JP2003075439A - Aggregate of string-like substrate - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a ligand immobilization substrate for continuously, speedily, and efficiently analyzing information on the combination of various kinds of ligands and the receptor. SOLUTION: In at least two string-like substrate aggregates, a plurality of different ligands are immobilized on a predetermined region on the surface. In the string-like aggregates, the combination of a ligand and a receptor having a combination property with the ligand is detected speedily. An apparatus detects the combination of the ligand using the string-like substrate and the receptor. In the detection method of the combination of the ligand and the receptor, the aggregate of the string-like substrate is used.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a substrate for obtaining a large amount of information on the binding of a ligand and its receptor useful in the field of biochemistry continuously and at high speed.

[0002]

2. Description of the Related Art The relationship between structure and activity of molecules is a fundamental matter in the study of biological systems, and some molecules are
It is known to interact and bind with other molecules.
Intermolecular binding having such specificity is known as a relationship between a receptor and a ligand. Many measurement methods are known for measuring the binding affinity between a ligand and a receptor (see, for example, JP-A-4-505763).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a ligand-immobilized substrate which enables continuous, high-speed, and high-efficiency analysis of information relating to the binding of various ligands and their receptors. To provide.

[0004]

SUMMARY OF THE INVENTION To outline the present invention, the first invention of the present invention is to provide two or more filamentous substrates each having a plurality of different ligands immobilized in a predetermined region on its surface. Regarding the aggregate.

In a first aspect of the present invention, there is provided an assembly of filamentous substrates in which different ligand groups are immobilized for each filamentous substrate. The filamentous substrates may be gathered in parallel or may be gathered in series. When gathered in parallel, they may be gathered in a flat plate shape, the filamentous substrates may be combined, or they may be separated from each other. A filamentous substrate having a plurality of different ligands immobilized on a predetermined region on its surface may be selected from a group of preliminarily produced filamentous substrates, and any filamentous substrate may be used in combination depending on the purpose. can do. The ligand is not particularly limited as long as it can be immobilized on the filamentous substrate, and examples thereof include nucleic acid, carbohydrate, peptide, protein, lipid or cell. The nucleic acid is not particularly limited as long as it is DNA or RNA, but double-stranded nucleic acid or single-stranded nucleic acid is exemplified. D
The NA may be a polynucleotide or an oligonucleotide.

In the first aspect of the present invention, there is provided an assembly of filamentous substrates capable of detecting the binding between a ligand and a receptor having a binding property to the ligand at a high speed. The receptor is not particularly limited as long as it can bind to a ligand, and examples thereof include nucleic acids, sugars, peptides, proteins, lipids and cells. A labeled receptor can be used as the receptor. As the substrate of the present invention, a substrate in which the distance between regions on the substrate is at least 1 mm or more, a substrate in which the number of regions on which the ligand is immobilized is 100 or more, the number of regions on the substrate An example is a substrate having 1000 or more.

The second invention of the present invention is a device for detecting the binding between a ligand and a receptor, comprising: a) a filamentous form in which a plurality of different ligands are immobilized in a predetermined region on its surface. An assembly of substrates; b) means for exposing said assembly of substrates to a receptor that binds at least one of said ligands; and c) means for detecting the ligand-receptor bound region on said substrate. To a device comprising.

In a second aspect of the present invention, there is provided a device for detecting the binding between a ligand and a receptor, comprising a filamentous substrate having a plurality of different ligands immobilized on a predetermined region on its surface. An apparatus is provided that comprises means for the rapid detection of ligand-receptor bound regions of an assembly.

In the device of the second invention of the present invention, it is preferred to use the substrate of the first invention aspect of the present invention, wherein the receptor is a labeled receptor, and The region where the ligand and the receptor are bound can be detected with a label. As the receptor, it is preferable to use a nucleic acid, a carbohydrate, a peptide or a protein which has a binding property to a nucleic acid, a carbohydrate, a peptide or a protein which is a ligand.

A third invention of the present invention is a method for detecting the binding between a ligand and a receptor, comprising the steps of: a) a plurality of ligands bound to at least one of the ligands in a predetermined region on its surface. A method for detecting binding between a ligand and a receptor, comprising: exposing a group of filamentous substrates having different ligands immobilized thereon; and b) detecting a region where the ligand and the receptor are bound on the substrate. Regarding

In the detection method of the third invention of the present invention, it is preferable to use the substrate of the aspect of the first invention of the present invention. The region where the ligand and the receptor are bound can be detected with a label. As the receptor, it is preferable to use a nucleic acid, a carbohydrate, a peptide or a protein which has a binding property to a nucleic acid, a carbohydrate, a peptide or a protein which is a ligand. [Detailed Description of the Invention] In the present invention, the ligand is not particularly limited as long as it is a molecule that is recognized and bound by a specific receptor, for example, a nucleic acid, a carbohydrate, a protein, a peptide,
Lipids, cells, etc. The ligand is not particularly limited, but examples thereof include agonists and antagonists for cell membrane receptors, toxins (toxin and venom), viral epitopes, hormones (eg, sedatives, opiates, steroids, etc.), hormone receptors, peptides, enzymes. , Enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, polynucleotides, nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.

