, and the linker compound is bound to one kind of ganglioside sugar chain (for example, GM1-Glc sugar chain). You may use only the made sugar chain ligand conjugate.

Further, one kind of linker compound (for example, a linker compound represented by the formula (30)) is mixed with two or more kinds of ganglioside sugar chains (for example, a GM1-Glc sugar chain and a sugar chain represented by the chemical formula 14). A sugar chain ligand complex in which different types of sugar chains are independently bonded to one type of linker compound (for example, a linker compound represented by the formula (30) in which a GM1-Glc sugar chain is bonded; And a mixture with a linker compound represented by the formula (30) to which a sugar chain is bonded may be prepared and used.

Furthermore, two or more types of linker compounds (for example, a linker compound represented by the formula (30) and a linker compound in which n ¹ and q are 0 in the structure represented by the general formula (26)) are used. The saccharide is linked to one type of ganglioside sugar chain (for example, GM1-Glc sugar chain) or two or more types of ganglioside sugar chains (for example, GM1-Glc sugar chain and the sugar chain represented by Chemical Formula 14). A chain ligand complex may be prepared and used.

As described above, the sugar chain ligand complex used in the present invention may consist of one kind of linker compound and one kind of ganglioside sugar chain. In this case, there is an advantage that a specific anti-ganglioside antibody (for example, anti-GM1 antibody) can be specifically detected with high sensitivity, simply and in a short time.

In addition, the sugar chain ligand complex used in the present invention may be a mixture of sugar chain ligand complexes obtained by combining two or more types of linker compounds and two or more types of ganglioside sugar chains. In this case, since two or more types of anti-ganglioside antibodies can be detected, for example, a plurality of anti-ganglioside antibodies (for example, anti-GM1 antibody and anti-GD1a antibody) involved in GBS can be detected simultaneously.

The sugar-ligand complex includes a sulfur atom (S). For example, the sulfur atom (S) includes cadmium (Cd) contained in the fluorescent nanoparticle and a metal-sulfur bond (Cd—S). Bond) and can bind to the fluorescent nanoparticles.

The linker compound that can be used in the present invention is mainly composed of a hydrocarbon structure containing an S—S bond or an SH group in that a metal-sulfur bond (eg, Cd—S bond) can be easily formed. It is preferable to provide at the other end of the chain.

Accordingly, the linker compound can be arranged by assembling sugar chain molecules on the fluorescent nanoparticles. Sulfur (S) in a disulfide bond (SS bond) or SH group, for example, forms a metal-sulfur bond (Cd-S bond) with cadmium (Cd) present on the fluorescent nanoparticles, The bond with can be strengthened.

In the sugar chain ligand complex, a glycoside sugar chain having a reducing end is introduced into the amino group of the linker compound. In other words, the sugar chain ligand complex has a structure in which the linker compound and a sugar chain having a reducing end are bonded via an amino group.

The introduction of the sugar chain can be performed, for example, by a reductive amination reaction between the amino group (—NH ₂ group) of the linker compound and the sugar chain. That is, an aldehyde group (—CHO group) or a ketone group (—CRO group, R is a hydrocarbon group) in the sugar chain generated by equilibrium reacts with the amino group of the linker compound. Then, by continuously reducing the Schiff base formed by this reaction, the sugar chain can be easily introduced into the amino group.

The “sugar chain having a reducing end” is a monosaccharide, oligosaccharide chain or polysaccharide chain in which the anomeric carbon atom is not substituted. That is, the sugar chain having the reducing end is a reducing sugar chain. The sugar chain having a reducing end may be a commercially available product or a natural product, or a product prepared by decomposing a commercially available and natural polysaccharide chain.

As the sugar chain having the reducing end, for example, the ganglioside sugar chain described above in (1-1. Sugar chain containing a ganglioside-derived sugar chain) can be used.

The linker compound contained in the sugar chain ligand complex has a sulfur atom capable of binding to a metal and an amino group capable of binding to a sugar chain molecule such as an oligosaccharide chain. Therefore, for example, the sugar chain ligand complex is fixed to the metal on the fluorescent nanoparticle by a metal-sulfur bond such as a Cd—S bond, so that the fluorescent nanoparticle according to the present invention is bonded to the fluorescent nanoparticle according to the present invention via the linker compound. The sugar chain molecules can be bound firmly and easily, and the fluorescent nanoparticles can be stabilized in a colloidal state.

Furthermore, the immobilization of the above-mentioned sugar chain ligand complex to the fluorescent nanoparticles can be performed by simply mixing the ligand complex treated with a reducing agent and the solution containing the fluorescent nanoparticles. It is possible to immobilize sugar chains. The reducing agent used for the reducing agent treatment is preferably sodium borohydride.

As a result, the S—S bond in the linker compound is reduced to be converted into a —SH group, and can be bonded to any metal by a metal-sulfur (S) bond, for example, a cadmium-sulfur (Cd—S) bond. it can.

In addition, the sugar chain ligand complex has a non-cyclic partial structure in which many hydroxyl groups are present at the binding site to the linker compound (that is, the reducing end sugar unit bonded to the linker compound has many hydroxyl groups. Therefore, the influence of non-specific interaction with the protein can be almost ignored. Therefore, by using the ligand complex having the linker compound, the interaction between the sugar chain and the anti-ganglioside antibody can be evaluated with good reproducibility.

Since the sugar chain ligand complex includes a linker compound and a sugar chain molecule, a metal-sulfur (S) bond such as cadmium-sulfur (Cd-S) is formed at the SS bond in the linker compound. ) It can be bonded to any metal by bonding. Thereby, for example, a sugar chain-immobilized fluorescent nanoparticle in which a sugar chain molecule is immobilized on the fluorescent nanoparticle via the Cd—S bond can be provided. Any metal can be used as long as it can bind to the sugar-ligand complex, and metals such as zinc, copper, silver, and indium can be used in addition to cadmium. Particularly, cadmium and zinc can be used. It is preferable to use it.

As will be described later, the sugar chain-immobilized fluorescent nanoparticle according to the present invention is a fluorescent nanoparticle in which a particle core composed of first and second metal components is coated with a layer composed of first and third metal components. Contains particles. In the sugar chain ligand complex, since the linker compound has a hydrocarbon structure containing a sulfur atom at the other end, the hydrocarbon structure is immobilized on the layer. That is, the S—S bond of the hydrocarbon structure forms a metal-sulfur (S) bond with the metal contained in the layer, so that the hydrocarbon structure is fixed to the layer.

(1-4. Immobilization of oligoethylene glycol)
The sugar chain-immobilized fluorescent nanoparticle according to the present invention contains the above-mentioned sugar chain ligand complex, but preferably further contains the following complex in addition to the sugar chain ligand complex. That is, it comprises an aminated oligoethylene glycol and a linker compound having a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, and the main chain of the linker compound has the aminated oligoethylene at one end thereof. It is preferable to include a complex having a carboxyl group bonded to glycol and having a hydrocarbon structure containing a sulfur atom at the other end.

That is, the ganglioside sugar chain-immobilized fluorescent nanoparticles according to the present invention also include fluorescent nanoparticles prepared by co-immobilizing oligoethylene glycol with the local density of the immobilized sugar chains. .

By further containing such a complex of an aminated oligoethylene glycol and the linker compound, the oligoethylene glycol can be immobilized on the fluorescent nanoparticle together with the sugar chain. The local density of the sugar chain immobilized on the nanoparticles can be suitably adjusted.

As a result, as shown in the examples described later, the detection sensitivity of the anti-ganglioside antibody can be increased. Therefore, detection of an anti-ganglioside antibody can be performed more easily.

The oligoethylene glycol is an alcohol obtained by dehydration polycondensation of 2 to 10 ethylene glycols. Of these, triethylene glycol, tetraethylene glycol, and pentaethylene glycol are preferably used, and tetraethylene glycol is particularly preferably used because it is easily available. The oligoethylene glycol used may be one type or two or more types.

A complex composed of an aminated oligoethylene glycol and a linker compound can be prepared by performing an amidation condensation reaction between the linker compound and the aminated oligoethylene glycol.

The linker compound has a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, and the re-main chain has a carboxyl group bonded to the aminated oligoethylene glycol at one end and a sulfur atom at the other end. It is preferable to have a hydrocarbon structure containing For example, a linker compound having a structure represented by the following general formula (31) can be used. The linker compound used may be one type or two or more types.

(Where n ¹ is an integer from 1 to 6)
A suitable molar ratio between the ganglioside sugar chain and the oligoethylene glycol is unclear because it varies depending on the type of ganglioside sugar chain used. For example, as shown in the Examples described later, when the ganglioside sugar chain is a GM1 sugar chain (Chemical Formula 14), the detection result was better when oligoethylene glycol was not included, but the ganglioside sugar chain was GM1. In the case of -Glc sugar chain (Chemical Formula 1), the best detection results are obtained when the molar ratio of GM1-Glc sugar chain to tetraethylene glycol is 5: 5.

Therefore, the molar ratio is preferably adjusted as appropriate depending on the type of ganglioside sugar chain used. As shown in the examples described later, when the ganglioside sugar chain is a GM1-Glc sugar chain, an aggregate that emits fluorescence even when the molar ratio of the ganglioside sugar chain to the oligoethylene glycol is 10: 0 to 3: 7. Since it is observed, it can be said that the molar ratio is an example of a suitable molar ratio.

(1-5. Fluorescent nanoparticles)
The fluorescent nanoparticle that is a constituent element of the sugar chain-immobilized fluorescent nanoparticle of the present embodiment is a layer in which particles (core) composed of first and second metal components are composed of first and third metal components ( It has a “core / shell” structure that is covered by a shell. As will be described later, since the fluorescent nanoparticles are heat-treated, the surface of the shell layer is homogenized, and the sugar chain ligand complex can be efficiently bound. As a result, on the fluorescent nanoparticles Can be provided with high stability.

The first metal component is preferably selected from the group consisting of cadmium, zinc, silver, indium and sulfur, and the second metal component is preferably selected from the group consisting of tellurium and sulfur. The third metal component is preferably selected from the group consisting of cadmium, sulfur and zinc. Examples of “core / shell” fluorescent nanoparticles that can be used in the present invention include, but are not limited to, CdTe / CdS and ZAIS / ZnS. ZAIS means Zn, Ag, In, and S.

As described above, the sugar chain-immobilized fluorescent nanoparticle disclosed in the present invention has a core / shell type quantum dot composed of Cd and Te as its basic structure as a fluorescent nanoparticle. The case where nanoparticles generally referred to as ZAIS composed of Ag, In, and S are also included in the applicable range.

In one aspect, the method for producing a fluorescent nanoparticle comprises mixing a first solution containing a first metal component and a second solution containing a second metal component under heating conditions, And the second solution is cooled to room temperature, and the particles composed of the first and second metal components are purified from the mixed solution. The solution mixed in the first step is the third metal component. In addition, the particles composed of the first and second metal components are covered with the layer composed of the first and third metal components.

In another aspect, the method for producing a fluorescent nanoparticle comprises mixing a first solution containing a first metal component and a second solution containing a second metal component under heating conditions, By cooling the mixed solution of the first solution and the second solution to room temperature, purifying the particles composed of the first and second metal components from the mixed solution, and heat-treating the aqueous solution containing the purified particles and the third metal component The particles composed of the first and second metal components are covered with the layer composed of the first and third metal components.

By performing the heat treatment after purifying the particle core, the homogenization of the surface of the shell layer is further improved, and the binding property of the sugar chain ligand complex to the fluorescent nanoparticle is remarkably improved. In this case, the heat treatment performed before purification is preferably performed for 30 minutes to 8 hours. As a result, sugar chain-immobilized fluorescent nanoparticles having a uniform particle size and high water dispersibility can be obtained.

In this specification, “room temperature” is a temperature in a normal laboratory and is preferably 20 to 30 ° C., but is not particularly limited as long as it is a temperature at which the reaction in the heat treatment can be stopped. It may be the temperature in the chamber (for example, 4 ° C.).

In the present embodiment, the first solution is a solution in which the salt or complex salt of the first metal component is dissolved, and the second solution is hydrophilic even if the salt or complex salt of the second metal component is dissolved. A solution in which the second metal component is dissolved may be used.

(1-6. Method for producing sugar chain-immobilized fluorescent nanoparticles)
The sugar chain-immobilized fluorescent nanoparticles (ganglioside sugar chain-immobilized fluorescent nanoparticles) according to the present invention can be produced as follows. Here, a case will be described in which the sugar chain represented by Chemical Formula 1 (GM1-Glc sugar chain) is used as the ganglioside sugar chain and the linker compound represented by the formula (30) is used as the linker compound. Also when using other linker compounds, sugar chain-immobilized fluorescent nanoparticles can be prepared according to the following method.

