JPH08261941A - Chemiluminescence detecting method and device - Google Patents

Abstract

PURPOSE: To provide the information on a polymer such as the accurate gene information by radiating low-energy radioactive rays to an accumulative phosphor sheet, and exposing the accumulative phosphor sheet via the chemiluminescence generated by the contact between the labeled polymer and a chemiluminescence material. CONSTITUTION: A line 1 of multiple dilutions of unmarked DNAs are prepared. The dilutions of the DNAs are dripped on a nylon membrane filter 2 to form spots 3. After the filter 2 is dried, ultraviolet rays are radiated to cross-link the DNAs and the filter 2, and the DNAs are fixed to the filter 2. The filter 2 transmits visible light, and it is stored in a sealable hybridization bag 4. A marker antibody solution is prepared and added into the bag 4, then it is discharged, and an AMPPD (adamantane-1,2-dioxane) solution is added into the bag 4. An accumulative phosphor sheet radiated with low-energy X-rays and the bag 4 are brought into face contact with each other in a dark room.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a chemiluminescence detection method and apparatus for obtaining positional information of a labeling substance in a polymer, and more specifically to chemiluminescence using a stimulable phosphor. The present invention relates to a detection method and device.

[0002]

2. Description of the Related Art A fixed polymer such as a protein or a nucleic acid sequence is selectively labeled with a labeling substance which is brought into contact with a chemiluminescent substance to cause chemiluminescence, and is selectively labeled with the labeling substance. By contacting a macromolecule with a chemiluminescent substance, and detecting chemiluminescence in the visible light wavelength range caused by the contact between the chemiluminescent substance and the labeling substance, information about the macromolecule such as gene information can be obtained. Chemiluminescent detection methods are known. This chemiluminescence detection method
Determination of nucleotide sequences of nucleic acids such as DNA and RNA by electrophoresis, screening of genes by electrophoresis and colony hybridization, generation of reference data of genes by dot blotting, simple detection of gene amount and expression level, It is widely used for protein separation, identification, and evaluation of molecular weight and properties. Conventionally, such chemiluminescence is detected by exposing and developing a photographic film by chemiluminescence emitted from a polymer.
This was done by detecting a visible image. However, since chemiluminescence generated by contact between a chemiluminescent substance and a labeling substance is weak light, it is necessary to use a high-sensitivity photographic film with a high γ value in order to reliably detect it with a photographic film. However, when using a high-sensitivity photographic film with a high γ value, it is difficult to make sure that the exposure is done using the straight line portion of the characteristic curve, and there are many exposure mistakes. There has been a problem that it is necessary to repeatedly expose the film by changing the sensitivity of the film or the exposure time, and the chemical treatment of development is indispensable, which makes the operation troublesome. Furthermore, it is difficult to detect information on macromolecules such as genetic information with high accuracy by visual analysis of the image obtained by exposing the photographic film. There is a problem in that it is necessary to scan an image with light, convert it into a digital signal, perform digital image processing, and analyze the image, which requires a complicated operation.

Further, when it is irradiated with light in the visible light wavelength range, its energy is accumulated, and when it is subsequently irradiated with electromagnetic waves such as visible light, ultraviolet rays and infrared rays, it is excited and accumulated. Generated by contact between a chemiluminescent substance and a labeling substance using a stimulable phosphor sheet having a stimulable phosphor layer containing a stimulable phosphor that emits light in proportion to the energy of light in the light wavelength region. The chemiluminescence is stored and recorded in the stimulable phosphor sheet, and then the stimulable phosphor sheet is scanned using electromagnetic waves such as visible light, ultraviolet rays, infrared rays, etc. A chemiluminescence detection method is known in which energy is emitted, the emitted light is converted into a digital signal, and the obtained digital signal is image-processed to obtain information on macromolecules such as genetic information (for example, And JP-A-4-232864 discloses.). According to the chemiluminescence detection method using such a stimulable phosphor sheet, the stimulable phosphor emits light proportional to the energy of light in the irradiated visible light wavelength region when excited by electromagnetic waves. Compared with the case of using a photographic film, it is simple in that there are few exposure errors and there is no need for chemical treatment such as development. Furthermore, information on macromolecules such as genetic information is digital. Since it can be obtained in the form of a signal, there is an advantage that necessary image processing can be performed to accurately analyze information on the polymer.