In the present invention, the receptor is not particularly limited as long as it shows a binding property to the ligand, and it may be a natural molecule or an artificial molecule. The receptor is not particularly limited, and examples thereof include nucleic acids, carbohydrates, proteins, peptides, lipids, cells such as enzymes, cell surface proteins (receptors), glycoproteins, antibodies and the like. That is,
In the present invention, the contact surface characteristics of the ligand and the receptor having the binding property to the ligand are complementary to each other.

In the present invention, the thread-like shape is a narrower width than the tape-like shape, and is a general term for string-like, thread-like and rope-like shapes. Any material can be used as the material of the filamentous substrate as long as it can immobilize the ligand in a predetermined region on its surface.

(1) Assembly of Filamentous Substrate of the Present Invention The filamentous substrate of the present invention is not particularly limited, but for example, a plurality of different ligands are immobilized in a predetermined region on the surface of the filamentous substrate. It is a thread-like substrate. The aggregate of the filamentous substrate of the present invention is not particularly limited, but for example, an aggregate of two or more filamentous substrates in which a plurality of different ligands are immobilized in a predetermined region on the surface of the filamentous substrate. Is.

In the aggregate of the filamentous substrate of the present invention, the aggregate of the filamentous substrate can be produced by selecting the filamentous substrate according to the purpose from the group of the filamentous substrates to which different ligand groups are immobilized. . The thread-like substrates may or may not be fixed to each other. Further, the filamentous substrate may be fixed on a flat plate.

The thread-like substrate of the present invention may be made of any material having softness or semi-softness, and it is desirable that a plurality of regions having ligands immobilized thereon be physically separated and arranged.

As the thread-like substrate of the present invention, a substantially insoluble material can be preferably used. Although not particularly limited, for example, a heteropolymer having a ligand covalently or non-covalently bonded, polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylene sulfide, polysiloxane, polyimide, polyacetate, polyvinyl chloride. , Nylon or other polymers that will become apparent after this disclosure, or paper, nylon, cellulose, cellulose derivatives such as nitrocellulose, and the like.

Materials used for synthetic fibers, semi-synthetic fibers, regenerated fibers, chemical fibers such as inorganic fibers, natural fibers and the like can be used for the filamentous substrate. Examples of the synthetic fiber material include nylon 6, nylon 66, aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polyester such as polylactic acid and polyglycol, and acrylic such as polyacrylonitrile.
Polyolefins such as polyethylene and polypropylene,
Polyvinyl alcohol type, polyvinyl chloride type, polyurethane type, phenol type, fluorine type such as polyvinylidene fluoride and polytetrafluoroethylene, polyalkylene paraoxybenzoate type, polymethyl methacrylate,
An optical system material such as polystyrene is exemplified. Examples of the semi-synthetic fiber-based material include protein-based materials such as diacetate, triacetate, chitin, chitosan, cellulose-based derivative, and promix. Furthermore, recycled fiber materials such as rayon, cupra, and polynosic are exemplified.

Inorganic fiber materials such as glass fiber and carbon fiber can also be preferably used. Furthermore, plant fiber materials such as cotton and flax, animal fibers such as wool and silk, and mineral fibers such as asbestos can be preferably used.

Further, the material of the filamentous substrate of the present invention may be a polymerized or non-polymerized material having a porous surface and a smooth surface. However, the carrier used in the present invention is not limited to these materials. The substrate may be, for example, one that can be used as a substrate for a DNA chip or a biosensor, as long as it can hold the DNA to be immobilized on the surface of the carrier. For example, a hydrophilic or hydrophobic functional group such as Those having a hydroxyl group, an amino group, a thiol group, an aldehyde group, a carboxyl group, an acyl group and the like can be suitably used. Further, the above-mentioned substrate includes a substrate in which the functional group is already present on the surface of the substrate as a characteristic of the material of the substrate. Furthermore, as long as the above-mentioned substrate does not have a hydrophilic or hydrophobic functional group on the surface of the carrier, it can have a hydrophilic or hydrophobic functional group on the surface of the substrate by surface treatment. There is no particular limitation.

As such a surface-treated product, for example,
Examples include those obtained by treating the surface of the substrate with a commercially available silane coupling agent such as aminoalkylsilane, those treated with a polycation such as polylysine and polyethyleneimine, and those treated with aminoalkylsilane and glutaraldehyde. Further, the filamentous substrate of the present invention can be suitably used in any of electrically negative, neutral and positive as long as the ligand can be efficiently immobilized. Further, the base may have a double structure of a ligand fixing part and a part supporting the fixing part, and it may be selected from the viewpoints of the ligand fixing efficiency and the strength of the filamentous base.

