JP2003014767A - Non-specific hybridization inhibitors, clinical test drugs and clinical test methods - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To increase the accuracy of the reaction at the time of hybridization and to suppress nonspecific hybridization, to suppress nonspecific hybridization in clinical test drugs and clinical tests, To provide a clinical test method capable of easily and accurately detecting a nucleic acid-related substance. The inhibitor and the clinical test agent of the present invention comprise a polymer (H) having a hydrophilic group in the molecule and having a nonspecific hybridization inhibitory function with a weight average molecular weight of 1,000 to 5,000,000, and a clinical test. The method includes a step of contacting a sample with a test agent that hybridizes to a predetermined nucleic acid-related substance in the presence of the inhibitor.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an inhibitor for suppressing non-specific hybridization when measuring a nucleic acid-related substance to be measured in a sample in the field of clinical tests, etc., and a clinical test drug using the same. And the clinical laboratory method.

[0002]

2. Description of the Related Art In the field of clinical examinations, a hybridization method is used for detecting nucleic acid-related substances such as DNA and RNA. The method involves contacting a labeled sample with a solid phase on which DNA having a sequence complementary to the nucleic acid-related substance to be measured is immobilized, and then washing to remove all but the nucleic acid-related substance to be measured. Is a method for measuring the activity of a labeling substance bound to a solid phase. In the field of clinical examination, when a nucleic acid-related substance to be measured is measured using a hybridization method, it is required to accurately recognize the sequence of the nucleic acid-related substance. Conventionally, when measuring a sample using a hybridization method, in order to suppress non-specific hybridization, sodium dodecyl sulfate (abbreviated as SDS), N-lauroyl sarcosine (abbreviated as N-LS), etc. Agent: It is widely known to add proteins such as bovine serum albumin (abbreviated as BSA) and casein. However, the surfactant and the protein have almost no effect of suppressing non-specific hybridization, and cannot accurately recognize the sequence of the nucleic acid-related substance. Also, Patrick et al. (Nature Biotechnolog
y, 17, 365-370 (1999)) have proposed a method of applying an electric current to the surface of a DNA chip to suppress SNPs of DNA. However, this method requires a dedicated DNA chip and cannot be applied to all existing DNA chips. On the other hand, studies have been conducted to use polymers having phosphorylcholine-like groups in the field of clinical examination. Phosphorylcholine group-containing polymer, due to the phospholipid-like structure derived from biological membrane,
It is known to have excellent biocompatibility such as blood compatibility, complement deactivation, nonadsorption of biological substances, and excellent properties such as antifouling property and moisturizing property. Therefore, research and development relating to the synthesis of polymers and the use thereof for the purpose of developing biomaterials that take advantage of these functions are being actively conducted. JP-A-7-5177, JP-A-7-83923, JP-A-10-11
In 4800 publication, a phosphorylcholine group-containing polymer,
The technology of highly accurate clinical test by suppressing the adsorption of protein to the container has been disclosed. However, it is not known that a phosphorylcholine group-containing polymer can be used to accurately recognize the sequence of a nucleic acid-related substance, that is, non-specific hybridization can be suppressed.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a non-specific hybridization inhibitor capable of increasing the accuracy of the reaction during hybridization and suppressing non-specific hybridization. Another object of the present invention is a clinical test drug capable of suppressing non-specific hybridization at the time of hybridization for the purpose of clinical test, efficiently and highly accurately detecting a nucleic acid-related substance to be measured. To provide. Another object of the present invention is to provide a clinical test method capable of suppressing non-specific hybridization in a clinical test and easily and highly accurately detecting a nucleic acid-related substance to be measured.

[0004]

According to the present invention, a non-specific polymer (H) having a hydrophilic group in the molecule and having a non-specific hybridization suppressing function having a weight average molecular weight of 1,000 to 5,000,000 is included. Hybridization inhibitors are provided. Further, according to the present invention, a clinical test drug containing the above-mentioned inhibitor and a test drug is provided. Furthermore, according to the present invention, a step of contacting a sample with a test agent that hybridizes to a predetermined nucleic acid-related substance under predetermined conditions in which the predetermined nucleic acid-related substance and the test agent hybridize in the presence of the inhibitor. There is provided a clinical test method including (1) and a step (2) of detecting a reaction product generated by hybridization with a test agent in the step (1).

[0005]

The present invention will be described in more detail below. The inhibitor of the present invention comprises a polymer (H) having a hydrophilic group in the molecule and having a non-specific hybridization inhibitory function of a specific molecular weight. Examples of the hydrophilic group of the polymer (H) include anionic groups, amphoteric charge-containing groups, cationic groups, amide groups, lactam groups and polyalkylene oxide groups. The polymer (H) has one or more kinds of these groups in the molecule. Polymer (H) is more effective in suppressing non-specific hybridization.
Preferably has an amphoteric charge-containing group, or both an amphoteric charge-containing group and an anionic group. Specifically, a phosphorylcholine-like group (abbreviated as PC group)
Or having a PC group and an anionic group such as a carboxyl group and / or a sulfone group.

Examples of the anionic group include a sulfone group, a carboxyl group and a phosphonic acid group. Examples of the amphoteric charge-containing group include betaine group and PC group. Examples of the cationic group include a primary amino group, a secondary amino group, a tertiary amino group, and a quaternary ammonium group. Examples of the amide group include an acrylamide group and a methacrylamide group. As the lactam group, for example, 4-butanelactam (2-pyrrolidone), 4-
Examples thereof include butanelactim (2-hydroxy-1-pyrroline) and 6-hexanelactam (ε-caprolactam) groups. Examples of the polyalkylene oxide group include a polyethylene oxide group, a polypropylene oxide group, or a block-type or random-type hydrophilic group of a polyethylene oxide group and a polypropylene oxide group. Among the above specific examples, the hydrophilic group is highly hydrophilic and highly effective in suppressing non-specific hybridization, and is one selected from the group consisting of a PC group, a lactam group, a 4-butanelactam group and a polyethylene oxide group. Alternatively, two or more kinds are preferable.