First, a ganglioside sugar chain structure having a protecting group (a sugar chain represented by Chemical Formula 14 having a protecting group) is obtained by chemical synthesis, and further, glucose protected at positions 1, 2, 3, and 4 is reacted. Finally, the protecting group is removed to synthesize a ganglioside sugar chain (sugar chain represented by Chemical Formula 1) having glucose at the reducing end.

Next, a uniquely developed fluorescent linker compound (Non-patent Document 8) represented by the formula (30) is introduced into the sugar chain by a reductive amination reaction, and the fact that the fluorescent linker compound exhibits fluorescence is utilized. To obtain a sugar chain ligand complex (ganglioside sugar chain ligand complex). The sugar chain ligand complex is immobilized on a core / shell type fluorescent nanoparticle by an existing method (Patent Document 1, Non-Patent Document 7) and purified using a centrifugal ultrafiltration method. Sugar chain-immobilized fluorescent nanoparticles can be produced.

Here, the existing method will be described. The sugar chain-immobilized fluorescent nanoparticles according to the present invention can be obtained by mixing a solution containing hydrophilic fluorescent nanoparticles subjected to heat treatment with the reducing agent-treated sugar chain ligand complex. Each S atom of the S—S bond of the ligand complex is bound by a metal-sulfur bond to a metal on the fluorescent nanoparticle. The resulting sugar chain-immobilized fluorescent nanoparticles are dispersed in an aqueous solution.

Specifically, for example, by adding a solution containing the above-mentioned sugar chain ligand complex treated with a reducing agent to a solution of fluorescent nanoparticles, the S—S bond of the sugar chain ligand complex is converted into a fluorescent nanoparticle. By converting the metal-sulfur bond on the particles, sugar chain-immobilized fluorescent nanoparticles can be obtained.

In addition, when the above-described complex of the aminated oligoethylene glycol and the linker compound is further contained, a solution containing the complex may be further added.

The conditions for the heat treatment of the hydrophilic fluorescent nanoparticles are not particularly limited, but are preferably performed at 50 to 200 ° C. in the presence of a thiol stabilizer, and 70 to 180 ° C. Is more preferable, and it is more preferable that it is 100 degreeC or more. The thiol stabilizer is not particularly limited, and examples thereof include thio compounds such as thioacetamide, 3-mercaptopropionic acid (3-MPA), thioglycolic acid (TGA), 4-mercaptobutanoic acid, cysteine, cystamine, and salts. It is done.

The reducing agent used for the reducing agent treatment is not particularly limited, and examples thereof include salts such as sodium borohydride and sodium cyanoborohydride, and salts having different cation components.

The solvent used in the solution containing the fluorescent nanoparticles and the sugar chain ligand complex is not particularly limited, and examples thereof include water, methanol, ethanol, propanol, and mixed solvents thereof.

Purification of the sugar chain-immobilized fluorescent nanoparticles can be performed, for example, by centrifugally filtering the sugar chain-immobilized fluorescent nanoparticles obtained by the above mixing and removing components such as low-molecular salts, in a solution state. And stable sugar chain-immobilized fluorescent nanoparticles can be obtained.

The mixing ratio of the metal nanoparticles used to prepare the sugar chain-immobilized fluorescent nanoparticles, the above-mentioned sugar chain ligand complex, and the reducing agent is not particularly limited, but when cadmium is included as a metal component The cadmium concentration in the solution containing the fluorescent nanoparticle and the sugar chain ligand complex is preferably 0.1 mM to 1 mM in the final concentration.

The concentration of the sugar chain ligand complex is preferably 0.1 mM to 10 mM as the final concentration in the solution containing the fluorescent nanoparticles and the sugar chain ligand complex.

In addition, when the above-described complex of aminated oligoethylene glycol and the linker compound is used, the concentration of the complex is preferably 0.1 mM to 10 mM as the final concentration in the solution.

The concentration of the reducing agent used is preferably 10 times the molar concentration of the sugar chain ligand complex as the final concentration in the solution.

Regarding the fact that sugar chains are immobilized on the fluorescent nanoparticles, for example, the sugar chains-immobilized fluorescent nanoparticles may be measured with a MALDI-TOF type mass spectrometer. At this time, the glycoside sugar chain ligand is reduced because the sugar chain ligand complex (ganglioside sugar chain ligand complex) immobilized with the fluorescent nanoparticles and sulfur-metal bond is reductively decomposed by sulfur-metal bond. When the complex is immobilized on the fluorescent nanoparticles, a molecular ion peak (m / Z) corresponding thereto is observed.

In addition, the ganglioside sugar chain-immobilized fluorescent nanoparticles are aggregated and centrifuged using a lectin (sugar chain-binding protein) that is known to specifically bind to the sugar chains that constitute the ganglioside sugar chain. What is necessary is just to investigate precipitation production.

That is, the above lectin and a solution containing fluorescent nanoparticles immobilized with sugar chains are mixed, and the sugar chains and proteins are allowed to interact and specifically bind to each other to confirm the formation of aggregates. It can be confirmed that sugar chains are immobilized on the nanoparticles.

Examples of the lectin include concanavalin A (ConA) and lentil lectin (LCA) which are proteins capable of recognizing glucose when the sugar chain located at the end of the sugar chain-immobilized fluorescent nanoparticle is glucose. Pea lectin (PSA) and the like can be used.

Similarly, when the sugar chain located at the end of the sugar chain-immobilized fluorescent nanoparticle is galactose, a bean lectin (RCA120), which is a protein that recognizes galactose, can be used. When the sugar chain located at the end of the sugar chain-immobilized fluorescent nanoparticle is N-acetylglucosamine, wheat germ lectin (WGA), which is a protein that recognizes N-acetylglucosamine, can be used.

Since the sugar chain-immobilized fluorescent nanoparticles produced by such a production method can easily immobilize antibodies on the surface thereof, antibody-immobilized fluorescent nanoparticles with improved specificity for living tissues can be easily obtained. Can be provided.

[2. (Method for detecting anti-ganglioside antibody)
The method for detecting an anti-ganglioside antibody according to the present invention was performed by mixing the sugar chain-immobilized fluorescent nanoparticle according to the present invention and a specimen, thereby immobilizing the sugar chain-immobilized fluorescent nanoparticle. The method includes a step of reacting a sugar chain containing a ganglioside-derived sugar chain with an anti-ganglioside antibody contained in the subject.

As described above, one or more ganglioside sugar chains are immobilized on the sugar chain-immobilized fluorescent nanoparticles. Therefore, when an anti-ganglioside antibody is contained in the subject, formation of an aggregate (aggregate) can be visually confirmed by an antigen-antibody reaction between the sugar chain and the antibody. At that time, the fluorescent color of the solution changes.

Therefore, the subject is preferably blood or serum, and particularly preferably serum. The blood or serum may be derived from a human or a mammal other than a human.

The mixing of the sugar chain-immobilized fluorescent nanoparticles with the analyte is performed by bringing the solution containing the sugar chain-immobilized nanoparticles close to the analyte, and the antigen-antibody reaction between the ganglioside sugar chain and the anti-ganglioside antibody. As long as it can provide a possible situation. For example, mixing can be performed by preparing a dilution series of a specimen on a microplate, a plastic tube, or the like, and adding a solution containing the sugar chain-immobilized fluorescent nanoparticles and leaving it to stand.

The above “solution containing a sugar chain-immobilized fluorescent nanoparticle” is intended to be a colloidal solution in which the sugar chain-immobilized fluorescent nanoparticle according to the present invention is dispersed in a liquid. As long as it contains sugar chain-immobilized fluorescent nanoparticles, other salts may be included. As the liquid, for example, water or a buffer solution can be used.

By performing the above mixing, if the specimen contains an anti-ganglioside antibody against a ganglioside sugar chain immobilized on a sugar chain-immobilized fluorescent nanoparticle, an antigen-antibody reaction occurs and emits fluorescence. Aggregates (aggregates) can be obtained.

For example, after the prepared ganglioside sugar chain-immobilized fluorescent nanoparticles (fluorescent nanoparticles with ganglioside sugar chains immobilized) are diluted with a buffer solution, patient serum is added and mixed in a plastic tube and left for several hours. Centrifuge. When autoantibodies that bind to the sugar chains of the ganglioside sugar chain-immobilized fluorescent nanoparticles are present in the serum, a fluorescent precipitate is obtained, so that the test result can be obtained easily and visually.

Therefore, it is possible to quickly and easily detect whether or not the anti-ganglioside antibody is suffering from immune peripheral neuropathy considered to be a pathogenic substance.

[3. Detection reagent for immune peripheral neuropathy
The ganglioside sugar chain-immobilized fluorescent nanoparticles of the present invention can be used as a rapid and simple detection reagent and diagnostic agent for terminal nerve paralysis such as Guillain-Barre syndrome.

Therefore, the detection reagent for immune peripheral neuropathy according to the present invention contains the sugar chain-immobilized fluorescent nanoparticles according to the present invention.

As described above, the sugar chain-immobilized fluorescent nanoparticle according to the present invention includes the above-described fluorescent nanoparticle, wherein a sugar chain ligand complex comprising one or more ganglioside sugar chains and the above-described linker compound. It is fixed to particles. Therefore, an anti-ganglioside antibody (autoantibody) in serum that binds to the ganglioside sugar chain can be detected quickly, simply, and with high accuracy.

Therefore, the reagent containing the sugar chain-immobilized fluorescent nanoparticles according to the present invention can be used as a detection reagent or diagnostic agent for immune peripheral neuropathy including Guillain-Barre syndrome.

Since the sugar chain-immobilized fluorescent nanoparticles are preferably used as a colloidal solution as described above, the detection reagent is in the form of a colloidal solution in which the sugar chain-immobilized fluorescent nanoparticles according to the present invention are dispersed in a liquid. Preferably there is. In the detection reagent for immune peripheral neuropathy according to the present invention, components other than the sugar chain-immobilized fluorescent nanoparticles according to the present invention include, for example, water, a buffer solution, and the aminated oligoethylene described above. Examples include a complex of glycol and the above linker compound.

In the colloid solution, the concentration of the sugar chain ligand complex constituting the sugar chain-immobilized fluorescent nanoparticle is preferably 0.1 mM to 10 mM as the final concentration in the solution as described above. In addition, the mixing ratio of the metal nanoparticles used for preparing the sugar chain-immobilized fluorescent nanoparticles, the sugar chain ligand complex, and the reducing agent is not particularly limited, but cadmium is included as a metal component. In this case, the cadmium concentration in the solution is preferably 0.1 mM to 1 mM in the final concentration. The concentration of the reducing agent used is preferably 10 times the molar concentration of the sugar chain ligand complex as the final concentration in the solution.

The present invention can also be configured as follows.

The ganglioside sugar chain-immobilized fluorescent nanoparticle according to the present invention is composed of metal nanoparticles emitting fluorescence composed of ganglioside GM1 sugar chain, cadmium and tellurium.

The method for detecting an autoantibody according to the present invention detects an autoantibody that reacts specifically with an immobilized ganglioside sugar chain by mixing the ganglioside sugar chain-immobilized fluorescent nanoparticles and patient serum. .

The ganglioside sugar chain-immobilized fluorescent nanoparticles according to the present invention are composed of ganglioside GM1 sugar chains and metal nanoparticles that emit fluorescence composed of zinc, silver, indium, and sulfur.

The method for detecting an autoantibody according to the present invention detects an autoantibody that reacts specifically with an immobilized ganglioside sugar chain by mixing the ganglioside sugar chain-immobilized fluorescent nanoparticles and patient serum. .

The ganglioside sugar chain-immobilized fluorescent nanoparticle according to the present invention is composed of metal nanoparticles emitting fluorescence composed of ganglioside GD1a sugar chain, cadmium and tellurium.

The method for detecting an autoantibody according to the present invention detects an autoantibody that reacts specifically with an immobilized ganglioside sugar chain by mixing the ganglioside sugar chain-immobilized fluorescent nanoparticles and patient serum. .

The ganglioside sugar chain-immobilized fluorescent nanoparticles according to the present invention are composed of ganglioside GD1a sugar chains and metal nanoparticles that emit fluorescence composed of zinc, silver, indium, and sulfur.

The method for detecting an autoantibody according to the present invention detects an autoantibody that reacts specifically with an immobilized ganglioside sugar chain by mixing the ganglioside sugar chain-immobilized fluorescent nanoparticles and patient serum. .