Further, the ligand-fixing part prepared in advance may be used by being attached to the part supporting the fixing part.
The shape of the ligand-immobilized portion is not limited, and the thread-shaped substrate may be prepared by using a material in which the ligand is immobilized in advance on the beads, a material in which the ligand is synthesized on the beads, or the like. Further, in the above-mentioned filamentous substrate, there is no particular limitation as long as it is a material that can sufficiently suppress the background to the signal when it is detected by the detector, and any of them can be preferably used. That is, the substrate of the present invention can be used either transparent, semitransparent or opaque.

The array of ligand-fixing sites on the surface of the filamentous substrate of the present invention is not particularly limited and can be varied according to the purpose. Although not particularly limited, for example, 1 to 100 ligands per 1 cm of the length of the filamentous substrate, 1 to 100 ligands per 1 cm ² of the area of the filamentous substrate,
It is sufficient to spot 1 to 100 ligands per 1 cm ³ of the volume of the filamentous substrate. For spotting the ligand on the substrate, it is convenient to use a commercially available spotter.
Further, depending on the kind of the ligand, it can be synthesized on the substrate.

As the substrate assembly of the present invention, a substrate assembly capable of detecting the binding of a ligand and a receptor having a binding property to the ligand at a high speed is suitable. Used to detect the binding of the ligand and the receptor having the binding property to the ligand at a high rate,
An assembly of substrates with a distance between the regions where the ligand is immobilized can be used to detect the binding of a large number of the ligand and the receptor having the binding property to the ligand in a short time. That is, even if an assembly of substrates that has a distance between the regions where the ligands are immobilized, which can be easily prepared, is used, by obtaining information on the substrates at a high speed, the high density ligands are immobilized. It is possible to obtain the same effect as that of the substrate having the formed region. Further, in the present invention, by making the filamentous substrate into a loop shape, it is possible to repeatedly measure the signal in the region where an arbitrary ligand is immobilized at high speed, so that the signal can be integrated, and S
It is possible to improve the / N ratio. Furthermore, by combining a time-resolved pulse measurement method at the time of detecting the signal, the S / N ratio can be improved and detection can be performed with high sensitivity. Further, in the present invention, it is possible to produce an aggregate of substrates having a distance between the regions where the ligands are immobilized, and the chance of contamination between the ligands and the like can be reduced in the production.

The present invention provides an assembly of filamentous substrates on which different groups of ligands are immobilized. This assembly enables efficient detection of ligand-receptor binding by selecting the filamentous substrate of interest from the group of filamentous substrates to which any different group of ligands has been immobilized. The different ligand groups are not particularly limited, but include, for example, disease-specific ligand groups, biological-type ligand groups, organ-specific, tissue-specific expression-specific ligand groups, such as disease morbidity-specific ligand groups, drug-sensitivity-specific ligand groups, and side effects to drugs. Related single nucleotide substitution polymorphism ligand group, gene expression regulatory unit ligand group, disease category ligand group, disease category ligand group, ligand group for analyzing differences between different cell types, specific biological environment Ligands to analyze the state of cells below, ligands to analyze the response of cells to specific stimuli, ligands to analyze pathological conditions, changes in gene expression with time A group of ligands for analysis is exemplified. That is, in one embodiment of the present invention, the ligand group that can be immobilized on the filamentous substrate is
It is possible to classify and select from genes having a difference in nucleotide sequence or expression level between the comparison targets, and to immobilize the gene or a fragment thereof in a predetermined region on the filamentous substrate.

In the present invention, the filamentous substrate in which the regions on which the ligands are immobilized are arranged in a specific region in a row can detect the binding between the target ligand and the receptor only by the primary scanning, Substrates containing a high density of ligand-immobilized regions required a secondary scan to detect binding between the target ligand and receptor A method of detecting binding is provided. Further, by using an aggregate of a plurality of filamentous substrates, the binding between the ligand and the receptor depending on the purpose can be detected more efficiently. Note that the single row is not limited to a straight line or a curved line.

A light transmission type detector or a light reflection type detector may be selected and used according to the material of the substrate of the present invention. The detector may be of a type in which an aggregate of bases is moved in a one-dimensional manner to the detector, or may be of a type in which the detector is moved in a one-dimensional manner on the base. An assembly of filamentous substrates comprising a plurality of ligands is exposed to at least one receptor, eg a labeled receptor, whose binding to the ligand is to be determined and the location of the label on the substrate is detected. Thus, the binding between the ligand and its receptor can be measured. The exposure of the receptor may be performed to the whole assembly of the substrates or may be sequentially performed from a part thereof. For example, in the case of a filamentous substrate, the tip may be exposed to the receptor.
In the case of an assembly of filamentous substrates, the binding between the ligand and its receptor is detected by exposing the labeled receptor from its short side and then detecting the location of the label on the assembly of the substrate. It can be measured continuously. In addition, in these steps, a washing step and a drying step of the aggregate of the filamentous substrates can be incorporated as needed.