In the polymer (H), the content ratio of the hydrophilic group is not particularly limited as long as it has a suppressing effect on non-specific hybridization, and it depends on the kind of the hydrophilic group and the molecular weight of the polymer (H). It can be selected as appropriate. The molecular weight of the polymer (H) is usually 1000 to 500000 in terms of weight average molecular weight.
It is 0, preferably 10000 to 2000000. The molecular weight is 1000
If it is less than 5, it is difficult to sufficiently suppress non-specific hybridization, and if it exceeds 5,000,000, the viscosity becomes too high and the hybridization may be inhibited, which is not preferable.

The polymer (H) is prepared, for example, by a monomer having a PC group (abbreviated as PC monomer), a monomer having a sulfone group (abbreviated as SA monomer), a monomer having a carboxyl group (abbreviated as CA monomer), an amide. A hydrophilic monomer such as a monomer having a group, or a hydrophilic monomer,
It can be obtained by polymerizing a hydrophobic monomer having a hydrophobic group and not a hydrophilic group by a known radical polymerization method or the like. Upon polymerization, one or more hydrophilic monomers can be used, and one or more hydrophobic monomers can also be used.

Examples of the PC monomer include the monomer represented by the formula (1).

[Chemical 3] In the formula (1), X represents a divalent organic residue, Y represents an alkyleneoxy group having 1 to 6 carbon atoms, Z is a hydrogen atom or R ⁵ O (C = O).
-Indicates. However, R ⁵ represents an alkyl group having 1 to 10 carbon atoms or a hydroxyalkyl group having 1 to 10 carbon atoms. R ¹ represents a hydrogen atom or a methyl group, and R ² , R ³ and R ⁴ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group. m is 0 or 1, and n is an integer of 1 to 4.

[0010] As the X is, for _{example, -C 6 H 4 -, -} C 6 H 10
-, - (C = O) O -, - O -, - CH 2 O -, - (C = O) NH -, - O (C = O) -, - O
(C = O) O -, - C 6 H 4 O -, - C 6 H 4 CH 2 O -, - C 6 H 4 (C = O) O- and the like. Examples of Y include a methyloxy group, an ethyloxy group, a propyloxy group, a butyloxy group, a pentyloxy group, and a hexyloxy group. Z
R ⁵ is, for example, a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group, a heptyl group, an octyl group, a nonyl group, an alkyl group having 1 to 10 carbon atoms such as a decyl group, or hydroxymethyl. Group, 2-hydroxyethyl group,
3-hydroxypropyl group, 2-hydroxypropyl group, 4-
Hydroxybutyl group, 2-hydroxybutyl group, 5-hydroxypentyl group, 2-hydroxypentyl group, 6-hydroxyhexyl group, 2-hydroxyhexyl group, 7-hydroxyheptyl group, 2-hydroxyheptyl group, 8-hydroxyoctyl group And a hydroxyalkyl group having 1 to 10 carbon atoms such as a group, a 2-hydroxyoctyl group, a 9-hydroxynonyl group, a 2-hydroxynonyl group, a 10-hydroxydecyl group and a 2-hydroxydecyl group.

Examples of the PC monomer include 2-((meth))
Acryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, 3-((meth) acryloyloxy) propyl-2'-(trimethylammonio) ethyl phosphate, 4-((meth) acryloyloxy) butyl-2 '-(Trimethylammonio) ethyl phosphate, 5-((meth) acryloyloxy) pentyl-2'-(trimethylammonio) ethyl phosphate, 6-((meth) acryloyloxy) hexyl-2 '-(trimethylammonio ) Ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2 '-(triethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2'-(tripropylammonio) ethyl phosphate, 2 -((Meth) acryloyloxy) ethyl-2 '-(tributylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2'-(tricyclohexylan NIO) ethyl phosphate, 2-
((Meth) acryloyloxy) ethyl-2 '-(triphenylammonio) ethyl phosphate, 2-((meth) acryloyloxy) ethyl-2'-(trimethanolammonio) ethyl phosphate, 2-((meth) Acryloyloxy) propyl-2 '-(trimethylammonio) ethyl phosphate, 2-
((Meth) acryloyloxy) butyl-2 '-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy) pentyl-2'-(trimethylammonio) ethyl phosphate, 2-((meth) acryloyloxy ) Hexyl-
2 '-(trimethylammonio) ethyl phosphate, 2- (vinyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate, 2- (allyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, 2- ( p-Vinylbenzyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, 2- (p-vinylbenzoyloxy) ethyl-2'-
(Trimethylammonio) ethyl phosphate, 2- (styryloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, 2- (p-vinylbenzyl) ethyl-2'-(trimethylammonio) ethyl phosphate, 2- (Vinyloxycarbonyl) ethyl-2 '-(trimethylammonio) ethyl phosphate, 2- (allyloxycarbonyl) ethyl-
2 '-(trimethylammonio) ethyl phosphate, 2- (acryloylamino) ethyl-2'-(trimethylammonio)
Ethyl phosphate, 2- (vinylcarbonylamino) ethyl-2 '-(trimethylammonio) ethyl phosphate, ethyl- (2'-trimethylammonioethylphosphorylethyl) fumarate, butyl- (2'-trimethylammonioethylphosphorylethyl) ) Fumarate, hydroxyethyl-
(2'-Trimethylammonioethylphosphorylethyl) fumarate, ethyl- (2'-trimethylammonioethylphosphorylethyl) malate, butyl- (2'-trimethylammonioethylphosphorylethyl) malate, hydroxyethyl- (2'- Trimethylammonioethylphosphorylethyl) malate. Among them, 2-((meth) acryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate is preferable, and further 2- (methacryloyloxy) ethyl-2'-(trimethylammonio) ethyl phosphate (2- (methacryloyloxy ) Ethylphosphorylcholine) (abbreviated as MPC) is more preferable in terms of availability. Here, (meth) acryloyl means methacryloyl and / or acryloyl.