The ganglioside sugar chain-immobilized fluorescent nanoparticle according to the present invention is composed of metal nanoparticles emitting fluorescence composed of ganglioside GQ1b sugar chain, cadmium and tellurium.

The method for detecting an autoantibody according to the present invention detects an autoantibody that reacts specifically with an immobilized ganglioside sugar chain by mixing the ganglioside sugar chain-immobilized fluorescent nanoparticles and patient serum. .

The ganglioside sugar chain-immobilized fluorescent nanoparticles according to the present invention are composed of ganglioside GQ1b sugar chains and metal nanoparticles that emit fluorescence composed of zinc, silver, indium, and sulfur.

The method for detecting an autoantibody according to the present invention detects an autoantibody that reacts specifically with an immobilized ganglioside sugar chain by mixing the ganglioside sugar chain-immobilized fluorescent nanoparticles and patient serum. .

The detection reagent or diagnostic agent for immune peripheral neuropathy including Guillain-Barre syndrome according to the present invention includes the sugar chain or nanoparticles according to the present invention.

In the present invention, based on the above knowledge, fluorescent nanoparticles were prepared by immobilizing the sugar chain part of ganglioside with the aim of developing a simple tool for a new GBS diagnostic test method.

The present invention includes the following 1) to 3) as medically or industrially useful methods and substances.
1) A sugar chain ligand complex having a structure in which a fluorescent linker is bound to the sugar chain part of ganglioside GM1.
2) A fluorescent nanoparticle having the above-mentioned sugar chain ligand complex immobilized thereon, having an average particle size of 8.9 nm.
3) The fluorescent nanoparticle is a semiconductor nanoparticle having cadmium and tellurium having a core-shell structure, or zinc, silver, indium, or sulfur as constituent elements.

The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention. Furthermore, a new technical feature can be formed by combining the technical means disclosed in each embodiment.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.

[Example 1: Synthesis of GM1-Glc sugar chain]
FIG. 1 is a diagram showing the chemical structure of a GM1-Glc sugar chain in which Glc is introduced into the sugar chain part of ganglioside GM1. Formula 1 shown in FIG. 1 shows the structure of the target GM1-Glc sugar chain.

FIG. 2 is a diagram showing an intermediate synthesis route when a GM1-Glc sugar chain is synthesized. First, as shown in FIG. 2, a tetrasaccharide structure (shown in Formula 4) serving as a GM1-core is converted into a disaccharide Galβ1-3GalN (shown in Formula 2) and NeuAcα2-3Gal (Formula 3) in the presence of NIS and TfOH. In a yield of 89%. Subsequently, the corresponding glucoside donor (shown in Formula 6) was derived from the tetrasaccharide structure in 5 steps.

FIG. 3 is a diagram showing a synthesis route of a disaccharide structure necessary for synthesizing a GM1-Glc sugar chain. Next, in accordance with FIG. 3, gentibiose (represented in Formula 11) was made reactive at the 4-position hydroxyl group by making the 4-position of the non-reducing terminal glucose serving as a sugar acceptor free and the protecting group be a benzyl group. Was prepared as follows.

That is, a glucose sugar donor (shown in Formula 7) and a sugar acceptor (shown in Formula 8) are reacted at 0 ° C. in dichloromethane in the presence of NIS and TfOH to produce a disaccharide (shown in Formula 9). ) Was obtained in 90% yield. The disaccharide protecting group represented by Formula 9 was converted from a benzoyl group to a benzyl group, and the sugar chain represented by Formula 10 was obtained in two steps with a yield of 88%. Then, the benzylidene group was selectively cleaved by treating with trimethylsilane and BF ₃ · OEt _{2 in} dichloromethane to obtain gentibiose represented by Formula 11 in a yield of 85%. The spectrum data of the gentibiose is as follows.
[Α] _D = −12.9 ° (c 1.0, CHCl ₃ ); ¹ H-NMR (600 MHz, CDCl ₃ ) δ 7.35-7.21 (m, 35 H, 7 Ph), 5 .01-4.69 (m, 10 H, 5 CHHPh), 4.59-4.51 (m, 3 H, H-1f, CHHPh), 4.46 (d, 1 H, J _1,2 = 9.6 Hz, H-1e), 4.19 (d, 1 H, J _gem = 11.0 Hz, H-6e), 3.74-3.58 (m, 6 H, H-6'e 3f, 4f, 5f, 6f, 6′f), 3.50-3.39 (m, 5 H, H-2f, 2e, 3e, 4e, 5e), 2.54 (s, 1 H, − OH); ¹³ C-NMR (150 MHz, CDCl ₃ ) δ 138.9, 138.7, 138.5, 138.5, 13 8.2, 138.0, 137.6, 128.6, 128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 128.0, 127. 9, 127.8, 127.7, 104.1, 102.7, 84.8, 84.2, 82.4, 81.6, 78.4, 77.3, 75.8, 75.4, 75.3, 75.1, 74.9, 74.8, 74.1, 73.8, 71.8, 71.3, 68.8, 29.8; MALDI MS: m / z: calcd for C ₆₁ H ₆₄ O ₁₁ Na: 995.43; found: 995.38 [M + Na] ⁺ .
FIG. 4 is a diagram showing a pathway showing glycosylation and deprotection for synthesizing the GM1-Glc sugar chain. Next, as shown in FIG. 4, the GM1 core sugar donor (shown in Formula 6) synthesized as described above and a disaccharide (shown in Formula 11) are reacted in dichloromethane in the presence of TMSOTf. The desired β-glycoside (shown in Formula 12) was obtained in 69% yield.

Next, the acyl-based protecting group was removed to obtain a sugar chain represented by Formula 13, and finally the benzyl group was subjected to hydrogenolysis to obtain a GM1-Glc sugar chain (Formula 1) almost quantitatively. The spectrum data of the GM1-Glc sugar chain shown in Formula 1 are as follows.
[Α] _D = + 0.1 ° (c 1.0, H ₂ O); ¹ H-NMR (600 MHz, CD ₃ OD) δ 5.16 (d, 1 H, J _1,2 = 3.7 Hz, H-1e), 4.79 (d, 1 H, H-1c), 4.57 (d, 1 H, J _1,2 = 8.0 Hz, H-1d), 4.49-4 .45 (m, 3 H, H-1a, 1b, 1f), 4.15-3.19 (m, 39 H, ring H), 2.62 (dd, 1 H, H-3beq), 99 and 1.96 (2 s, 6 H, 2 Ac), 1.87 (m, 1 H, H-3bax), ¹³ C-NMR (150 MHz, CD ₃ OD) δ 175.0, 174.8 , 174.1, 106.1, 105.7, 105.5, 104.1, 104.0, 103.4, 101.8, 9 7.0, 95.3, 94.2, 93.0, 91.5, 84.4, 81.2, 78.1, 77.6, 76.5, 75.1, 74.8, 74. 5, 74.3, 73.9, 73.5, 73.4, 72.6, 72.1, 71.5, 70.8, 70.2, 68.9, 68.0, 67.1, 61.2, 61.0, 60.7, 59.7, 59.3, 58.8, 52.5, 51.6, 48.8, 47.5, 28.7, 25.9, 23. _{5; MALDI MS: m / z} : calcd for C 43 H 72 N 2 O: 1160.40; found: 1159.75 [M-H] -.
[Example 2: Synthesis and fractionation of GM1-Glc-immobilized sugar chain ligand complex]
The GM1 ganglioside sugar chain (GM1-Glc, 1.0 mg, 0.86 μmol) having 6-glucose at the reducing end synthesized in Example 1 was dissolved in 20 μL of ultrapure water, and the original development shown in the above formula (30) To a 30 μL solution of N, N-dimethylformamide in a fluorescent linker compound (referred to as “f-mono”, 0.28 mg, 1.1 μmol), and further 6 μL of acetic acid was added.

After standing at 40 ° C. for 6 hours, a solution prepared by dissolving NaBH ₃ CN (1.5 mg, 24 μmol) in 10 μL of ultrapure water was added, left at 40 ° C. for 3 days, and lyophilized. This lyophilized residue was dissolved in ultrapure water and purified with an ODS column (elution solvent: water / methanol 1/1 (v / v)). The eluted fraction was lyophilized to obtain a GM1-Glc-immobilized sugar chain ligand complex (hereinafter abbreviated as “GM1-Glc-f-mono”) shown in FIG. 5 as a white powder. FIG. 5 shows the chemical structure of GM1-Glc-f-mono. The yield of GM1-Glc-f-mono was 0.69 mg (yield 56%). The spectrum data of GM1-Glc-f-mono is as follows.
MS calcd. for: C ₅₆ H ₉₂ N ₅ O ₃₄ S ₂ : 1442.51, Found: m / z 1442.73 [M−H] ⁻ .
[Example 3: Preparation of fluorescent nanoparticles having immobilized GM1-Glc sugar chains]
FIG. 6 is a schematic view showing a method for preparing GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP).

First, CdCl ₂ (9.17 mg, 50.0 μmol) and 3-mercaptopropionic acid (3-MPA, 5.45 μL, 63 μmol) were dissolved in 10 mL of ultrapure water, and the pH of the solution was adjusted to 9 with 1M NaOH. After bubbling argon gas for 30 minutes with stirring, the solution was heated to 105 ° C. with vigorous stirring.

In a separate flask, tellurium powder (16.0 mg, 0.125 mmol) and NaBH ₄ (18.9 mg, 0.500 mmol) were dissolved in ultrapure water (2 mL) degassed under argon, and at room temperature. Stir for 1.5 hours. The obtained solution (NaHTe solution, 200 μL) was added to the above solution heated and stirred at 105 ° C., stirred at the same temperature for further 2 hours, and returned to room temperature.

2-Propanol was added to the solution to precipitate CdTe quantum dots (QD), which was filtered and then added again to dissolve it. The solution was allowed to stand at 4 ° C. for 10 hours, after which thioacetamide (0.27 μL, 1.33 μM) was further added.

Then, the mixture was stirred at 105 ° C. for 57 hours and returned to room temperature to obtain a CdTe / CdS core / shell QD solution.

Next, as shown in FIG. 6, GM1-Glc-f-mono (1 mM, 50 μL) prepared in Example 2 was mixed with an aqueous solution of NaBH ₄ (10 mM, 50 μL) at room temperature and allowed to stand for 10 minutes. Thereafter, 100 μL of a solution obtained by diluting the CdTe / CdS core / shell QD solution 5 times with ultrapure water was added to the solution allowed to stand for 10 minutes and stirred at room temperature in the dark for 24 hours. Unreacted sugar-ligand complex was removed by centrifugal ultrafiltration (14000 × g, 5 min) using Amicon Ultra 10 K (Millipore, MA, USA), followed by washing with ultrapure water three times. A GM1-Glc sugar chain-immobilized fluorescent nanoparticle (GM1-Glc-FNP) solution was prepared by finally suspending the precipitate in PBS.

FIG. 7 is a graph showing the particle size distribution of GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP) by DLS measurement. As shown in FIG. 7, according to the DLS measurement (device used: Zetasizer Nano ZS90, Malvern Instruments, Worcestershire, UK), the average particle diameter of the nanoparticles (GM1-Glc-FNP) was 8.9 nm.

[Example 4: Confirmation of immobilized ganglioside sugar chain ligand complex by MALDI-TOF / MS]
1 μL of PBS solution of GM1-Glc sugar chain-immobilized fluorescent nanoparticles prepared in Example 3 was mixed with 10 μL of saturated DHBA solution (water / methanol 1/1 solution), and 1 μL was placed on a measurement plate and allowed to air dry. . The plate was put into the measuring section of Voyager-DE-PRO (Applied Biosystems, CA, USA) and subjected to mass spectrometry.

FIG. 8 is a diagram showing a result of MALDI-TOF / MS analysis by POS1-mode of GM1-Glc-FNP, and FIG. 9 shows a result of MALDI-TOF / MS analysis by Negative mode of GM1-Glc-FNP. FIG.

As shown in FIGS. 8 and 9, a peak (m / Z value) having the same mass number as that of the GM1-Glc-immobilized sugar chain ligand complex prepared in Example 2 was obtained. It was confirmed that the Glc sugar chain was immobilized.

[Example 5: Confirmation of protein binding ability of GM1-Glc sugar chain-immobilized fluorescent nanoparticles]
Protein Concanavalin A (ConA), Jaccalin, Peant agglutinin (PNA), Ricin communis agglutinin I (RCA120), Bovine serum albumin (BSA), and GM1-Glc sugar chain-immobilized fluorescent nanoparticle immobilized fluorescent protein . Other than BSA, it is a sugar chain binding protein (lectin).