When the binding between the ligand and its receptor is measured, the passage speed of the aggregate of filamentous substrates passing through the detector may be set appropriately, for example, even at a constant speed. Alternatively, the speed may be arbitrarily changed in the inspection area. For example, when passing at a constant speed, for example, when the resolution is 10 μm, the passing speed is preferably 15 seconds or less per 1 cm ² , and more preferably 10 seconds or less. Further, when the resolution is 80 μm, it is preferably 1 second or less per 1 cm ² , and more preferably 0.5 second or less. Further, the whole or the probed region of the aggregate of the filamentous substrate may be repeatedly passed through the detector, and the moving direction of the medium may be repeated so as to integrate the obtained signal data for detection.

In the present invention, by using the aggregate of the bases which has been subjected to the step of previously binding the ligand and the receptor, the step of detecting the binding between the ligand and the receptor can be carried out at high speed, Even when an aggregate of substrates having a low density region where the ligand is immobilized is used, the same effect as that of a high density substrate where the region where the ligand is immobilized can be obtained at high speed. Further, since the low-density substrate can immobilize the ligand accurately, easily, and inexpensively, the aggregate of the substrate of the present invention is industrially useful in that the region where the ligand is immobilized is equivalent to or higher than that of the high-density substrate. Have sex.

In the assembly of the thread-like substrates of the present invention, it is also included in the embodiment of the present invention that the binding of the ligand and the receptor is performed for each thread-like substrate and the detection is performed by the assembly of the thread-like substrates.

In one preferred embodiment of the invention,
A linker molecule is provided on the substrate. The ligand reacts with the exposed functional group on the linker molecule. In a preferred embodiment, the ligand has an amino or carboxyl group at its end and the linker molecule has a reactive amino or carboxylic acid group at the end of the ligand.

The resulting substrate assembly has a variety of uses including, for example, screening multiple receptors for biological activity. In order to screen for biological activity, an assembly of filamentous substrates, to which different groups of ligands are immobilized, is used for one or more receptors such as antibodies,
Exposure to whole cells, receptors on vesicles, lipids, or various other receptors. The receptor is preferably labeled, eg with a fluorescent label, a radioactive label, or a labeled antibody reactive with the receptor. The position of the label on the substrate is detected by, for example, a photon detection method or an autoradiography method. Although not particularly limited, for example, when the above fluorescent label is used, detection can be performed using one type of fluorescent label. In this case, each of the aggregates of the filamentous substrate of the present invention is allowed to react with a plurality of specimen samples labeled with one type of fluorescence, and then the fluorescence signal on the aggregate of the filamentous substrates is detected at high speed. The presence of the receptor can be comparatively analyzed. Further, the above analysis can be performed using two or more kinds of fluorescent labels.

Further, the binding between the ligand immobilized on the substrate and the receptor can be detected by detecting the magnetic field of the magnetic substance that labels the receptor. That is, according to the present invention, the binding between the ligand and the receptor immobilized in a predetermined region on the assembly of the substrate is detected by detecting the magnetic field of the magnetic substance that labels the receptor. A detection method is provided. For example, in the method:
The receptor is labeled with demagnetized magnetic microparticles bound or coated with a linker molecule having a functional group that binds to the receptor. After exposing the receptor labeled with demagnetized magnetic microparticles to the ligand, the ligand and unbound receptor are washed away. The magnetic fine particles of the receptor bound to the ligand are re-magnetized by exposure to a magnetic field of moderate strength. As a result, the binding between the ligand and the receptor can be detected by detecting the magnetic field of the magnetized magnetic fine particles again. In the present invention, a hard magnetic material can be used as the magnetic material fine particles. The hard magnetic material is not particularly limited, but magnetite (Fe ₃ O ₄ ) or maghemite (γ-Fe) is used.
A magnetic powder obtained by growing fine particles of ₂ O ₃ ) in a needle shape can be used. Highly accurate detection can be achieved by using a hard magnetic material having a high magnetic saturation point or a hard magnetic material that is less susceptible to thermal demagnetization.
In some cases, a magnetic substance may be bound to the receptor by a hydrophobic bond, and the magnetic substance may be used without being demagnetized. Alternatively, a magnetic substance derived from a microorganism can be used.

Through knowledge of the ligand at the position where binding is detected, it is possible to rapidly determine which ligand binds to the receptor, and thus this technique can be used to screen large numbers of receptors. it can.
Other possible uses of the invention include diagnostics, where:
Various ligands for a specific receptor are placed on a substrate, and the presence or absence of the receptor in a sample diagnoses a disease associated with the abnormality of the receptor.