The PC monomer can be produced by a known method.
For example, as disclosed in JP-A-54-63025, 2-hydroxyethyl methacrylate and 2-bromoethylphosphoryl dichloride are reacted in the presence of a tertiary base, and the resulting compound is reacted with a tertiary amine. Method, JP-A-58-154591
After the reaction of the hydroxyl group-containing polymerizable monomer with the cyclic phosphorus compound, which is disclosed in Japanese Patent Publication No. 2003-242242, the compound can be produced according to a known method of ring opening with a tertiary amine.

Examples of the SA monomer include 2-methylpropanesulfonic acid, 2-dimethylpropanesulfonic acid, styrenesulfonic acid and 2-sulfoethylmethacrylate. Preferably, 2-methylpropanesulfonic acid,
2-Dimethylpropanesulfonic acid and styrenesulfonic acid may be mentioned. Examples of the CA monomer include (meth) acrylic acid, 3-pentenoic acid, 4-pentenoic acid, 3-acryloxypropionic acid, and 2- (meth) acryloyloxyethyl phthalic acid. Preferred are methacrylic acid and acrylic acid.

Examples of the hydrophobic monomer include the monomer represented by the formula (2).

[Chemical 4] In the formula (2), R ⁶ represents a hydrogen atom or a methyl group, L ¹ is -C ₆ H ₄
-, -C ₆ H ₁₀ -,-(C = O) O-, -O-,-(C = O) NH-, -O (C = O)-or
-O (C = O) O- is shown, L ² is a hydrogen atom,-(CH ₂ ) gL ³ or ((C
Shows the _{H 2) p -O) h -L} 3. g and h are 1 to 24, p is an integer of 3 to 5, L ³ represents a hydrogen atom, a methyl group, -C ₆ H _5, the -OC ₆ H _5.

Examples of the hydrophobic monomer include methyl.
Linear or branched alkyl (meth) acrylate such as (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate; cyclohexyl ( Cyclic alkyl (meth) acrylates such as (meth) acrylate; aromatic rings such as benzyl (meth) acrylate and phenoxyethyl (meth) acrylate
(Meth) acrylate; polypropylene glycol (meth)
Polyalkylene glycol (meth) acrylates such as acrylates; styrene-based monomers such as styrene, methylstyrene, chloromethylstyrene; vinyl ether-based monomers such as methyl vinyl ether and butyl vinyl ether; vinyl ester-based monomers such as vinyl acetate and vinyl propionate. To be Preferred are butyl methacrylate and lauryl methacrylate.

The polymer (H) is preferably a polymer having a PC group (abbreviated as PC polymer), and more preferably a PC polymer containing both a PC group and an anionic group. polymer
As (H), for example, a hydrophilic monomer 40 to 100 mol%,
And a polymer obtained by polymerizing a monomer composition comprising 0 to 60 mol% of a hydrophobic monomer, preferably a hydrophilic monomer
A polymer obtained by polymerizing a monomer composition composed of 50 to 100 mol% and a hydrophobic monomer of 0 to 50 mol% can be mentioned.
When the hydrophilic monomer content is less than 40 mol%, the effect of the hydrophilic group is not sufficient, which is not preferable. Furthermore, as the PC polymer, for example, a PC monomer is composed of 5 to 100 mol%, a monomer having an anionic group, preferably at least one of a carboxyl group and a sulfone group, 0 to 95 mol%, and a hydrophobic monomer, 0 to 60 mol%. A polymer obtained by polymerizing the monomer composition may be mentioned. Specifically, PC monomer is usually 5
~ 95mol%, preferably 10-90mol%, and SA monomer and / or CA monomer is usually 5-95mol%, preferably 10-90
Polymer obtained by polymerizing a monomer composition containing mol%, or PC monomer is usually 5 to 90 mol%, preferably
Monomer composition containing 10 to 85 mol%, SA monomer and / or CA monomer usually 5 to 90 mol%, preferably 10 to 85 mol%, and hydrophobic monomer usually 5 to 60 mol%, preferably 5 to 50 mol% A polymer obtained by polymerizing
When the PC monomer content is less than 5 mol%, the effect of the PC group is not sufficient, which is not preferable. Such PC polymer can also be produced by ordinary radical copolymerization.

The inhibitor of the present invention may contain the polymer (H). In use, the polymer (H) can be added and mixed in the system at the time of hybridization so as to be usually 0.0001 to 20% by weight, preferably 0.001 to 10% by weight. If the polymer (H) is less than 0.0001% by weight, the effect of suppressing non-specific hybridization may not be sufficiently exerted, and if it exceeds 20% by weight, the viscosity of the system may become too high, which may hinder the hybridization reaction. Absent. The inhibitor of the present invention may contain other components such as a surfactant, a solvent and an antiseptic as long as the effects of the present invention are not impaired. The inhibitor of the present invention may be in the form of powder or solution.

The clinical test drug of the present invention is a test drug kit containing the inhibitor of the present invention and a test drug. The test agent is not particularly limited as long as it is a nucleic acid-related substance that hybridizes with a predetermined nucleic acid-related substance that is a measurement target, and depending on the measurement target,
For example, a test agent used as a known clinical test agent can be used.

A nucleic acid-related substance is one in which one or more molecules of phosphate are bound to a carbohydrate molecule, for example, pentose or hexose, and the molecule is, for example, from a purine such as adenine or a pyrimidine such as thymine. Means any group of one or more compounds that each bind to a base derived from In particular, naturally-occurring nucleic acid molecules include genomic DNA, genomic RNA and various different types of RNA, such as mRNA, tRNA and rRNA, including cDNA, and also synthetic or naturally-occurring DNA. It also includes hybrids with synthetic DNA.