ConA, RCA120, Jaccalin and BSA were dissolved in PBS to a concentration of 10 μM and PNA at a concentration of 3.6 μM, respectively, and 5 μL thereof was transferred to a 200 μL capacity plastic tube.

Thereto, 5 μL of 0.2 μM PBS solution of GM1-Glc sugar chain-immobilized fluorescent nanoparticles prepared in Example 3 was added, stirred with a vortex mixer, and left in a dark place at 4 ° C. for a certain time (12 hours). And centrifuged at 14000 G for 5 minutes. The tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 10 is a diagram showing the results of fluorescence emission of precipitates after centrifugation in an aggregation experiment between GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP) and a sugar chain-binding protein; FIG. 11 is a diagram showing the fluorescence spectrum of the supernatant after the centrifugation.

As shown in FIG. 10, only PNA produced a precipitate emitting fluorescence that was visually distinguishable. Moreover, as shown in FIG. 11, only the fluorescence intensity of the supernatant of PNA significantly decreased. 10 and 11, it can be seen that the sugar chains of the GM1-Glc sugar chain-immobilized fluorescent nanoparticles specifically bound to PNA, and as a result, formed aggregates.

[Example 6: Reaction of GM1-Glc sugar chain-immobilized fluorescent nanoparticles with patient serum]
5 μL of serum from patients with immune peripheral neuropathy, including patients with Guillain-Barre syndrome, was transferred to a plastic tube with a volume of 200 μL. Thereto was added 5 μL of a 0.1 μM PBS solution of GM1-Glc sugar chain-immobilized fluorescent nanoparticles prepared in Example 3, and the mixture was stirred with a vortex mixer and left in a dark place at 4 ° C. for a certain time (12 hours). And centrifuged at 14000 G for 5 minutes. The tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 12 shows the results of fluorescence emission of precipitates after centrifugation in an aggregation experiment between GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP) and sera of patients with immune peripheral neuropathy. FIG. FIG. 13 shows the results of centrifugation at different incubation times in an aggregation experiment between GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP) and sera of patients with immune peripheral neuropathy. It is a figure which shows the result of fluorescence emission of the subsequent deposit.

The numbers in the figure are sample numbers, 13912 and 13923 are the results of using anti-GM1 antibody negative and anti-GD1a antibody negative sera by ELISA, and 13882 and 13934 are anti-GM1 antibody negative and anti-antigen by ELISA. 14078, 14134, 14151, 14192, and 14614 show the results using GD1a antibody-positive sera, and the results using anti-GM1 antibody-positive sera by ELISA, respectively.

As shown in FIG. 12, formation of a fluorescent precipitate was confirmed only when anti-GM1 antibody-positive serum was used in the existing ELISA method, and the fluorescence intensity of the supernatant of the tube was significantly reduced. On the other hand, in the case of anti-GM1 antibody-negative and anti-GD1a antibody-negative sera and anti-GM1 antibody-negative and anti-GD1a antibody-positive sera, formation of a fluorescent precipitate is not confirmed, and the solution in the tube There was no change in the fluorescence intensity. This change (formation of the precipitate) was confirmed 3 hours after mixing the GM1-Glc sugar chain-immobilized fluorescent nanoparticles into the serum sample as shown in FIG.

[Example 7: Identification of protein in patient serum formed aggregate with GM1-Glc sugar chain-immobilized fluorescent nanoparticles]
The presence of the antibody in the fluorescent precipitate obtained in Example 6 was confirmed by SDS-PAGE of the precipitate. The precipitate obtained using the serum of sample number 14151 was washed three times with PBS, the precipitate was collected and dispersed again in PBS, and SDS-PAGE sample preparation buffer (reducing conditions and non-reducing conditions) Was applied to 10% polyacrylamide gel under reducing conditions and 8% polyacrylamide gel under non-reducing conditions, and silver stained after electrophoresis.

FIG. 14 shows the result of silver staining using an SDS buffer containing 2-mercaptoethanol as the sample preparation buffer. FIG. 15 shows an SDS buffer containing no 2-mercaptoethanol as the sample preparation buffer. The results of silver staining using are shown. In the figure, GM1-Glc-FNP is the GM1-Glc sugar chain-immobilized fluorescent nanoparticle, ppt is the precipitate obtained in Example 6, Serum sample is the result of using the serum of sample number 14151, The left lane is a molecular weight marker.

14 and 15, protein bands corresponding to IgG heavy chain, light chain, and Fc were observed, and it was found that GM1-Glc sugar chain-immobilized fluorescent nanoparticles were selectively bound to antibodies in serum. .

The precipitate was also subjected to western blotting. As an antibody, goat anti-human IgG (heavy chain and light chain) HRP complex was used. FIG. 16 is a diagram showing the result of Western blotting confirming the presence of the antibody in the fluorescent precipitate obtained in Example 6. In the figure, ppt indicates the result obtained by using the precipitate obtained in Example 6, and Serum sample indicates the serum of sample number 14151. FIG. 16 also shows that the sugar chain-immobilized fluorescent nanoparticles are selectively bound to the serum antibodies.

[Example 8: Competitive inhibition using GM1 sugar chain and GM1-Glc sugar chain]
The detection of anti-GM1 antibody when the GM1 sugar chain represented by Chemical Formula 14 and the GM1-Glc sugar chain were simultaneously present in the serum sample was examined.

Serum 2.5 μL of sample No. 14151 used in Example 5 was placed in a 200 μL plastic tube, and 2.5 μL of GM1 sugar chain in PBS (0 mM, 1 mM, 2 mM, 4 mM, 8 mM, 16 mM) was carried out. 5 μL of GM1-Glc-FNP in PBS (0.1 μM) prepared in Example 3 was added, stirred with a vortex mixer, allowed to stand at 4 ° C. in the dark for 6 hours, and centrifuged at 14000 G for 5 minutes. . Next, the tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 17 is a diagram showing the results of fluorescence emission of precipitates after centrifugation when GM1 sugar chains and GM1-Glc sugar chains are simultaneously present in a serum sample. FIG. 18 is a diagram showing a fluorescence spectrum of a supernatant after centrifugation in the case where a GM1 sugar chain and a GM1-Glc sugar chain are simultaneously present in a serum sample.

As shown in FIG. 17, the size of the aggregates decreased as the concentration of the GM1 sugar chain increased. Further, as shown in FIG. 18, it was found that the fluorescence spectrum of the supernatant increases as the concentration of the GM1 sugar chain increases.

These results indicate that competitive inhibition occurs when GM1 sugar chain and GM1-Glc-FNP are simultaneously present in serum, and that anti-ganglioside antibody binds to GM1 sugar chain of GM1-Glc-FNP. Is clear. However, as shown in FIG. 17, since the aggregate has been confirmed, it is possible to detect an anti-ganglioside antibody even if GM1 sugar chain and GM1-Glc-FNP are present in the serum at the same time. It can be said that.

[Example 9: Preparation of fluorescent nanoparticles on which tetraethylene glycol and GM1-Glc sugar chains are immobilized]
FIG. 19 shows a method for preparing fluorescent nanoparticles (hereinafter referred to as “TEG-containing GM1-Glc-FNP”) in which tetraethylene glycol (hereinafter also referred to as “TEG”) and GM1-Glc sugar chain are immobilized. FIG.

First, tetraethylene glycol is bonded to a linker compound in which n ^{1 of the} linker compound represented by the general formula (31) is 4 by the following method, and from aminated tetraethylene glycol and the linker compound, To obtain a complex (hereinafter referred to as “TEG-mono”).

That is, TEG is reacted with tetraethylene glycol and TsCl in the presence of pyridine in dichloromethane at 0 ° C. under argon to convert one end of the ethylene glycol chain to a tosyl group, and further to an azide group, whereby TEG -Mono precursor was obtained in two steps with a yield of 73%.

Then, the azide group was converted to an amino group by treatment with palladium in methanol in a hydrogen atmosphere, and stirred in argon in the presence of thioctic acid and DCC in dichloromethane for 6 hours to obtain FIG. The TEG-mono shown was obtained in 39% yield in two steps. The spectrum data of TEG-mono is as follows.
¹ H-NMR (600 MHz, CD ₃ OD) δ 3.73-3.59 (19H, m, H, —CH ₂ CH ₂ O—, —SSCH ₂ CH ₂ CH =), 3.17 (1H, ddd , -SSCH ₂ CH ₂ CH =), 3.11 (1H, ddd, -SSCH ₂ CH ₂ CH =), 2.50 (1H, dddd, -SSCH ₂ CH ₂ CH =), 2.18 (2H, t, —NHCOCH ₂ —), 1.91 (1H, dddd, —SSCH ₂ CH ₂ CH═), 1.75-1.62 (4H, m, —NHCOCH ₂ CH ₂ —, —NHCOCH ₂ CH ₂ CH _{2 CH 2 -), 1.52-1.41 (} 2H, m, -NHCOCH 2 CH 2 CH 2 -); ESI MS: m / z: calcd for C 16 H 31 NO 5 S 2: 381.16; found : 404.12 [M + Na] ⁺ .
In a mixed solution of GM1-Glc-f-mono (1 mM, 12.5 μL) prepared in Example 2 and the above TEG-mono (1 mM, 12.5 μL), an aqueous solution of NaBH ₄ (10 mM, 25 μL) was added at room temperature. Mix and leave for 10 minutes. In this case, the molar ratio of GM1-Glc to TEG is 5: 5. In addition, the molar ratio of GM1-Glc sugar chain to TEG was adjusted to 10: 0, 7: 3, 3: 7.

50 μL of a solution obtained by diluting the CdTe / CdS core / shell QD solution prepared in Example 3 five times with ultrapure water was added to the solution left standing for 10 minutes, and the mixture was stirred at room temperature in the dark for 24 hours. Unreacted glycan ligand complex was removed by performing centrifugal ultrafiltration (14000 × g, 5 min) using Amicon Ultra 10 K (Millipore, MA, USA) three times, and the precipitate was finally added to PBS. By suspension, a solution of fluorescent nanoparticles (TEG-containing GM1-Glc-FNP) in which GM1-Glc sugar chains and tetraethylene glycol were immobilized was prepared.

FIG. 20 is a schematic diagram showing the structure of TEG-containing GM1-Glc-FNP. By immobilizing TEG-mono in addition to GM1-Glc-f-mono, the density of sugar chains immobilized on the fluorescent nanoparticles can be controlled.

[Example 10: Aggregation experiment of serum sample using TEG-containing GM1-Glc-FNP]
In this example, the concentration of TEG with respect to the GM1-Glc sugar chain was changed, and an agglutination experiment of a serum sample using TEG-containing GM1-Glc-FNP was performed.

As serum samples, sample numbers 13923 and 13938 (anti-GM1 antibody-negative and anti-GD1a antibody-negative sera by ELISA method), 13934 (anti-GM1 antibody-negative and anti-GD1a antibody-positive serum by ELISA method), 14078, 14151 , 14192 (Serum positive for anti-GM1 antibody by ELISA) was used.

The 5 μL serum sample was transferred to a 200 μL plastic tube. Thereto was added 5 μL of a TEG-containing GM1-Glc-FNP PBS solution (0.1 μM) prepared in Example 9. The TEG-containing GM1-Glc-FNP was prepared so that the molar ratio of the GM1-Glc sugar chain to the TEG was 10: 0, 7: 3, 5: 5, 3: 7.

Next, the tube was stirred with a vortex mixer and then left in a dark place for 1 hour, followed by centrifugation at 14000 G for 5 minutes. Then, the tube was irradiated with ultraviolet light, and the fluorescence of the precipitate was observed to take a photograph.

FIG. 21 is a diagram showing the results of a serum sample agglutination experiment using TEG-containing GM1-Glc-FNP while changing the TEG concentration relative to the GM1-Glc sugar chain. (A) to (d) of FIG. 21 show the results when the molar ratio of GM1-Glc sugar chain to TEG is 10: 0, 7: 3, 5: 5, 3: 7, respectively. .

In FIG. 21 (a) using GM1-Glc-FNP without addition of TEG, no aggregates are observed in sample number 14078. On the other hand, as shown in FIGS. 21B and 21C, when the molar ratio of GM1-Glc sugar chain to TEG is 7: 3, 5: 5, aggregates are also observed in sample number 14078. ing. When the molar ratio was 3: 7, as shown in FIG. 21 (d), although aggregates were observed at 14078 and 14192, the fluorescence was weak.