(2) Detection Method and Device Using the Assembly of Filamentous Substrate of the Present Invention By using the device for measuring the binding between the ligand and the receptor of the present invention, the ligand and its receptor are separated. The binding measurement can be automated. The apparatus uses an assembly of filamentous substrates in which a plurality of different ligands are immobilized in a predetermined area on the surface thereof, and detects the binding between the ligand on the assembly of the substrates and the receptor in the sample. A) means for exposing said substrate to a receptor that binds at least one of said ligands; and b) for detecting a ligand-receptor bound region on an assembly of said substrates. Any device may be included. Examples of the apparatus include a means for transferring the aggregate of filamentous substrates, a prehybridization tank, a prehybridization tank, a labeled receptor tank, a washing tank, a drying means, a ligand / receptor binding detection means, and a ligand / receptor binding data analysis device. It can consist of a computer. Further, each of the above tanks may be one or plural.

FIG. 1 shows the structure of an example of the detection device of the present invention. The filamentous substrate aggregate 10 on which the ligand is immobilized is first transferred to the pre-hybridization pre-tank 30 by the transfer means 20 for the filamentous substrate aggregate, which is driven by the roller 21, and is pretreated. The pretreated substrate assembly is transferred to the pre-hybridization tank 40 and then to the labeled receptor tank 50.
Ligand-receptor binding occurs in the labeled receptor reservoir. The assembly of substrates is washed in wash tank I60 and wash tank II70. After washing, the substrate is transferred by the drying transfer means 80 and dried by the drying means 90. Next, the aggregate of the substrates is transferred to the ligand / receptor binding detection means 1.
00, and the binding between the ligand and the receptor is detected by the ligand / receptor binding detection means 110.
Information on the detected binding is analyzed by the computer 120 for analyzing ligand / receptor binding data. Examples of embodiments of the aggregate of the thread-like substrate of the present invention are shown in FIGS. That is, FIGS. 2 and 3 are enlarged views of a portion transferred by the ligand / receptor binding detection transfer means 100, and the binding between the ligand and the receptor is detected by the ligand / receptor binding detection means 110. Black circles represent the thread-like substrate of the present invention. FIG. 2 shows a mode in which there is a distance between a plurality of thread-shaped substrates, and FIG. 3 shows a mode in which a plurality of thread-shaped substrates are substantially in contact with each other.

As an example of the case where each of the above tanks is one,
In a container with an inlet and an outlet that function as a tank,
An example is a cassette in which an assembly of filamentous substrates and a winding means are housed. In this case, for example, the above-mentioned steps from pretreatment to cleaning are performed in the cassette while exchanging the liquid. Then attach the cassette to the drying / detection means,
Drying of the assembly of substrates and detection of ligand-receptor binding are performed while moving the assembly of substrates by winding or rotating means.

By using the device for measuring the binding between the ligand and the receptor of the present invention, the measurement of the binding between the ligand and the receptor can be automated.
The apparatus uses an assembly of filamentous substrates in which a plurality of different ligands are immobilized in a predetermined area on the surface thereof, and detects the binding between the ligand on the assembly of the substrates and the receptor in the sample. A device for detecting the region where the ligand and the receptor are bound on the aggregate of the substrate. When the device is used, it is convenient to use a substrate which has been previously subjected to the step of binding the ligand and the receptor on the substrate. In addition, it is convenient to use an aggregate of substrates which has been subjected to a step of binding a ligand and a receptor on the aggregate of substrates in advance.
In addition, after performing a step of binding a ligand and a receptor on a substrate or on an assembly of substrates, a region in which each ligand is immobilized is used to prepare an assembly of filamentous substrates, and the region is set to the above device. It may be detected by. In these devices, the measurement time can be arbitrarily adjusted by adjusting the moving speed of the aggregate of filamentous substrates on the device, and the binding measurement of the ligand and the receptor can be speeded up and made more efficient. it can.

In accordance with the present invention, improved detection devices and methods are disclosed. This detection method and apparatus uses an assembly of filamentous substrates in which a plurality of ligands are immobilized at known positions on the surface of the substrate. The assembly of filamentous substrates is, for example, sequentially exposed to a receptor, such as a fluorescently labeled receptor, which binds to one or more ligands. Further, in the case where a group of ligands is repeatedly immobilized on the filamentous substrate in a group unit, it may be exposed to a plurality of receptors in a group unit. That is,
If the apparatus has a plurality of labeled receptor tanks, each labeled receptor can be exposed in group units. The assembly of filamentous substrates is then successively placed in a detection device, for example a microscope detection device, for the purpose of locating the position where the binding took place. The microscope detection device includes a light source for directing light at the substrate, for example means for detecting fluorescence from the substrate, and means for determining the location of the fluorescence. Further, the detection device may be one that detects the binding between the ligand and the receptor electrically and timely. Furthermore, the detected signal is
It is also possible to improve the signal-to-background ratio (SN ratio) by measuring the aggregate of the filamentous substrates a plurality of times. Examples of means for detecting the fluorescence on the aggregate of the thread-like substrate include a photon counter. The means for determining the binding position of the ligand and its receptor may be fixed as the assembly of filamentous substrates passes through and may include x / y migration for migration.
The collection of data on the assembly of filamentous substrates passing through the detector is recorded and analyzed by a suitably programmed digital computer.