The nucleic acid-related substance may be either single-stranded or double-stranded, linear or circular, for example, a plasmid, or an oligonucleotide or a polynucleotide. In addition, the number of bases from about 10 bases or base pairs to 2000 bases
It also contains about 0 bases or base pairs (20 kb). In addition to these naturally occurring substances, for example, those obtained from the sources of the ATCC or Gene Bank libraries, as well as synthetic compositions, ie nucleic acid analogues are included. Synthetic nucleic acids can be prepared by a variety of solution or solid phase methods. Solid phase synthesis is generally preferred,
Phosphite triester, phosphotriester and H-
Nucleic acid synthesis by phosphonate chemistry and the like has been widely performed (Itakura USP 4,458,066 specification, USP 4,500,707 specification; Garuters et al. Genetic Engineering, 4: 1-17 (1982); Jo.
nes Chapter 2, Atkinson et al. Chapter 3 and Sproat et al. Chapter 4 Oligonu
cleotids Synthesis: A Practical Approach, Gait (ed.),
IRL Press, Washington DC (1984)).

In the clinical test drug of the present invention, the ratio of the inhibitor of the present invention can be appropriately selected according to the method of clinical test and the measurement object, and the polymer (H) in the inhibitor is usually 0.0001 in the hybridization system. ~ 20% by weight, preferably 0.0
The proportion may be 01 to 10% by weight. The clinical test drug of the present invention may contain other compounds necessary for the test drug, and may contain various additives, solvents and the like which are usually used for clinical test drugs. The method of using the clinical test agent is not particularly limited, and for example, the clinical test method of the present invention described later can be used.

In the clinical test method of the present invention, first, a specimen and a test agent that hybridizes to a predetermined nucleic acid-related substance are hybridized with a predetermined nucleic acid-related substance and the test drug in the presence of the inhibitor of the present invention. The step (1) of contacting soybeans under predetermined conditions is performed. The step (1) can be performed using the inhibitor of the present invention or the clinical test agent of the present invention. Step (1), for example, by adding the inhibitor of the present invention in a system containing a sample and a test agent, a method of maintaining a predetermined condition, maintaining a system containing a sample and a test agent in a predetermined condition, A method of adding the inhibitor of the present invention into the system, or a method of previously adding the inhibitor of the present invention to a sample and / or a test agent, and then contacting the sample and the test agent under predetermined conditions, etc. Can be done by.

The step (1) can be carried out, for example, when the test agent is immobilized on the carrier or after the test agent is immobilized on the carrier and the inhibitor of the present invention is added to hybridize with the sample. At this time, the form of the inhibitor of the present invention is:
It may be in a solution state or a powder state, but a solution state which becomes promptly homogeneous with the reaction system is preferable. Examples of the carrier include, but are not limited to, a film such as a nitrocellulose film, a nylon film, and a PVDF film; a plate or a flat plate made of polystyrene, polypropylene, glass, metal, or the like. Examples of the method of fixing to the carrier include an electrostatic binding method, a method utilizing ionicity, a method of covalently bonding to a carrier with ultraviolet rays and the like, and a method of covalently bonding with a crosslinking reagent. In addition to the method using the carrier, the step (1) can be performed in a solution. Examples of the method include the polymerase chain reaction method (PCR
Method), a sequence reaction method, an RNA-dependent DNA synthesis reaction method using a reverse transcriptase, and various transcription reaction methods using a DNA polymerase used in genetic engineering.

In step (1), the predetermined conditions under which the predetermined nucleic acid-related substance and the test agent hybridize are, for example, when the predetermined nucleic acid-related substance to be measured is DNA, appropriate conditions in Southern hybridization, When the predetermined nucleic acid-related substance to be measured is RNA, it can be appropriately selected from the appropriate conditions in Northern hybridization.

In the clinical test method of the present invention, the step (2) of detecting the reaction product generated by the hybridization with the test agent in the step (1) is performed. In step (2), the detection of the reaction product can be carried out, for example, by labeling with a known labeling technique for detecting the reaction product and measuring the activity of the label. As a labeling method, for example, ³² P,
Examples include methods using radioisotopes such as ¹⁴ C and ³ H, methods using fluorescent dyes, methods using enzyme proteins, and methods using antibodies to various antigenic substances. Examples of the preparation of these labeled compounds include a method of obtaining the labeled compound with a DNA polymerase as a substrate, a method of chemically reacting the labeled compound, and the like. On the other hand, the detection of the reaction product can be appropriately selected depending on the labeling method. For example, in the case of a method using a radioisotope, it can be detected by a scintillation counter or autoradiography, in the case of a method using a fluorescent dye, it can be detected by a detection device using a laser or the like, in the case of a method using an enzyme protein or various antigens. Can be detected by chemiluminescence, fluorescence detection, coloring of an enzyme substrate, or the like.

The clinical test method of the present invention comprises the steps (1) and
Any method may be used as long as it includes at least (2), and various known methods can be easily used. A specific example using Southern blot hybridization will be described below. Although the inhibitor of the present invention is not described in the specific examples, for example, the clinical test method of the present invention can be carried out by appropriately adding the above-mentioned preferred ratios at the above-mentioned various addition times.