Thus, it has been clarified that by adjusting the content of TEG, the detection of aggregates can be made easier, that is, the detection sensitivity of anti-ganglioside antibodies can be increased.

[Example 11: Relationship between antibody concentration and reaction time and aggregate detection sensitivity]
In this example, a dilution series of a serum sample was prepared and the reaction time between the serum sample and GM1-Glc-FNP was varied to examine the relationship between the antibody concentration and reaction time and the detection sensitivity of the aggregate.

Sample number 14152 was used as a serum sample. Dilute the sample with PBS and take 5 μL each of the dilution ratios of 1, 1/2, 1/4, 1/8, 1/16, 1/32 in a 200 μL capacity plastic tube. Moved.

Thereto, 5 μL of a TEG-containing GM1-Glc-FNP PBS solution (0.1 μM) prepared in Example 10 and having a molar ratio of GM1-Glc sugar chain to TEG of 5: 5 was added. The tube was stirred with a vortex mixer, allowed to stand in the dark at 4 ° C. for 1 hour or 12 hours, and centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light, and the fluorescence of the precipitate was observed to take a photograph.

FIG. 22 is a diagram showing the results of examining the relationship between antibody concentration and reaction time and aggregate detection sensitivity using TEG-containing GM1-Glc-FNP. 22A shows the result when the standing time is 1 hour, and FIG. 22B shows the result when the standing time is 12 hours. From FIG. 22, it can be seen that the aggregates increased depending on the antibody concentration and the reaction time, and as a result, the antibody detection sensitivity was improved.

[Example 12: Detection of anti-GM1 IgG antibody using TEG-containing GM1-Glc-FNP]
In this example, anti-GM1 IgG antibody detection by TEG-containing GM1-Glc-FNP was examined using 50 samples each of anti-GM1 antibody-positive sera by ELISA and anti-GM1 antibody-negative sera by ELISA.

Each serum sample was put into a plastic tube having a capacity of 15 μL and 200 μL, respectively, and the TEG-containing GM1-Glc-FNP PBS prepared in Example 10 and having a molar ratio of GM1-Glc sugar chain to TEG of 5: 5 was used. 15 μL of the solution (0.1 μM) was added. The tube was stirred with a vortex mixer, allowed to stand in the dark at 4 ° C. for 3 hours, and centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light and photographed.

FIG. 23 shows the detection results of anti-GM1 IgG antibody by TEG-containing GM1-Glc-FNP when 50 samples of anti-GM1 IgG antibody-positive serum were used by ELISA. FIG. 24 shows the results of detection of anti-GM1 IgG antibody by TEG-containing GM1-Glc-FNP when 50 samples of anti-GM1 IgG antibody-negative serum were used by ELISA. The numbers in the figure indicate sample numbers.

As shown in FIG. 23, among the 50 anti-GM1 antibody-positive sera, 37 samples circled in the figure confirmed the fluorescence of the precipitate. That is, the positive rate was 37/50 = 74%. On the other hand, as shown in FIG. 24, when anti-GM1 IgG antibody-negative serum was used, no fluorescence of the precipitate was confirmed. That is, the positive rate was 0% and the negative rate was 100%.

23 and 24, it can be seen that anti-GM1 IgG antibody in serum can be detected easily and with high sensitivity in a short time by TEG-containing GM1-Glc-FNP.

Next, the same experiment as in the above-described example was performed except that 15 μL of each of the 13 serum samples in which fluorescence was not confirmed in FIG. 23 was used and the standing time after stirring by the vortex mixer was set to 12 hours. Were irradiated with ultraviolet light and photographed.

FIG. 25 shows anti-GM1 antibody-positive sera obtained by ELISA, and when the mixture was allowed to stand for 3 hours after mixing with TEG-containing GM1-Glc-FNP, the serum of the precipitate was not confirmed. It is a figure which shows the detection result of the anti-GM1IgG antibody at the time of leaving still for a time.

As shown in FIG. 25, when the standing time is 3 hours, the fluorescence can be confirmed for 2 samples (sample numbers: 10511 and 10751) out of 13 samples in which the fluorescence of the precipitate could not be confirmed. It was.

[Example 13: Preparation of GM1 sugar chain-immobilized ligand complex and preparation of fluorescent nanoparticles having immobilized GM1 sugar chain]
The GM1 sugar chain represented by the above chemical formula 14 (purchased from Oligotech. 1.0 mg, 1.0 μmol) was dissolved in 60 μL of ultrapure water, and the fluorescent linker compound (f-mono, 1.. 98 mg, 6.65 μmol) of N, N-dimethylformamide in 90 μL solution, and further 18 μL of acetic acid was added.

After standing at 40 ° C. for 6 hours, a solution in which NaBH ₃ CN (0.63 mg, 10 μmol) was dissolved in 30 μL of ultrapure water was added, left at 40 ° C. for 3 days, and lyophilized. This lyophilized residue was dissolved in ultrapure water and purified with an ODS column (elution solvent: water / methanol 1/1 (v / v)). The elution fraction was lyophilized to obtain a GM1 sugar chain-immobilized sugar chain ligand complex (hereinafter abbreviated as “GM1-f-mono”) shown as Formula 32 in FIG. 26 as a white powder. FIG. 26 is a diagram showing a synthesis route of GM1-f-mono. The yield of GM1-f-mono was 0.37 mg (yield 28.9%). The spectrum data of GM1-f-mono is as follows.
MS calcd. for: C ₅₁ H ₈₃ N ₄ O ₂₉ S ₂ : 1278.45, Found: m / z 1277.82 [M−H] ⁻ .
Next, a GM1 sugar chain-immobilized fluorescent nanoparticle (GM1-FNP) solution was prepared by the same method as in Example 3 except that GM1-f-mono was used instead of GM1-Glc-f-mono.

[Example 14: Confirmation of protein binding ability of GM1-FNP]
Protein Concanavalin A (ConA), Wheat germ agglutinin (WGA), Sambucus nigra agglutinin (SNA), Peanut agglutinin (PNA), Ricin communis aglutin 120 The binding activity of the conductive nanoparticles was investigated.

As for the binding sugar chains of these proteins, ConA is αGlc, RCA120 is βGal, PNA is Galβ1-3GalNAC, WGA is GlcNAc, SNA is SA (sialic acid), and BSA has the ability to bind to sugar chains. Absent.

Each protein was dissolved in PBS at a concentration of 3.6 μM, and 5 μL thereof was transferred to a plastic tube having a capacity of 200 μL. Thereto was added 5 μL of a GM1 sugar chain-immobilized fluorescent nanoparticle (GM1-FNP) PBS solution (0.25 μM) prepared in Example 13 and stirred with a vortex mixer, and left in a dark place at 4 ° C. for 12 hours. And centrifuged at 14000 G for 5 minutes. The tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 27 is a diagram showing the fluorescence emission results of the precipitate after centrifugation in an aggregation experiment of GM1-FNP and a sugar chain binding protein, and FIG. 28 shows the fluorescence spectrum of the supernatant after the centrifugation. FIG.

As shown in FIG. 27, only when PNA was used, a precipitate emitting fluorescent light that was visually identifiable. Further, as shown in FIG. 28, only the fluorescence intensity of the supernatant of PNA greatly decreased. 27 and 28, it can be seen that the sugar chain of the GM1 sugar chain-immobilized fluorescent nanoparticle (GM1-FNP) specifically bound to PNA, and as a result, an aggregate was formed.

[Example 15: Reaction of GM1-FNP with patient serum]
5 μL of serum from patients with immune peripheral neuropathy, including patients with Guillain-Barre syndrome, was transferred to a plastic tube with a volume of 200 μL. Thereto was added 5 μL of a 0.1 μM PBS solution of GM1 sugar chain-immobilized fluorescent nanoparticles (GM1-FNP) prepared in Example 13, and the mixture was stirred with a vortex mixer and allowed to stand in the dark at 4 ° C. for 12 hours. And centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 29 shows the results of fluorescence emission of precipitates after centrifugation in an aggregation experiment between GM1 sugar chain-immobilized fluorescent nanoparticles (GM1-FNP) and sera of patients with immune peripheral neuropathy. . FIG. 30 is a diagram showing the results of measuring the fluorescence spectrum of the supernatant after the centrifugation.

The numbers in 5 digits in FIGS. 29 and 30 are sample numbers. 13923 and 13938 are the results of using anti-GM1 antibody negative and anti-GD1a antibody negative sera by ELISA, and 13934 is anti-GM1 antibody negative by ELISA. In addition, 14078, 14151 and 14192 show the results using anti-GD1a antibody-positive sera, and the results using anti-GM1 antibody-positive sera by the ELISA method, respectively.

As shown in FIG. 29, formation of a fluorescent precipitate was confirmed only when anti-GM1 antibody-positive serum was used in the ELISA method (14078, 14151 and 14192). As shown in FIG. 30, the fluorescence intensity of the supernatant when using 14078, 14151 and 14192 was greatly reduced.

Based on the above results, the case of using GM1 sugar chain-immobilized fluorescent nanoparticles (GM1-FNP) is the same as the case of using GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1-Glc-FNP). It was revealed that anti-GM1 antibody can be detected.

[Example 16: Comparison between sugar chain-immobilized fluorescent nanoparticles and sugar chain-immobilized gold nanoparticles]
In this example, PNA is used using sugar-chain immobilized fluorescent nanoparticles (SFNP) and sugar-chain immobilized gold nanoparticles (SGNP). Agglutination experiments were performed and the results were compared.

GM1-Glc sugar chain-immobilized fluorescent nanoparticles prepared in Example 3 (GM1-Glc-FNP), GM1 sugar chain-immobilized fluorescent nanoparticles (GM1-FNP) prepared in Example 13, and GM1 sugar chain Immobilized gold nanoparticles (GM1-GNP) were subjected to the experiment.

The GM1-GNP was prepared as follows. That is, an aqueous solution of HAuCl ₄ (1.25 mM, 80 μL) was transferred to a 1.5 mL plastic tube. Thereto was added an aqueous solution of NaBH ₄ (50 mM, 10 μL), and the mixture was stirred with a vortex mixer and allowed to stand for 10 minutes. GM1-f-mono (5 mM, 10 μL) prepared in Example 13 was added to the solution allowed to stand for 10 minutes and allowed to stand for 30 minutes. Unreacted sugar-ligand complex was removed by performing centrifugal ultrafiltration (14000 × g, 5 min) using Amicon Ultra 10 K (Millipore, MA, USA) three times, and the precipitate was finally added to PBS. By suspending, a solution of sugar chain-immobilized gold nanoparticles (GM1-GNP) in which the GM1 sugar chain was immobilized was prepared. The average particle diameter of the obtained GM1-GNP was measured by the DLS method and found to be 8.1 nm.

For each of the above GM1-Glc-FNP, GM1-FNP and GM1-GNP, TEG-containing GM1-Glc-FNP, TEG-containing GM1-FNP and TEG-containing GM1-GNP in which tetraethylene glycol was also immobilized were prepared. The preparation method is according to the method described in Example 9. The molar ratio of sugar chain to TEG was 10: 0, 7: 3, 5: 5, 3: 7, 1: 9. When the molar ratio is 10: 0, TEG is not contained. However, in this example, for convenience sake, this case is also referred to as “TEG-containing GM1-Glc-FNP” or the like hereinafter.

5 μL of TEG-containing GM1-Glc-FNP, TEG-containing GM1-FNP, and TEG-containing GM1-GNP in PBS (0.1 μM) were each taken and transferred to a 200 μL plastic tube. Thereto, 5 μL of PNA was added, and the tube was stirred with a vortex mixer and allowed to stand in the dark for 3 hours. PNA having a concentration of 0 μM, 0.11 μM, 0.225 μM, 0.45 μM, 0.9 μM, 1.8 μM, 3.6 μM was used.

Next, the tube containing TEG-containing GM1-GNP was centrifuged at 5000 G for 1 minute, and the other was centrifuged at 14000 G for 5 minutes. Then, the tube was irradiated with ultraviolet light, and the fluorescence of the precipitate was observed to take a photograph.

31, 32, and 33 show TEG-containing GM1-Glc-FNP (GM1-Glc: TEG = 5: 5), TEG-containing GM1-FNP (GM1: TEG = 10: 0), and TEG-containing GM1-GNP ( It is a figure which shows the result of mixing GM1: TEG = 5: 5) with PNA of the said density | concentration and confirming the aggregation of PNA. (A) of each figure shows the result which confirmed the fluorescence of the deposit, (b) of each figure is a figure which shows the change of the fluorescence intensity of a supernatant liquid.