EXAMPLES The present invention will be described in more detail with reference to the following examples.
The invention is not limited to the scope of the examples.

Example 1 (1) For each of the cytokine-related gene, the gene affected by endocrine disruptor and the cancer-related gene, an assembly of the filamentous substrates of the present invention was prepared, and the gene expression of cells was performed using the substrate. investigated. That is, the cytokine-related genes selected from Tables 1 and 2 below, the genes affected by endocrine disruptors selected from Tables 3 and 4 below, and the cancer-related genes selected from Tables 5 and 6 below or DNA fragments thereof are Was prepared as follows.

[0042]

[Table 1]

[Table 2]

[0043]

[Table 3]

[Table 4]

[0044]

[Table 5]

[Table 6]

First, the nucleotide sequence information of the gene to be fixed or the gene of the gene product registered under a specific accession number (Accession No.) is obtained from GeneBank, and oligo primer is used based on this information. Analysis software (OLIGO ^TM Primer Analy
sis Software, manufactured by Takara Shuzo Co., Ltd., so as to obtain a DNA fragment of about 100 b to about 1 kb, which is optimum for the method of the present invention, parameters of the software (primer mode for PCR, primer length: 20 to 25 mer, string) Gentle parameter: very high to high) was set to construct a primer pair for PCR amplification. Next, it was synthesized using a DNA synthesizer based on the nucleotide sequences of the obtained primers. Regarding the above genes, Human UniG
If there was a primer for the gene in the ene DNA (Research Genetics) set, it was used. Using the primer pair synthesized above, a target PCR amplified fragment was obtained using genomic DNA, a genomic DNA library, or a cDNA library as a template according to the standard protocol conditions attached to a commercially available PCR kit. The obtained DNA fragment was purified using SUPREC-02 (Takara Shuzo). Nucleotide sequence analysis of the amplified cDNA was performed to confirm that it was the target fragment, and the amplified fragment was recovered by the ethanol precipitation method.
It was dissolved in 0 mM carbonate buffer (pH 9.5) to a concentration of 1 μM. In addition, a β-actin gene, a housekeeping gene, was prepared as a positive control in the same manner as in the above method. The plasmid pBR322 DNA was used as a negative control.

A portion of the above purified DNA fragment was added to 50 mM.
After dissolving in a carbonate buffer (pH 9.5), the concentration is measured by measuring the absorbance at 260 nm.
Those diluted in 5 steps within the range of 0.5 mg / ml were prepared. Affymetrix this DNA solution
417 Arrayer (manufactured by Affymetrix) was used to spot a cytokine-related gene, an endocrine disrupter-affected gene, and a cancer-related gene on separate filamentous substrates. Furthermore, one filamentous substrate on which the above-mentioned three groups of genes were spotted was also prepared. Three types of nylon fibers, polyester fibers, and cotton fibers were used as the filamentous substrate. Here, each spot was spotted with a diameter of 0.15 mm so that the distance between the spots was 1 mm or more. That is, 30 kinds of DNA fragments for each of the cytokine-related gene, the gene affected by endocrine disruptor and the cancer-related gene, and a group diluted in 5 steps for the positive and negative controls (35 × 5 =
175 spots) were spotted repeatedly.
The filamentous substrate on which the above DNA fragment was spotted had a temperature of 3 at room temperature.
After leaving it for 0 minutes, it was washed with 0.5 M Tris-hydrochloric acid (pH 7.5) containing 0.5 M sodium chloride, and then rinsed with 2 × SSC (Wako Pure Chemical Industries, Ltd.). Then, the DNA was fixed by UV irradiation for about 2 minutes using a transilluminator (manufactured by Cosmo Bio Inc.). In this way, a DNA array, which is a filamentous substrate, was prepared.

(2) Next, 10% fetal bovine serum (FBS)
Breast cancer cells MCF- grown in DME medium containing
After 7 was trypsinized, 2 × 10 ⁶ cells were put into a dish having a diameter of 10 cm and cultured in a DME medium containing 5% fetal bovine serum from which steroid hormones were removed by activated carbon-dextran treatment for 24 hours. After removing the medium, the cells were cultured for 2 hours in the same medium containing 10 nM diethylstilbestrol (DES). Also,
As a control, a substance in the absence of the aforementioned chemical substances was similarly prepared. The cells after each treatment were collected, and total RNA was prepared using Trizol reagent (manufactured by Gibco BRL).