First, a genomic DNA is prepared from a biological tissue such as blood using ISOGEN (manufactured by Nippon Gene Co., Ltd.). Then, the obtained DNA is separated by 0.7% agarose gel electrophoresis. After electrophoresis, the gel is placed in 0.25N hydrochloric acid solution at room temperature for 15
Soak in 0.4N sodium hydroxide solution for 30 minutes at room temperature with gentle shaking. On the blot table prepared in advance, the gel, Gene Screen Plus membrane (D
upont), 3MM filter paper (Whatman) Kim towel and weights are sequentially stacked and left overnight, and the DNA in the gel is screened by GeneScreen.
Transfer to Plus membrane. After the transfer is completed, the Gene Screen Plus membrane is immersed in 0.5 M Tris buffer for 15 minutes at room temperature. After drying at room temperature, baking is further performed at 80 ° C for 2 hours. The resulting membrane is 2
× SSC (0.3M sodium chloride, 0.03M sodium citrate)
Pre-hybridization is carried out by shaking in, then immersing in a rapid hybridization buffer (manufactured by Amersham Pharmacia) and shaking at 65 ° C. for 15 minutes. Beforehand [γ
^The desired DNA probe solution ( ³² P label) prepared by the nick translation method using ³² P] -dCTP is added to the hybridization buffer, and hybridization is performed at 65 ° C. for 2 hours. Shake with 0.1 x SSC (0.015M sodium chloride, 0.0015M sodium citrate) at 65 ° C for 15 minutes. Repeat this operation twice to wash out unnecessary probes. After removing excess water on the filter paper, -70 ℃, 24 hours
The radiation emitted from the probe is printed on the X-ray film. One method is to develop an X-ray film and quantify the band of interest with a densitometer (Molecular Cloni
ng A LABORATORY MANUAL 2nd EDITION, Cold Spring Ha
rbor Laboratory Press (1989)).

[0028]

EFFECT OF THE INVENTION Since the inhibitor of the present invention has an inhibitory effect on non-specific hybridization, the accuracy of the reaction during hybridization can be increased. In the clinical test agent and clinical test method of the present invention, since the inhibitor of the present invention is used, non-specific hybridization at the time of hybridization for the purpose of clinical test is suppressed, and the measurement target can be measured efficiently and with high accuracy. A nucleic acid-related substance can be detected.

[0029]

EXAMPLES The present invention will be described in more detail with reference to Synthesis Examples, Examples and Comparative Examples, but the present invention is not limited thereto. Synthesis Example 1 MPC 19.4 g and methacrylic acid (abbreviated as MA) 0.6 g were dissolved in 40 g of water and placed in a four-necked flask, and after blowing nitrogen for 30 minutes, 1.6 g of succinic acid peroxide was added at 60 ° C. to obtain 8 The polymerization reaction was carried out for a time. The polymerization solution was added dropwise to 3 L of acetone while stirring, the deposited precipitate was filtered, and vacuum dried at room temperature for 48 hours to give a polymer powder (abbreviated as polymer (P1)) 14.6.
got g. The molecular weight of the polymer (P1) was analyzed by gel permeation chromatography (GPC). The analysis conditions were 20 mM phosphate buffer (pH 7.4) as an eluent, polyethylene glycol as a standard substance, and detection by refractive index. Further, the composition ratio and the hydrophilic group of the polymer (P1) were measured by ¹ H-NMR. The results are shown in Table 1.

Synthetic Examples 2 to 5 Polymer powders were prepared in the same manner as in Synthetic Example 1 except that the types and composition ratios of the monomers used in Synthetic Example 1 were changed as shown in Table 1. Each polymer powder obtained is polymer (P2) ~
(P5), the same analysis as in Synthesis Example 1 was performed. The results are shown in Table 1.

Synthesis Examples 6 and 7 The types and composition ratios of the monomers used in Synthesis Example 1 were changed as shown in Table 1, and 120 g of isopropanol was used instead of 40 g of water.
Was used, and a polymer powder was prepared in the same manner as in Synthesis Example 1 except that 0.7 g of azobisisobutyronitrile (abbreviated as AIBN) was used instead of 1.6 g of succinic acid peroxide. The obtained polymer powders were designated as polymers (P6) and (P7), and synthesis examples
The same analysis as in 1 was performed. The results are shown in Table 1. In addition, in Table 1, BMA represents butyl methacrylate.

Synthesis Examples 8 to 10 The kinds and composition ratios of the monomers used in Synthesis Example 1 were changed as shown in Table 2, and the amount of succinic acid peroxide used was changed to 0.65.
A polymer powder was prepared in the same manner as in Synthesis Example 1 except that g was replaced. Each polymer powder obtained is polymer (P8) ~ (P10)
Then, the same analysis as in Synthesis Example 1 was performed. The results are shown in Table 2. In Table 2, MPs represents 2-methylpropanesulfonic acid.

Synthesis Examples 11 to 13 Except that the types and composition ratios of the monomers used in Synthesis Example 1 were changed as shown in Table 2, the amount of water used was changed to 120 g, and the amount of succinic acid peroxide used was changed to 0.23 g. A polymer powder was prepared in the same manner as in Synthesis Example 1. The obtained polymer powders were designated as polymers (P11) to (P13), and the same analysis as in Synthesis Example 1 was performed. The results are shown in Table 2. In Table 2, Am is acrylamide, Vp is N-vinyl-2-pyrrolidone, E4 is methoxypoly (ethylene oxide (4)) monomethacrylate, MAN
a shows sodium methacrylic acid salt, respectively.