When TEG-containing GM1-Glc-FNP (GM1-Glc: TEG = 5: 5) was used, fluorescence was detected when 0.11 μM or more of PNA was used ((a) in FIG. 31). When TEG-containing GM1-GNP (GM1: TEG = 5: 5) was used, fluorescence was detected when 0.45 μM or more of PNA was used (FIG. 32 (a)). When TEG-containing GM1-GNP (GM1: TEG = 5: 5) was used, fluorescence was detected when 1.8 μM or more of PNA was used ((a) in FIG. 33). Thus, it was confirmed that the binding properties of TEG-containing GM1-Glc-FNP, TEG-containing GM1-GNP, and TEG-containing GM1-GNP to PNA are different.

A similar experiment was performed by changing the standing time from 3 hours to 12 hours. Figure fluorescence was confirmed in the sediment are not shown, as shown in Table 1 summarized the detection limit concentration minimum and PNA with a K _D value in this case. The results shown in FIGS. 31 to 33 (stationary time is 3 hours) are summarized in Table 2.

From Tables 1 and 2, when GM1-Glc is used as the sugar chain, the molar ratio of GM1-Glc and TEG is optimally 5: 5, and when GM1 is used as the sugar chain, GM1 and TEG It can be seen that a molar ratio of 10: 0 is optimal.

From the above results, anti-ganglioside that can be performed with 1 mg of GM1-Glc-immobilized sugar chain ligand complex (GM1-Glc-f-mono) and 1 mg of GM1-immobilized sugar chain ligand complex (GM1-f-mono). The number of antibody tests is as shown in Table 3 when the binding characteristics of the anti-ganglioside antibody are the same as those of PNA.

In Table 3, the number of testable times is the number of times when 15 μL of sugar chain-immobilized fluorescent nanoparticles or sugar chain-immobilized gold nanoparticles are used per test.

As shown in Table 3, when the sugar chain-immobilized fluorescent nanoparticles according to the present invention are used, the anti-ganglioside antibody has better detectability than the case where the sugar chain-immobilized gold nanoparticles are used, and the number of examinations It can be seen that it is much more.

[Example 17: Comparison of TEG-containing GM1-Glc-FNP, TEG-containing GM1-FNP, and GM1-FNP in serum aggregation experiments]
In Example 16, TEG-containing GM1-Glc-FNP and GM1-FNP had different binding properties to PNA. Therefore, in this example, whether or not the binding properties to anti-ganglioside antibodies were different was examined.

TEG-containing GM1-Glc-FNP (GM1-Glc: TEG = 5: 5), GM1-FNP (GM1: TEG = 10: 0), and TEG used in Example 16 were used as the sugar chain-immobilized fluorescent nanoparticles. Containing GM1-FNP (GM1: TEG = 5: 5) was used. As a serum sample, sample number 14151, which is anti-GM1 antibody-positive serum by ELISA, was used.

Serum sample 14151 5 μL was transferred to a 200 μL plastic tube. Then, a dilution series is prepared using PBS so that the serum dilution ratio is 1/4, 1/8, and 1/16, and each of the sugar chain-immobilized fluorescent nanoparticles is added to each dilution series. 5 μL was added, and the mixture was stirred with a vortex mixer and allowed to stand in the dark at 4 ° C. for 3 hours. Subsequently, the tube was centrifuged at 14000 G for 5 minutes, the tube was irradiated with ultraviolet light, and the fluorescence of the precipitate was observed to take a picture.

FIG. 34 shows TEG-containing GM1-Glc-FNP (GM1-Glc: TEG = 5: 5), GM1-FNP (GM1: TEG = 10: 0), and TEG-containing GM1-FNP (GM1: TEG = 5: 5). ) Is a diagram showing the results of a serum agglutination test.

Although the TEG-containing GM1-Glc-FNP and GM1-FNP had different binding properties to PNA, as shown in FIG. 34, the binding properties to the anti-GM1 antibody were three sugar chain-immobilized fluorescent nanoparticles. There was no big change.

[Example 18: Detection of anti-GM1 IgG antibody using GM1-FNP]
In this example, detection of anti-GM1 IgG antibody by GM1-FNP prepared in Example 13 was examined using 50 samples each of sera positive for anti-GM1 antibody by ELISA and anti-GM1 antibody negative by ELISA.

Each serum sample was placed in a plastic tube having a capacity of 15 μL and 200 μL, respectively, and 15 μL of a GM1-FNP PBS solution (0.1 μM) was added thereto. The tube was stirred with a vortex mixer, allowed to stand in the dark at 4 ° C. for 3 hours, and centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light, a photograph was taken, and the fluorescence spectrum of the supernatant was measured (Ex. 360 nm, Em. 650 nm).

FIG. 35 is a diagram showing the detection results of anti-GM1 IgG antibody by GM1-FNP when 50 samples of anti-GM1 IgG antibody-positive serum were used by ELISA. FIG. 36 is a diagram showing the fluorescence intensity of the supernatant in each tube shown in FIG.

The numbers shown in FIG. 35 are obtained by adding a serial number from 1 to 50 to the sample number. Further, in FIG. 35, the determination criterion of the detection result is described using + and − signs. That is, “+++” allows visual observation of aggregates and the fluorescence intensity of the supernatant is less than 150 (positive), “++” enables visual observation of aggregates, and The fluorescence intensity of the supernatant is 150 or more and less than 300 (positive), “+” indicates that the aggregate can be visually observed, and the fluorescence intensity of the supernatant is 300 or more (positive). “-” Indicates that the aggregate can be observed by magnifying the photograph shown in FIG. 35 (false positive), and “-” indicates that no aggregate was observed (negative).

35, the ratio (positive rate) occupied by “++++”, “++”, and “+” was 40/50 = 80%. Further, the ratio of “+/−” (false positive rate) was 3/50 = 6%.

FIG. 37 is a diagram showing the detection results of anti-GM1 IgG antibody by GM1-FNP when 50 samples of anti-GM1 IgG antibody-negative serum were used by ELISA. FIG. 38 is a diagram showing the fluorescence intensity of the supernatant in each tube shown in FIG.

As shown in FIG. 37, all the determination results were “−”. That is, the positive rate was 0% and the negative rate was 100%. As shown in FIG. 38, the fluorescence intensity of the supernatant was almost the same level in 50 samples.

Next, among the 50 anti-GM1 IgG antibody-positive sera positive by ELISA, 7 samples that were negative were used, and the standing time after stirring with a vortex mixer was changed from 3 hours to 12 hours. The same experiment as in the example was performed, and the tube was irradiated with ultraviolet light to take a photograph.

FIG. 39 shows the anti-GM1 IgG antibody-positive sera by ELISA, which was judged to be negative for 3 hours after mixing with GM1-FNP. It is a figure which shows the detection result of an aggregate. As shown in FIG. 39, no new aggregate was formed even when the standing time was 12 hours.

Furthermore, the same experiment as in the above-described example was performed except that 3 samples that were false positive among 50 anti-GM1 IgG antibody-positive sera samples by ELISA were used and the standing time was changed from 3 hours to 12 hours. And the tube was irradiated with ultraviolet light to take a picture.

FIG. 40 shows a serum positive for anti-GM1 IgG antibody by ELISA, and when the mixture was allowed to stand for 3 hours after mixing with GM1-FNP, the determination result was a false positive. It is a figure which shows the detection result of an aggregate.

As shown in FIG. 40, aggregates that were clearly visible were formed in all three samples that were false positive when the standing time (reaction time) was 3 hours. Thus, the positive rate could be improved to 43/50 = 86% by performing the reaction overnight.

Here, the results of Example 12 and the results of this example are shown in Table 4 when compared with the results of the ELISA method. The numbers in the table are the number of samples. For example, the column in which the glycan-immobilized fluorescent nanoparticles are positive in TEG-containing GM1-Glc-FNP is shown in 37 samples out of 50 sera positive in anti-GM1 antibody in ELISA. It shows that the fluorescence of the precipitate was confirmed. The false positive was included as a positive because an aggregate was formed.

In Example 12 using TEG-containing GM1-Glc-FNP (GM1-Glc sugar chain to TEG molar ratio of 5: 5) as sugar chain-immobilized fluorescent nanoparticles, as described above, the standing time ( When the reaction time was 3 hours, the fluorescence of the precipitate was confirmed in 37 samples out of 50 sera positive for anti-GM1 antibody by ELISA (positive rate: 74%). When the standing time was 12 hours (overnight reaction), the positive rate improved to 39/50 = 78%. In Table 4, the numbers shown in parentheses represent the results for the overnight reaction.

From the results shown in Table 4, when the sugar chain-immobilized fluorescent nanoparticles are TEG-containing GM1-Glc-FNP, when the reaction time is 3 hours, the χ ² value is 3.438 (<3.841), the p value. Is 0.0637 (> 0.05), and in the case of overnight reaction, the χ ² value is 2.4496 (<3.841) and the p value is 0.118 (> 0.05). When the sugar chain-immobilized fluorescent nanoparticles are GM1-FNP, when the reaction time is 3 hours, the χ ² value is 0.985 (<3.841), and the p value is 0.321 (> 0.05). ) Note that the χ ² distribution when the degree of freedom is 1 has χ ² values of 2.706, 3.841, and 6.635, respectively, when the significance level is 0.1, 0.05, 0.01, and 0.001. 10.828.

From the above results, it was clarified that the result of detecting the anti-ganglioside antibody using the sugar chain-immobilized fluorescent nanoparticles according to the present invention was not different from that of ELISA. That is, it has been clarified that the detection has a detection sensitivity equivalent to that of ELISA. As mentioned above, detection of anti-ganglioside antibodies by ELISA is very time consuming and usually requires sending the patient's serum to the testing company and waiting for a week or more before the results arrive. On the other hand, when the sugar chain-immobilized fluorescent nanoparticles according to the present invention are used, a sufficient detection result can be obtained with a reaction time as short as 3 hours. Therefore, it can be said that the present invention is very useful.

[Example 19: Preparation of GD1a sugar chain-immobilized ligand complex and preparation of fluorescent nanoparticles having immobilized GD1a sugar chain]
The GD1a sugar chain represented by the above chemical formula 15 (purchased from Oligotech. 1.04 mg, 0.78 μmol) was dissolved in 80 μL of ultrapure water, and the fluorescent linker compound (f-mono, 0. 35 mg, 1.16 μmol) of N, N-dimethylformamide in 90 μL solution, and further 18 μL of acetic acid was added.

After standing at 40 ° C. for 6 hours, a solution in which NaBH ₃ CN (0.49 mg, 7.75 μmol) was dissolved in 10 μL of ultrapure water was added, left at 40 ° C. for 3 days, and lyophilized. This lyophilized residue was dissolved in ultrapure water and purified with an ODS column (elution solvent: water / methanol 1/1 (v / v)). The elution fraction was lyophilized to obtain a GD1a sugar chain-immobilized sugar chain ligand complex (hereinafter abbreviated as “GD1a-f-mono”) shown as Formula 33 in FIG. 41 as a white powder. FIG. 33 is a diagram showing a synthesis route of GD1a-f-mono. The yield of GD1a-f-mono was 0.51 mg (yield 41.8%). The spectral data of GD1a-f-mono is as follows.
MS calcd. for: C ₆₂ H ₉₇ N ₅ NaO ₃₇ S ₂ : 1591.53, Found: m / z 1591.75 [M−H] ⁻ .
Next, a GD1a sugar chain-immobilized fluorescent nanoparticle (GD1a-FNP) solution was prepared by the same method as in Example 3 except that GD1a-f-mono was used instead of GM1-Glc-f-mono. .

[Example 20: Confirmation of protein binding ability of GD1a-FNP]
The binding activity of GD1a sugar chain-immobilized fluorescent nanoparticles (GD1a-FNP) to ConA, WGA, SNA, PNA, RCA120 lectins and BSA was examined.

Each protein was dissolved in PBS at a concentration of 3.6 μM, and 5 μL thereof was transferred to a plastic tube having a capacity of 200 μL. Thereto was added 5 μL of the GD1a-FNP PBS solution (0.25 μM) prepared in Example 19, stirred with a vortex mixer, left in the dark for 12 hours, and centrifuged at 14000 G for 5 minutes. The tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 42 is a diagram showing the fluorescence emission results of the precipitate after centrifugation in the aggregation experiment of GD1a-FNP and sugar chain binding protein, and FIG. 43 shows the fluorescence spectrum of the supernatant after the centrifugation. FIG.