(3) D of total RNA prepared in (2) above
Nase treatment was performed. About 100 to about 300 μg of total RNA described above, 10 μl of 10 × DNaseI buffer (Takara Shuzo) and 10 U of DNaseI (Takara Shuzo)
Was prepared and incubated at 37 ° C. for 10 minutes, followed by phenol / chloroform extraction twice, followed by ethanol precipitation. Total R obtained
A part of NA was taken and the concentration was measured.

(4) A reverse transcription reaction was carried out using the total RNA prepared in (3) above. The composition of the reaction solution is shown below. Reaction solution A: about 130 μg of total RNA described above, 300 pmo
l Oligo dT primer (Takara Shuzo) and 20 μl
Water treated with diethylpyrocarbonate (DEPC, manufactured by Wako Pure Chemical Industries, Ltd.). Reaction solution B: 12 μl of 5 × AMV RTase buffer (manufactured by Life Science), 0.5 mM dATP, dCTP, dGTP and 0.2 mM dTT
P, 60 U RNase inhibitor (Takara Shuzo),
0.1 mM Cy3-labeled dUTP (Amersham Pharmacia). After holding the reaction solution A at 70 ° C. for 10 minutes,
Cooled on an ice bath. Then, the reaction solution B was added, and the mixture was kept at 42 ° C. for 5 minutes. After that, further AMV RTase
About 60 U (manufactured by Life Science) was added to make the reaction solution volume 60 μl. This RT reaction solution was kept at 42 ° C. for 70 minutes. 5. 500 mM EDTA solution in this reaction solution
Add 5 μl of 1 M sodium hydroxide 15 μl and add 6
It was kept at 0 ° C for 1 hour to decompose the template RNA. After cooling to room temperature, add 1 M Tris-hydrochloric acid (pH 7.5) to 3
7.5 μl was added. This solution is Microcon ^TM- 30
Concentrate to 20 μl with (Millipore) and 1 mM ED
After 200 μl of 10 mM Tris-hydrochloric acid containing TA was added, the solution was concentrated again to 20 μl. This Cy3 labeled cDN
Solution A was used for subsequent hybridization.

(5) The filamentous substrate prepared by the above method (1) was impregnated with 2 × SSC for 1 minute, and then 4 × SSC,
Prehybridization was performed at 25 ° C. for 1 hour in a solution containing 0.2% SDS and 1 mg / ml salmon sperm DNA. Next, after subjecting the Cy3-labeled target DNA prepared in (4) to heat denaturation, 4 × SSC, 0.2% SDS and 100 μ were added so that the target DNA would be about 10 μg / ml.
The suspension was suspended in a hybridization solution containing g / ml salmon sperm DNA, and the DNA array on the filamentous substrate was incubated at 60 ° C. overnight in the hybridization solution. After the hybridization, a washing operation at room temperature for 5 minutes was performed twice with a solution containing 2 × SSC and 0.1% SDS. Furthermore, 0.1 × SSC and 0.1%
A washing step at 68 ° C. for 15 minutes was performed twice with a solution containing SDS. Then, the fluorescence signal of each spot was measured by applying a fluorescence detector capable of continuously measuring the fluorescence signal on the substrate from the short side, and the binding between the labeled receptor and the ligand was analyzed. Furthermore, in the present invention, when any of nylon fiber, polyester fiber, and cotton fiber is used, a significant signal of expression for each of cytokine-related gene, gene affected by endocrine disruptor and cancer-related gene It was possible to observe the fluctuation of

Example 2 (1) An automatic detection device using an aggregate of a plurality of thread-like substrates was examined. The apparatus comprises a transfer means capable of processing a plurality of filamentous substrates in parallel, a prehybridization tank, a prehybridization tank, a labeled receptor tank, a washing tank I, a washing tank II, a drying means, a ligand / receptor binding detection means, An apparatus consisting of a computer for analyzing ligand / receptor binding data was prepared, and the filamentous substrate prepared in Example 1 (1) was continuously moved using the apparatus to continuously bond the ligand and the receptor. It was measured. A 2 × SSC solution was placed in the pre-hybridization tank. 4 × SSC, 0.2% for prehybridization tank
A solution containing SDS and 100 μg / ml salmon sperm DNA was added. Furthermore, in the labeled receptor tank, Example 1 was used.
After heat denaturing the Cy3-labeled target DNA prepared in (4), it was adjusted to about 10 μg / ml with 4 × SS.
C, 0.2% SDS and 1 mg / ml salmon sperm DNA
What was suspended in the hybridization solution containing was added. 2 x SSC and 0.1% S in the washing tanks I and
A solution containing DS was added. 0.1 x S in the cleaning tank II
A solution containing SC and 0.1% SDS was added.