[0034]

[Table 1]

[0035]

[Table 2]

Example 1-1 An immobilization oligo DNA (abbreviated as PUC-NH ₂ ) having the following DNA sequence modified at the 5 ′ end with an amino group, and a sequence complementary to the PUC-NH ₂ Full match oligo DNA having 5'end modified with biotin (abbreviated as PUC), oligo DNA having mismatch 5'end having biotin modified with SNPs of the PUC
(Abbreviated as PUC 'or PUC'') was used. In addition, PUC-NH ₂ , PUC, P
UC 'and PUC''used were manufactured by Espec Oligo Service Co., Ltd. PUC-NH ₂ : 5'-ACTGGCCGTCGTTTTACAACGTCGTGACTGGG-3 'PUC: 5'-CCCAGTCACGACGTTGTAAA-3' PUC ': 5'-CCCAGTCACCACGTTGTAAA-3' PUC '': 5'-CCCAGTCACGTCGTTGTAAA-3 '1mM EDTA added 50mM phosphorus Acid buffer solution (pH 8.5) (E-NaP
A 25 pmol / ml PUC-NH ₂ solution dissolved in B)
100 μl / well was added to a bind 96-well plate (Corning) and incubated overnight at 4 ° C. (immobilization of PUC-NH ₂ ). Then removed PUC-NH ₂ solution from each well, DULC
The plate was washed 3 times with eco's PBS (abbreviated as D-PBS). Next, 3% BSA
200 μl / well of E-NaPB solution (abbreviated as BE-NaPB) to which was added was added, and the mixture was incubated at 37 ° C. for 1 hour (blocking operation-1). Then, remove BE-NaPB from each well and remove PUC.
The -NH _{2-immobilized} plate was prepared.

3.2% by weight of polymer (P1), PUC, PUC 'and PU
C '' and 0.2 pmol / ml each and 75 mM sodium citrate solution containing 750 mM NaCl (abbreviated as 5 × SSC) were added to the PUC-NH ₂ -immobilized plate prepared above at 100 μl / well, and 55
Hybridization was carried out by incubating at 1 ° C for 1 hour. Each solution was then removed from each well and 55
C, 30 mM sodium citrate solution containing 300 mM NaCl (2
X SSC) and washed twice. Then, add BE-NaPB to 200μ
After adding 1 / well and incubating at 37 ° C. for 30 minutes (blocking operation-2), BE-NaPB was removed from each well. Furthermore, avidin-modified horseradish peroxidase (SIGMA) diluted 10000 times with BE-NaPB (abbreviated as Avdin-HRP)
The solution was added at 100 μl / well and incubated at 37 ° C. for 30 minutes (avidin-biotin reaction). Then, after washing 3 times with D-PBS, 100 μl / well of the substrate solution of the HRP kit was added and incubated at 25 ° C. for 10 minutes (POD-substrate reaction).
Next, add 100 μl / well of the reaction stop solution from the HRP kit,
The resulting absorbance at 450 nm was measured with SPECTRA MAX250 (microplate reader, manufactured by Molecular Device). The HRP kit is a color development kit T for peroxidase.
(Sumitomo Bakelite Co., Ltd.) was used. From the measurement results, (P
(UC absorbance) / (PUC 'absorbance) and (PUC absorbance) / (P
The absorbance of UC '' was determined. The results are shown in Table 3.

Examples 1-2 to 1-13 Each measurement was carried out in the same manner as in Example 1-1 except that the polymers (P2) to (P13) were used instead of the polymer (P1). The results are shown in Table 3.

Comparative Example 1-1 Polymer (P1) 3.2 wt%, PUC, PUC 'and PUC''0.2%
PUC, PUC 'and P instead of 5 x SSC solution containing pmol / ml
The measurement was performed in the same manner as in Example 1-1, except that a 5 × SSC solution containing UC ″ at 0.2 pmol / ml was used. The results are shown in Table 3.

Comparative Example 1-2 3.2 wt% of polymer (P1) and 0.2% of PUC, PUC 'and PUC'', respectively.
Instead of 5 × SSC solution containing pmol / ml, 1.0% casein (SIGMA) and 0.1% N-LS (Wako Pure Chemical Industries, Ltd.) are included.
The measurement was performed in the same manner as in Example 1-1 except that 5 × SSC was used. The results are shown in Table 3.

Comparative Example 1-3 3.2 wt% of polymer (P1) and 0.2% of PUC, PUC 'and PUC''respectively
Instead of 5 × SSC solution containing pmol / ml, PUC, PUC 'and PUC''are 0.2 pmol / ml each, 10 mg / ml Ficol400, 1
6mg / ml polyvinylpyrrolidone, 10mg / ml bovine serum albumin and 0.5% SDS in 6xSSC solution (Denhard's
(s solution) was used, and the measurement was performed in the same manner as in Example 1-1. The results are shown in Table 3.

[0042]

[Table 3]

From Table 3, in comparison with the comparative example, in the example, the absorbance of full match (absorbance of PUC) / absorbance of mismatch
It can be seen that the ratio of (PUC ', PUC''absorbance) is high. That is, it does not suppress specific hybridization,
It can be seen that non-specific hybridization is suppressed and more accurate hybridization is performed.

Production Example 1-1 0.2 mg / ml poly (lysine-phenylalanine) dissolved in 5 × SSC was added to a 96-well multi-plate (Nunc, white) to give 15
0 μl / well was added and the mixture was allowed to stand at room temperature for 20 hours. After washing with 50 mM phosphate buffer (pH 8.3), 200 mM phosphate buffer (pH 8.
650 μM 2-iminothiolane hydrochloride dissolved in
After adding μl / well and allowing to stand at room temperature for 2 hours, it was washed with 10 mM phosphate buffer (pH 7.0). Oligonucleotide for immobilization with amino linker modified at the 5'end (Amersham Pharmacia) (sequence: 5'-CATTAGGGATCCAGCCGTGAATTCGTC
ACT-3 ') was dissolved in 50 mM phosphate buffer (pH 7.0), followed by N-succinimidyl 4-maleimidobutyric acid (abbreviated as GMBS) dissolved in dimethylformamide (abbreviated as DMF), an oligonucleotide. After reacting at 37 ° C. for 90 minutes, a non-reacted GMBS was removed to obtain a maleimidated oligonucleotide. This maleimidized oligonucleotide
To the 96-well multiplate treated with 2-iminothiolane hydrochloride, 25 pmol / well was added and reacted at room temperature for 90 minutes. After the reaction, discard the supernatant and add 0.8 mM iodoacetamide solution to 250
μl / well was added and left at room temperature for 20 hours. Then, 10 mM
After washing with phosphate buffer (pH 7.0), 50 mM Tris-50 mM
Add 250 μl / well of sodium chloride-1% BSA buffer,
The plate was left standing at room temperature for 20 hours to prepare an oligonucleotide-immobilized plate.