42. As shown in FIG. 42, only when WGA was used, a precipitate emitting fluorescence that was visually distinguishable was generated. Moreover, as shown in FIG. 43, only the fluorescence intensity of the supernatant of WGA significantly decreased. 42 and 43, it can be seen that the GD1a sugar chain of the GD1a sugar chain-immobilized fluorescent nanoparticle (GD1a-FNP) specifically bound to WGA, and as a result, an aggregate was formed.

[Example 21: Reaction of GD1a sugar chain-immobilized fluorescent nanoparticles (GD1a-FNP) with patient serum]
5 μL of serum from patients with immune peripheral neuropathy, including patients with Guillain-Barre syndrome, was transferred to a plastic tube with a volume of 200 μL. Thereto was added 5 μL of a 0.1 μM PBS solution of GD1a sugar chain-immobilized fluorescent nanoparticles (GD1a-FNP) prepared in Example 19, stirred with a vortex mixer, and left in a dark place at 4 ° C. for 12 hours. And centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 44 is a diagram showing the results of fluorescence emission of precipitates after centrifugation in an agglutination experiment between GD1a-FNP and serum of a patient with immune peripheral neuropathy. FIG. 45 is a diagram showing the results of measuring the fluorescence spectrum of the supernatant after the centrifugation.

The numbers in FIGS. 44 and 45 are sample numbers, and 13923 and 13938 are the results of using anti-GM1 antibody negative and anti-GD1a antibody negative sera by ELISA, and 13882 and 13934 are anti-GM1 antibody negative by ELISA. In addition, 14078, 14151 and 14192 show the results using anti-GD1a antibody-positive sera, and the results using anti-GM1 antibody-positive sera by the ELISA method, respectively.

As shown in FIG. 44, when anti-GD1a antibody-positive sera (13882 and 13934) were used in the ELISA method, formation of a fluorescent precipitate was confirmed. In addition, when 14151 which is anti-GM1 antibody positive serum was used, formation of a fluorescent precipitate was confirmed. Then, as shown in FIG. 45, when 13882 and 14151 were used, the fluorescence intensity of the supernatant was particularly greatly reduced.

From the above results, it was revealed that anti-GD1a antibody can be detected even when GD1a sugar chain-immobilized fluorescent nanoparticles (GD1a-FNP) are used. According to the results using 14151, anti-GD1a antibody may also be present in anti-GM1 antibody-positive serum.

[Example 22: Aggregation experiment using anti-GM1 IgG antibody-positive serum and other sugar chain-immobilized fluorescent nanoparticles]
In this example, anti-GM1 IgG antibody-positive serum was mixed with sugar chain-immobilized fluorescent nanoparticles to which sugar chains other than GM1 sugar chains were immobilized, thereby confirming the presence or absence of cross-reactivity.

As sera positive for anti-GM1 IgG antibody by ELISA, sera that were negative as a result of reaction with GM1-FNP in Example 18 (7 samples with “-” in FIG. 35; “SFNP” in FIGS. 46 and 47) And three samples labeled with “+/−” in FIG. 35 and “SFNP” in FIGS. 46 and 47. And a "false positive sample").

Each serum sample was put in a plastic tube having a capacity of 5 μL and 200 μL, respectively, and 5 μL of GD1a-FNP PBS solution (0.1 μM) prepared in Example 19 was added thereto. The tube was stirred with a vortex mixer, allowed to stand in the dark at 4 ° C. for 3 hours, and centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light and photographed.

FIG. 46 is a graph showing the results of an agglutination experiment in which GD1a-FNP was mixed with serum that was positive for anti-GM1 IgG antibody by ELISA and negative or false positive as a result of reaction with GM1-FNP in Example 18. It is. As shown in FIG. 46, no aggregate was observed. That is, GD1a-FNP did not show cross-reactivity with anti-GM1 IgG antibody.

Next, the GD1a-GM1 sugar chain-immobilized fluorescent nanoparticle (GD1a-GM1 sugar chain-immobilized fluorescent nanoparticle (the GD1a-GM1 sugar chain-immobilized fluorescent nanoparticle) was used by using the sugar chain of GD1a and the sugar chain of GM1 at a ratio of 50 mol%. GD1a-GM1-FNP) was prepared and subjected to the same experiment.

GD1a-GM1-FNP was prepared by the following method. That is, to the mixed solution of GM1-f-mono (1 mM, 12.5 μL) prepared in Example 13 and GD1a-f-mono (1 mM, 12.5 μL) prepared in Example 19, an aqueous solution of NaBH ₄ ( 10 mM, 25 μL) was mixed at room temperature and allowed to stand for 10 minutes. In this case, the molar ratio between GM1 and GD1a is 5: 5. 50 μL of a solution obtained by diluting the CdTe / CdS core / shell QD solution prepared in Example 3 five times with ultrapure water was added to the solution allowed to stand for 10 minutes, and stirred at room temperature in the dark for 24 hours. Unreacted sugar-ligand complex was removed by performing centrifugal ultrafiltration (14000 × g, 5 min) using Amicon Ultra 10 K (Millipore, MA, USA) three times, and the precipitate was finally added to PBS. By suspending, a solution of fluorescent nanoparticles (GD1a-GM1-FNP) in which the GM1 sugar chain and the GD1a sugar chain were immobilized was prepared.

FIG. 47 shows the results of an agglutination experiment in which GD1a-GM1-FNP was mixed with serum that was positive for anti-GM1 IgG antibody by ELISA and negative or false positive as a result of reaction with GM1-FNP in Example 18. FIG. As shown in FIG. 47, no aggregates were observed. That is, GD1a-GM1-FNP did not show cross-reactivity with anti-GM1 IgG antibody.

[Example 23: Synthesis of GQ1b-Glc sugar chain]
In this example, a sugar chain represented by Chemical Formula 16 (GQ1b-Glc sugar chain) in which one glucose was further bonded to the reducing end of the GQ1b sugar chain was synthesized.

FIG. 48 is a diagram showing an intermediate synthesis route for synthesizing the GQ1b-Glc sugar chain. That is, in FIG. 48, a heptasaccharide structure (shown in Formula g) that forms GQ1b-core was prepared. First, the NeuAcα2-8 NeuAcα2-3Gal trisaccharide structure (shown in Formula a) was introduced into the corresponding glycoside donor (shown in Formula b) in 4 steps. On the other hand, in the presence of Cp ₂ ZrCl ₂ and AgOTf, a galactosamine derivative (shown in formula c) and the above NeuAcα2-8NeuAcα2-3Gal trisaccharide structure (shown in formula a) are condensed to give NeuAcα2-8NeAcα2-3 [ The GalNβ1-4] Gal tetrasaccharide structure (shown in formula d) was prepared in 85% yield. This was subsequently led to the tetrasaccharide glycosyl acceptor (shown in formula e) in two steps. Next, the trisaccharide glycosyl donor obtained above (shown in formula b) and the tetrasaccharide glycosyl acceptor (shown in formula e) are condensed in the presence of TMSOTf to form the GQ1b-core heptasaccharide. The structure (shown in formula f) was obtained. Furthermore, from this heptasaccharide structure, the corresponding glycosyl donor (shown in formula g) was derived in 4 steps.

FIG. 49 is a diagram showing a pathway showing glycosylation and deprotection for synthesizing the GQ1b-Glc sugar chain. The aforementioned GQ1b-core glycosyl donor with a heptasaccharide structure (shown in formula g) and a gentiobiose acceptor (shown in formula 11) are reacted in dichloromethane in the presence of TMSOTf to give the desired β-glycoside (formula h Was obtained in 80% yield.

Next, after removing the acyl protecting group, the benzyl group was finally hydrocracked to obtain the GQ1b-Glc sugar chain (Formula 16) in a yield of 83% (2 steps).

The spectrum data of GQ1b-Glc sugar chain shown in Formula 16 is as follows.
[Α] _D = −3.7 ° (c 0.4, H ₂ O); ¹ H-NMR (600 MHz, D ₂ O) δ 5.20 (d, 1 H, J _1,2 = 3. 4 Hz, H-1 of Glc unit), 4.71 (m, anomer H), 4.63 (d, 1 H, J _1,2 = 8.2 Hz, H-1 of Glc unit), 4. 58 (m, anomer H), 4.52 (d, 1 H, J _1,2 = 8.2 Hz, anomer H), 4.50 (d, 1 H, J _1,2 = 7.5 Hz, anomer H), 4.47 (d, 1 H, J _1,2 = 8.2 Hz, anomer H), 3.51 (dd, 1 H, J _1,2 = 3.4 Hz, J _2,3 = 10.3 Hz, H-2 of Glc unit), 3.44 (t, 1 H, J 2,3 = J 3,4 = 8 9 Hz, H-3 of Glc unit), 3.40-3.30 (m, 3 H, 3 H-2), 3.22 (near t, 1 H, J 1,2 = 8.2 Hz, J _2,3 = 8.9 Hz, H-2), 2.76-2.62 (m, 4 H, 4 H-3 eq of Neu5Ac unit), 2.04, 2.03, 2.02, 2 .00 (4 s, 15 H, 5 NAc), 1.78-1.69 (m, 4 H, 4 H-3ax of Neu5Ac unit), ¹³ C-NMR (150 MHz, D ₂ O— (CD ₃ ) ₂ CO = 10: 1) δ 165.9, 164.8-163.9, 95.3, 93.8, 93.6, 93.5, 92.0-90.9, 87.0, 83 .1, 71.0, 69.2, 69.1, 66.7, 66.4, 65.9, 65. , 65.8, 65.6, 65.3, 65.2, 65.1, 65.1, 64.8, 63.7, 62.7, 62.4, 61.4, 60.6, 60 .5, 60.4, 60.2, 59.8, 59.6, 59.4, 59.3, 59.2, 59.0, 58.6, 53.6, 52.5, 52.4 52.0, 52.0, 51.6, 51.0, 43.3, 43.2, 42.8, 42.1, 31.6, 31.5, 30.7, 30.2, 13 .6, 13.4, 13.3, 13.1; ESI MS: m / z: calcd for C ₇₆ H ₁₂₃ N ₅ O ₅₈ : 507.4143; found: 507.4145 [M-4H ⁺ ] ⁴⁻ .
[Example 24: Preparation of GQ1b-Glc-immobilized sugar chain ligand complex, preparation of fluorescent nanoparticles with immobilized GQ1b-Glc sugar chain]
FIG. 50 is a diagram showing a synthesis route for a GQ1b-Glc-immobilized sugar chain ligand complex (GQ1b-Glc-f-mono). The GQ1b sugar chain having 6-glucose at the reducing end (GQ1b-Glc, 1.00 mg, 0.49 μmol) synthesized in Example 23 was dissolved in 20 μL of ultrapure water, and the original development shown in the above formula (30) was performed. A fluorescent linker compound (f-mono: 0.16 mg, 0.54 μmol) was added to a 30 μL solution of N, N-dimethylformamide, and 6 μL of acetic acid was further added.

After standing at 40 ° C. for 6 hours, a solution in which NaBH ₃ CN (0.31 mg, 4.91 μmol) was dissolved in 10 μL of ultrapure water was added, left at 40 ° C. for 3 days, and lyophilized. This lyophilized residue was dissolved in ultrapure water and purified with an ODS column (elution solvent: water / methanol 1/1 (v / v)). The eluted fraction was lyophilized to obtain a GQ1b-Glc-immobilized sugar chain ligand complex (hereinafter abbreviated as “GQ1b-Glc-f-mono”) shown as Formula 34 in FIG. 50 as a white powder. The yield of GQ1b-Glc-f-mono was 0.57 mg (yield 50%).

Next, GQ1b-Glc sugar chain-immobilized fluorescent nanoparticles (GQ1b-Glc-FNP) were prepared in the same manner as in Example 3 except that GQ1b-Glc-f-mono was used instead of GM1-Glc-f-mono. PBS solution was prepared.

FIG. 51 is a diagram showing the results of measuring the fluorescence and UV-Vis spectrum of the GQ1b-Glc-FNP solution. FIG. 52 is a diagram showing the particle size distribution of GQ1b-Glc-FNP measured by DLS. As shown in FIG. 52, the average particle diameter of GQ1b-Glc-FNP was 9.7 nm as a result of measurement using the same equipment as in Example 3.

[Example 25: Confirmation of GQ1b-Glc-FNP by MALDI-TOF / MS]
1 μL of PBS solution of GQ1b-Glc-FNP prepared in Example 24 was mixed with 10 μL of saturated DHBA solution (water / methanol 1/1 solution), and 1 μL was placed on a measuring plate and allowed to air dry. The plate was put into the measuring section of Voyager-DE-PRO (Applied Biosystems, CA, USA) and subjected to mass spectrometry.