(2) Three filamentous substrates on which the cytokine-related gene, the gene affected by endocrine disruptor and the cancer-related gene prepared in Example 1 (1) are spotted are set in the above apparatus, and the filamentous substrate is prepared. Was passed through a pre-hybridization bath set at room temperature. next,
It was passed through a prehybridization bath set at 25 ° C. and kept for 1 hour. Next, it was passed through a labeled receptor tank containing Cy3-labeled target DNA (receptor) set at 60 ° C. and kept overnight. Then, it wash | cleaned through the washing tank I set to room temperature, and also passed through the washing tank II set to 68 degreeC. The washed filamentous substrate was passed through the fluorescence detector so that it became one filament at the entrance of the fluorescence detector, the fluorescence signal of each spot was measured, and the binding between the labeled receptor and the ligand was analyzed. As a result, the same result as the data obtained in Example 1 could be obtained.

[0053]

INDUSTRIAL APPLICABILITY The present invention provides a substrate and a measuring device for continuously and rapidly measuring the binding between a ligand and a receptor, which is useful in various fields of biochemistry and fields such as diagnosis. An immobilized filamentous substrate, a carbohydrate-immobilized filamentous substrate, a protein-immobilized filamentous substrate, a peptide-immobilized filamentous substrate and the like are provided. Further, by using the filamentous substrate, the binding between the ligand and the receptor can be measured at high speed without increasing the density of the ligand per unit area on the thread. That is, according to the present invention, it becomes possible to detect the binding between the ligand and the receptor at a high speed, the detection can be performed at high speed, and the density on the substrate can be increased, and the cost can be reduced.
It is possible to obtain an effect equal to or higher than that of the high-density substrate by using a substrate having a low density region where the ligand is immobilized, which can be produced accurately and easily. Further, in the present invention, since the ligands are arranged in a line, the detector only needs to detect the presence or absence of the binding between the ligand and the receptor, and there is no need for the secondary scanning of the detector in detecting the labeled substance. This is the remarkable simplicity of the present invention.

[0054]

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a structure of a detection device using an assembly of thread-shaped substrates of the present invention.

FIG. 2 is a diagram showing an example of an enlarged view of a detection portion of a detection device using the assembly of thread-shaped substrates of the present invention.

FIG. 3 is a diagram showing an example of an enlarged view of a detection portion of a detection device using the assembly of thread-shaped substrates of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 37/00 102 G01N 37/00 102 C12N 15/00 F

Claims (14)

[Claims] 1. An aggregate of two or more filamentous substrates having a plurality of different ligands immobilized on a predetermined region on the surface thereof. 2. The assembly of filamentous substrates according to claim 1, wherein different ligand groups are immobilized for each filamentous substrate. 3. The filamentous bases are gathered in parallel.
Or the assembly of the thread-like substrates according to 2. 4. The assembly of thread-like substrates according to claim 1, wherein the thread-like substrates are assembled in a flat plate shape. 5. The filamentous substrate is assembled in series.
Or the assembly of the thread-like substrates according to 2. 6. A filamentous substrate having a plurality of different ligands immobilized on a predetermined region on the surface thereof, which is a filamentous substrate selected from a group of previously produced filamentous substrates. An aggregate of the filamentous substrate according to any one of items. 7. The ligand according to claim 1, which is a nucleic acid, a carbohydrate, a peptide, a protein, a lipid or a cell.
Item 2. An assembly of thread-like substrates according to item. 8. The filamentous substrate assembly according to claim 7, wherein the nucleic acid is a double-stranded DNA or a single-stranded DNA. 9. The filamentous substrate assembly according to claim 8, wherein the DNA is a polynucleotide or an oligonucleotide. 10. The aggregate of the filamentous substrate according to claim 1, wherein the binding between the ligand and the receptor having the binding property to the ligand can be detected at a high speed. 11. The assembly of filamentous substrates according to claim 10, wherein the receptor is a nucleic acid, a carbohydrate, a peptide, a protein, a lipid or a cell. 12. The assembly of filamentous substrates according to claim 11, wherein the receptor is a labeled receptor. 13. A device for detecting the binding between a ligand and a receptor, comprising: a) an assembly of filamentous substrates having a plurality of different ligands immobilized in predetermined regions on the surface thereof; b). An apparatus comprising: means for exposing a population of the substrate to a receptor that binds at least one of the ligands; and c) means for detecting the ligand-receptor bound region on the substrate. 14. A method for detecting the binding between a ligand and a receptor, comprising: a) a filamentous substrate in which a plurality of different ligands are immobilized in a predetermined region on the surface of the receptor that binds to at least one of the ligands. And b) detecting a region where the ligand and the receptor are bound on the substrate, and a method of detecting binding between the ligand and the receptor.