Production Example 1-2 4.8 mg of anti-AFP antibody clone No. 9 which reacts with AFP was reacted with 39 μg of pepsin at 37 ° C. for 30 minutes, and then purified by gel filtration column chromatography to obtain F (ab ′). ) 2 fractions 2.4 mg were obtained. Further, 0.2M 2-mercaptoethanolamine was added to the obtained fraction in a 1/20 volume of the F (ab ') 2 fraction, and the Fab' fraction was prepared by reduction reaction. Next, a full-matching oligonucleotide (Amersham Pharmacia, sequence: 5'-AGTGACGAATTCACGGCTGGA) with a 5'-terminal modified with an amino linker.
TCCCTAATG-3 ') and mismatched oligonucleotide (Amersham Pharmacia, Sequence: 5'-GATTTTAGCTCTTCT
TTGGAGAAAGTGGTG-3 ') each with 50 mM phosphate buffer (pH 7.0)
GMBS dissolved in DMF and added in 5 times the molar amount of the GMBS dissolved in DMF was reacted at 37 ° C. for 90 minutes, and then unreacted GMBS was removed to obtain a maleimidated oligonucleotide.
The above-mentioned anti-AFP was added to the obtained maleimidized oligonucleotide.
Anti-Fab'-full-match oligonucleotide complex (abbreviated as FM complex) and anti-Fab'-mismatch oligonucleotide by performing a reaction by adding 0.2 times the molar amount of Fab 'fraction of the antibody to the oligonucleotide and performing a desalting operation. A complex (abbreviated as MM complex) was prepared.

Example 2-1 FM composite solution and polymer containing 0.2 wt% of polymer (P1)
The MM complex solution containing 0.2 wt% of (P1) was added to the oligonucleotide-immobilized plate prepared in Production Example 1-1 at 100 μl / well and incubated at 37 ° C. for 30 minutes. Tris-TritonX
After washing with (hereinafter abbreviated as washing solution (X)), 100 ng / ml AFP
100 μl of the antigen diluent was added and incubated at 37 ° C. for 1 hour. After washing with the washing solution (X), 200 μl of luminescence substrate AMPPD solution
Was added and incubated at 37 ° C. for 5 minutes, and the amount of luminescence was measured by a fluorescent plate reader ARVOsx (Wallac Beltold). From the measurement result (the amount of light emitted from the FM complex) /
(Amount of luminescence of MM complex) was determined. The results are shown in Table 4.

Examples 2-2 to 2-13 Operations and measurements were performed in the same manner as in Example 2-1 except that the polymers (P2) to (P13) were used instead of the polymers (P1). The results are shown in Table 4.

Comparative Example 2-1 FM composite solution and polymer containing 0.2 wt% of polymer (P1)
Operation and measurement were performed in the same manner as in Example 2-1 except that the FM complex solution and the MM complex solution were used instead of the MM complex solution containing (P1) 0.2 wt%. The results are shown in Table 4.

[0049]

[Table 4]

From Table 4, Comparative Examples 2-1 to Examples 2-1 to 2
-13 shows that the ratio of the luminescence amount of the FM complex / the luminescence amount of the MM complex is high. That is, it can be seen that even at 37 ° C., specific hybridization is not suppressed, non-specific hybridization is suppressed, and more accurate hybridization is performed.

Example 3-1 An immobilization oligo DNA (abbreviated as PUC2-NH ₂ ) having the following DNA sequence modified at the 5 ′ end with an amino group, and a sequence complementary to the PUC2-NH ₂ A PUC having, a mismatched 5'-terminal PUC 'or PUC''having SNPs of the PUC was used. PUC2-NH ₂ is Espec Oligo Service Co., Ltd.
Used. PUC2-NH ₂ : 5 '-(CH ₂ ) ₁₂ TTTACAACGTCGTGACTGGG-3' A solution of 25 pmol / ml of PUC2-NH ₂ dissolved in E-NaPB was added to DNA-
100 μL / well was added to a bind 96-well plate (manufactured by Corning) and incubated overnight at 4 ° C. (immobilization of PUC2-NH ₂ ). Then, the solution was removed from each well, washed 3 times with D-PBS, 200 μl / well of BE-NaPB was added, and 37 ° C, 1
Incubated for an hour (blocking operation-1). BE-NaPB was removed from each well to prepare a PUC2-NH ₂ immobilized plate.

3.2% by weight of polymer (P1), PUC, PUC 'and PU
5 × SSC containing C ″ at 0.2 pmol / ml was added to the PUC2-NH ₂ -immobilized plate at 100 μl / well and incubated at 25 ° C. for 1 hour. Then, each oligo DNA solution was removed from each well and washed twice with a 30 mM sodium citrate solution containing 0.1% SDS and 300 mM NaCl at 25 ° C. Next, add BE-NaPB to 200μ
After adding 1 / well and incubating at 37 ° C. for 30 minutes (blocking operation-2), BE-NaPB was removed from each well. Furthermore, add Avdin-HRP solution at 100 μl / well, and add 37
After incubating at 30 ° C for 30 minutes (avidin-biotin reaction), the cells were washed 3 times with D-PBS. Subsequently, 100 μl / well of the substrate solution of the HRP kit was added and incubated at 25 ° C. for 10 minutes (POD-substrate reaction). Next, add 100 μl / well of the reaction stop solution from the HRP kit, and measure the resulting absorbance at 450 nm by SPECT.
It was measured with RA MAX250 (microplate reader, manufactured by Molecular Devices). As the HRP kit, a coloring kit T for peroxidase (Sumitomo Bakelite Co., Ltd.) was used.
From the measurement results, (PUC absorbance) / (PUC 'absorbance) and (PUC's
Absorbance of /) (absorbance of PUC '') was determined. The results are shown in Table 5.