FIG. 53 is a diagram showing a result of MALDI-TOF / MS analysis by GQ1b-Glc-FNP Positive mode, and FIG. 54 is a diagram showing a result of MALDI-TOF / MS analysis by Negative mode of GQ1b-Glc-FNP. FIG.

As shown in FIGS. 53 and 54, a peak (m / Z value) having the same mass number as that of the GQ1b-Glc-immobilized sugar chain ligand complex prepared in Example 24 was obtained, and GQ1b was observed on the fluorescent nanoparticles. -It was confirmed that the Glc sugar chain was immobilized.

[Example 26: Confirmation of protein binding ability of GQ1b-Glc-FNP]
The binding activity of GQ1b-Glc-FNP to ConA, PNA, RCA120, jackalin, WGA, and von Willebrand factor (vWF) was examined. The binding sugar chain of jackalin is αGal.

ConA, RCA120, and WGA were dissolved in PBS to a concentration of 20 μM, jackalin 10 μM, and PNA 3.6 μM, respectively, and 5 μL thereof was transferred to a 200 μL capacity plastic tube. Thereto, 5 μL of PBS solution (0.1 μM) of GQ1b-Glc-FNP prepared in Example 24 was added, stirred with a vortex mixer, left in the dark at 4 ° C. for 12 hours, and centrifuged at 14000 G for 5 minutes. It was. The tube was irradiated with ultraviolet light, the fluorescence of the precipitate was observed, and a photograph was taken. Then, the fluorescence spectrum of the supernatant was measured.

FIG. 55 is a diagram showing the results of fluorescence emission of the precipitate after centrifugation in an aggregation experiment of GQ1b-Glc-FNP and a sugar chain binding protein, and FIG. 56 shows the fluorescence spectrum of the supernatant after centrifugation. FIG.

As shown in FIG. 55, only when WGA was used, a precipitate emitting fluorescence that was visually distinguishable was generated. Also, as shown in FIG. 56, only the fluorescence intensity of the supernatant of WGA was greatly reduced. 55 and 56, it can be seen that the sugar chain of GQ1b-Glc-FNP specifically bound to WGA, and as a result, an aggregate was formed.

[Example 27: Reaction of GQ1b-Glc-FNP with patient serum]
5 μL of serum from patients with immune peripheral neuropathy, including patients with Guillain-Barre syndrome, was transferred to a plastic tube with a volume of 200 μL. Thereto was added 5 μL of 0.1 μM PBS solution of GQ1b-Glc sugar chain-immobilized fluorescent nanoparticles (GQ1b-Glc-FNP) prepared in Example 13 and stirred with a vortex mixer at 4 ° C. in the dark. The mixture was allowed to stand for 3 hours or 12 hours, and centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light, and the fluorescence of the precipitate was observed to take a photograph.

FIG. 57 shows the results of fluorescence emission of precipitates after centrifugation in an agglutination experiment between GQ1b-Glc-FNP and sera of patients with immune peripheral neuropathy. FIG. 57 (a) shows the result when left for 3 hours, and FIG. 57 (b) shows the result when left for 12 hours. The numbers in FIG. 57 are sample numbers.

13923 and 13138 show the results of using anti-GM1 antibody-negative and anti-GD1a antibody-negative sera by ELISA method, 13934 shows the results of using anti-GM1 antibody-negative and anti-GD1a antibody-positive serum by ELISA method, 14078, 14151 and 14192 show the results of using the anti-GM1 antibody-positive sera by the ELISA method, and 14992, 15056 and 15090 show the results of using the anti-GQ1b antibody-positive sera by the ELISA method, respectively.

As shown in FIG. 57, aggregates as shown in Example 15 and the like were not observed. This suggests that the anti-GQ1b antibody recognizes the entire ganglioside, or the sugar chain and the ceramide part, and does not recognize the sugar chain part unlike the anti-GM1 antibody or the anti-GD1a antibody. ing. That is, if there is an antibody that recognizes only a sugar chain, it is considered that an aggregate is formed with ganglioside sugar chain-immobilized fluorescent nanoparticles, as in GM1 and GD1a. Alternatively, even if there is an antibody that recognizes the sugar chain portion, it is considered that the affinity with the sugar chain is so low that no aggregate is formed.

[Example 28: Preparation of fluorescent nanoparticles in which GM1 sugar chain is immobilized on ZAIS / ZnS core / shell nanoparticles having ZAIS as a core]
FIG. 58 is a schematic diagram showing a method for preparing fluorescent nanoparticles (GM1-FNP2) in which GM1 sugar chains are immobilized on ZAIS / ZnS core / shell nanoparticles having ZAIS as a core.

First, sodium N, N-diethyldithiocarbamate (563 mg, 2.50 mmol) was transferred to an Erlenmeyer flask and dissolved in 50 mL of ultrapure water. In another Erlenmeyer flask, AgNO ₃ (95.6 mg, 0.563 mmol), In (NO ₃ ) ₃ .3H ₂ O (200 mg, 0.563 mmol), Zn (NO ₃ ) ₂ .6H ₂ O (37.2 mg). 2.50 mmol) was dissolved in 50 mL of ultrapure water, and 50 mL of the solution prepared above was added dropwise under shading and stirred for 5 minutes. The obtained metal salt was centrifuged (3500 rpm, 5 min), and the precipitate was washed four times with ultrapure water and twice with methanol, and then dried under reduced pressure. 50 mg of the dried powder was transferred to a two-necked round bottom flask, heated at 180 ° C. for 30 minutes under argon, added with 3 mL of oleylamine, and heated at 180 ° C. for 5 minutes under argon. After standing to cool, the solution was centrifuged (3500 rpm, 5 min), the supernatant was membrane filtered (0.45 μm), and methanol was added to the filtrate to precipitate ZAIS nanoparticles. The precipitate was suspended in 2 mL of oleylamine and heated at 180 ° C. for 30 minutes under argon. After allowing to cool, anhydrous zinc acetate (10.3 mg, 56.3 μmol) and thioacetamide (4.22 mg, 56.3 μmol) were added to the solution, and the mixture was heated at 180 ° C. for 30 minutes under an argon atmosphere. Methanol was added to the solution, and the precipitate was resuspended in 3 mL of chloroform.

Then, 0.5 mL of the solution was transferred to a glass centrifuge tube, 1.5 mL of chloroform was added, and then 1 mL of 3-MPA ethanol solution (0.2 M) and 1 mL of potassium hydroxide ethanol solution (0.3 M) were added. The mixture was stirred overnight (12 hours) at 0 ° C. in the dark.

The solution was centrifuged (3500 rpm, 5 min), and the precipitate was resuspended in 2 mL of ultrapure water. Separately, zinc acetate anhydrous (20.6 mg, 112 μmol) and thioacetamide (8.44 mg, 112 μmol) were added to a two-necked round bottom flask, dissolved in 2 mL of ultrapure water, and TGA (7.96 μL, 112 μmol) 2 mL of a solution obtained by diluting the above solution 5-fold was added and heated at 80 ° C. for 5 hours. Unreacted reagents are removed by centrifugal ultrafiltration (14000 × g, 5 min) using Amicon Ultra 10 K (Millipore, MA, USA), followed by washing 6 times with ultrapure water. Finally, ZAIS / ZnS core / shell nanoparticles were obtained by suspending in ultrapure water.

Next, as shown in FIG. 58, an aqueous solution of NaBH ₄ (100 mM, 12.5 μL) was mixed with GM1-f-mono (10 mM, 12.5 μL) prepared in Example 13 at room temperature and left for 10 minutes. . Thereafter, 100 μL of a solution obtained by diluting the ZAIS / ZnS core / shell nanoparticle solution 8 times with ultrapure water was added to the solution allowed to stand for 10 minutes, and heated at 50 ° C. for 2 hours in the dark. Unreacted sugar-ligand complex was removed by centrifugal ultrafiltration (14000 × g, 5 min) using Amicon Ultra 10 K (Millipore, MA, USA), followed by washing 4 times with ultrapure water. GM1 sugar chain-immobilized fluorescent nanoparticle (GM1-FNP2) solution was prepared by finally suspending the precipitate in PBS.

[Example 29: Reaction of GM1a-FNP2 with patient serum and protein]
Sera of patients with immune peripheral neuropathy including patients with Guillain-Barre syndrome and PNA dissolved in PBS at a concentration of 4 μM, 5 μL were transferred to a plastic tube having a volume of 200 μL. Thereto, 5 μL of 3 μM PBS solution of GM1 sugar chain-immobilized fluorescent nanoparticle (GM1-FNP2) having ZAIS / ZnS core / shell nanoparticles prepared in Example 28 as a core was added, and stirred with a vortex mixer. It was left in the dark at 4 ° C. overnight (12 hours), and centrifuged at 14000 G for 5 minutes. Next, the tube was irradiated with ultraviolet light, and the fluorescence of the precipitate was observed to take a photograph.

FIG. 59 is a diagram showing the results of fluorescence emission of precipitates after centrifugation in an agglutination experiment between GM1-FNP2 and sera and proteins of patients with immune peripheral neuropathy.

In FIG. 59, the 5-digit number is the sample number, and 13939 is the result of using anti-GM1 antibody negative and anti-GD1a antibody negative sera by ELISA, and 13934 is anti-GM1 antibody negative and anti-GD1a by ELISA. The results using antibody-positive sera, 14078 and 14192, respectively, show the results using anti-GM1 antibody-positive sera by ELISA.

As shown in FIG. 59, formation of a fluorescent precipitate was confirmed when anti-GM1 antibody-positive serum was used in the ELISA method and in PNA (14078 and 14192).

From the above results, even when fluorescent nanoparticles (GM1-FNP2) in which GM1 sugar chains are immobilized on ZAIS / ZnS core / shell nanoparticles are used, GM1-Glc sugar chain-immobilized fluorescent nanoparticles (GM1- It was revealed that anti-GM1 antibody can be detected as in the case of using (Glc-FNP).

From the above results, the sugar chain-immobilized fluorescent nanoparticles according to the present invention in which the sugar chain part of ganglioside is immobilized are used for rapid and simple detection of immune peripheral neuropathy such as Guillain-Barre syndrome and Fisher syndrome. As a means or diagnostic method, it is highly expected to be available in the medical field.

Claims (9)

1 type or 2 types or more of sugar chains containing a ganglioside-derived sugar chain, and 1 type or 2 types or more of linker compounds having a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, A sugar chain ligand complex having a hydrocarbon structure in which the main chain has an amino group bonded to the sugar chain at one end and a sulfur atom at the other end;
A fluorescent nanoparticle in which a particle core composed of first and second metal components is coated with a layer composed of first and third metal components;
A sugar chain-immobilized fluorescent nanoparticle, wherein the hydrocarbon structure is immobilized on the layer. The sugar chain-immobilized fluorescent nanoparticles according to claim 1, wherein the ganglioside is one or more gangliosides selected from the group consisting of GM1, GD1a, and GQ1b. The sugar chain immobilization according to claim 2, wherein the one or more sugar chains containing the ganglioside-derived sugar chain are selected from sugar chains represented by the following chemical formulas 1 and 14 to 16. Fluorescent nanoparticles.

An aminated oligoethylene glycol and a linker compound having a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, wherein the main chain of the linker compound has the aminated oligoethylene glycol at one end thereof A composite having a bonded carboxyl group and having a hydrocarbon structure containing a sulfur atom at the other end;
The sugar chain-immobilized fluorescent nanoparticles according to any one of claims 1 to 3, wherein the hydrocarbon structure is immobilized on the layer. The sugar chain-immobilized fluorescent nanoparticles according to any one of claims 1 to 4, wherein the first metal component is selected from the group consisting of cadmium, zinc, silver, indium, and sulfur. 6. The sugar chain-immobilized fluorescent nanoparticles according to any one of claims 1 to 5, wherein the second metal component is selected from the group consisting of tellurium and sulfur. The sugar chain-immobilized fluorescent nanoparticles according to any one of claims 1 to 6, wherein the third metal component is selected from the group consisting of cadmium, sulfur and zinc. A ganglioside-derived glycanid immobilized on the sugar chain-immobilized fluorescent nanoparticle by mixing the sugar chain-immobilized fluorescent nanoparticle according to any one of claims 1 to 7 and a subject. A method for detecting an anti-ganglioside antibody, comprising a step of reacting a sugar chain containing a sugar chain with an anti-ganglioside antibody contained in the subject. A detection reagent or diagnostic agent for immune peripheral neuropathy, comprising the sugar chain-immobilized fluorescent nanoparticles according to any one of claims 1 to 7.

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