Examples 3-2 to 3-13 The same operations and measurements were performed as in Example 3-1, except that the polymers (P2) to (P13) were used instead of the polymers (P1).
The results are shown in Table 5.

Comparative Example 3-1 3.2 wt% of polymer (P1) and 0.2 pmol of PUC, PUC 'and PUC''
, PUC, PUC 'and PU instead of 5 x SSC solution containing
Operation and measurement were performed in the same manner as in Example 3-1, except that 5 × SSC solution containing 0.2 pmol / ml of each C ″ was used. The results are shown in Table 5.

Comparative Example 3-2 3.2 wt% of polymer (P1) and 0.2 pmol of PUC, PUC 'and PUC''
1.0% casein (SIGM
5 x SSC containing A) and 0.1% N-LS (Wako Pure Chemical Industries, Ltd.)
Operations and measurements were performed in the same manner as in Example 3-1 except that was used. The results are shown in Table 5.

Comparative Example 3-3 3.2 wt% of polymer (P1) and 0.2% of PUC, PUC ′ and PUC ″ respectively
Instead of 5 × SSC solution containing pmol / ml, PUC, PUC 'and PUC''are 0.2 pmol / ml each, 10 mg / ml Ficol400, 1
6mg / ml polyvinylpyrrolidone, 10mg / ml bovine serum albumin and 0.5% SDS in 6xSSC solution (Denhard's
(s solution) was used, and the measurement was performed in the same manner as in Example 3-1. The results are shown in Table 5.

[0057]

[Table 5]

From Table 5, in comparison with the comparative example, in the example, the absorbance of full match (absorbance of PUC) / absorbance of mismatch (PU
It can be seen that the ratio of C'and PUC '' is higher.
That is, it can be seen that the specific hybridization is not suppressed, the non-specific hybridization is suppressed, and more accurate hybridization is performed.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yoshihiro Ashihara             691-17 Nakago Shimogo, Hanno City, Saitama Prefecture (72) Inventor Mitsuo Isomura             874-10 Sand, Kawagoe City, Saitama Prefecture F-term (reference) 4B063 QA01 QA18 QQ42 QQ52 QR41                       QR55 QR82 QS34 QX02 QX07

Claims (10)

[Claims] 1. A nonspecific hybridization inhibitor comprising a polymer (H) having a hydrophilic group in the molecule and having a weight average molecular weight of 1000 to 5000000 and a nonspecific hybridization inhibition function. 2. The hydrophilic group is one or two selected from the group consisting of an anionic group, an amphoteric charge-containing group, a cationic group, an amide group, a lactam group and a polyalkylene oxide group.
The inhibitor according to claim 1, which is one or more groups. 3. The inhibitor according to claim 1, wherein the polymer (H) contains at least one type of anionic group and amphoteric charge-containing group as a hydrophilic group. 4. The inhibitor according to claim 2, wherein the amphoteric charge-containing group is a phosphorylcholine-like group and the anionic group is at least one of a carboxyl group and a sulfone group. 5. The inhibitor according to claim 4, wherein the phosphorylcholine-like group is a constituent group based on a phosphorylcholine-like group-containing monomer represented by the formula (1). [Chemical 1] (In the formula, X represents a divalent organic residue, Y represents an alkyleneoxy group having 1 to 6 carbon atoms, Z is a hydrogen atom or R ⁵ O (C = O).
-Indicates. However, R ⁵ represents an alkyl group having 1 to 10 carbon atoms or a hydroxyalkyl group having 1 to 10 carbon atoms. R ¹ represents a hydrogen atom or a methyl group, and R ² , R ³ and R ⁴ are the same or different groups and represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a hydroxyalkyl group. m is 0 or 1, and n is an integer of 1 to 4. ) 6. The polymer (H) comprises 5 to 100 mol% of a phosphorylcholine-like group-containing monomer, and at least one of a carboxyl group-containing monomer and a sulfo group-containing monomer.
A polymer obtained by polymerizing a monomer composition consisting of ˜95 mol% and a hydrophobic monomer of 0 to 60 mol%.
Inhibitor as described in 1. 7. The phosphorylcholine-like group-containing monomer is 2
-((Meth) acryloyloxy) ethyl-2 '-(trimethylammonio) ethyl phosphate, the carboxyl group-containing monomer is (meth) acrylic acid, the sulfone group-containing monomer is 2-methylpropanesulfonic acid, 7. The inhibitor according to claim 6, wherein the hydrophobic monomer is a monomer represented by the formula (2). [Chemical 2] (In the formula, R ⁶ represents a hydrogen atom or a methyl group, and L ¹ represents -C ₆ H
_4- , -C ₆ H ₁₀ -,-(C = O) O-, -O-,-(C = O) NH-, -O (C = O)-or -O (C = O) O- are shown, L ² is hydrogen atom, - (CH ₂₎ gL ^3, or ((C
Shows the _{H 2) p -O) h -L} 3. g and h are 1 to 24, p is an integer of 3 to 5, L ³ represents a hydrogen atom, a methyl group, -C ₆ H _5, the -OC ₆ H _5. ) 8. A clinical test drug containing the inhibitor according to claim 1 and a test drug. 9. A sample and a test agent that hybridizes to a predetermined nucleic acid-related substance are contacted in the presence of the inhibitor according to claim 1 under predetermined conditions such that the predetermined nucleic acid-related substance and the test agent hybridize. A clinical test method comprising a step (1) of performing and a step (2) of detecting a reaction product generated by hybridization with a test agent in the step (1). 10. In the step (1), the amount of the inhibitor used is such that the polymer in the inhibitor is in the hybridizing system.
The clinical test method according to claim 9, wherein (H) is present in an amount of 0.0001 to 20% by weight.