[0004]

However, in such a chemiluminescence detection method, the stimulable phosphor has the property of accumulating it when irradiated with light in the visible light wavelength range. It is always necessary to block visible light, which is inconvenient to handle. On the other hand, for the same purpose as in the chemiluminescence detection method, a radiolabel is attached to a tissue of an organism and / or a substance derived from the organism, and the tissue of the organism and / or a substance derived from the organism to which the radiolabel is attached An autoradiographic measurement method is known in which the position information of a radiolabeled substance in a medium containing is absorbed, and the energy of radiation is absorbed and accumulated, and then electromagnetic waves such as visible light and infrared light are used. When excited, the stimulable phosphor layer containing a stimulable phosphor having a characteristic of emitting a stimulable amount of light in accordance with the amount of irradiated radiation energy is used to label a stimulable phosphor sheet with a radioactive label. An autoradiography measuring method used as a detection material for obtaining positional information of a substance has been proposed (for example, Japanese Patent Publication No. 1-60784 and Japanese Patent Publication No. 1).
No. -60782, Japanese Patent Publication No. 4-3952, etc. ).

Since this autoradiographic measuring method is used for the same purpose as the chemiluminescence detecting method, the same stimulable phosphor sheet is used, depending on the object, or the autoradiographic method. Using the measurement method,
Alternatively, it is desirable for users to be able to obtain information about macromolecules such as genetic information using chemiluminescent detection methods. However, in the chemiluminescence detection method, it is necessary to detect chemiluminescence in the visible light wavelength range caused by contact between the chemiluminescent substance and the labeling substance, while in the autoradiography measurement method, radiation is detected. Therefore, the same stimulable phosphor sheet could not be used for both chemiluminescence detection method and autoradiographic measurement method.

[0006]

An object of the present invention is to efficiently and efficiently use a stimulable phosphor sheet that is easy to handle and can be used in both chemiluminescence detection methods and autoradiography measurement methods. Another object of the present invention is to provide a chemiluminescence detection method capable of accurately obtaining information on macromolecules such as gene information. A second object of the present invention is to use a stimulable phosphor sheet that is easy to handle and can be used for both chemiluminescence detection methods and autoradiography measurement methods, and is efficient and accurate for gene analysis. It is an object of the present invention to provide a chemiluminescence detection device capable of obtaining information about a polymer such as information.

[0007]

According to the first aspect of the present invention, the energy of radiation can be accumulated, and the energy of the accumulated radiation is excited as light by the light in the visible light wavelength range. A stimulable phosphor layer containing a stimulable phosphor to be emitted is uniformly irradiated with low-energy radiation on a stimulable phosphor sheet, so that the energy of the radiation is uniformly accumulated, resulting in chemiluminescence. A macromolecule is selectively labeled with a labeling substance that is brought into contact with a substance to generate chemiluminescence,
The stimulable phosphor sheet is obtained by contacting the polymer labeled with the labeling substance with the chemiluminescent substance, and chemiluminescence generated by the contact between the polymer labeled with the labeling substance and the chemiluminescent substance. Is exposed to light by a chemiluminescence detection method.
In the present invention, radiation includes X-rays, α-rays, β-rays, γ-rays and electron beams. In the preferred embodiment of the first aspect of the invention, X-rays having an energy of 20 KV or less are selected as the radiation. In another preferred embodiment of the first aspect of the present invention, the radiation is 100
An electron beam having an energy of KV or less is selected.

[0008] In a further preferred embodiment of the first aspect of the present invention, the method for detecting chemiluminescence further comprises scanning the stimulable phosphor sheet exposed by chemiluminescence with electromagnetic waves to excite the stimulable phosphor sheet, The step of photoelectrically converting the light emitted from the phosphor sheet to obtain image data is provided. In a further preferred embodiment of the first aspect of the present invention, the chemiluminescent substance contains a sensitizer capable of converting the emission wavelength of the chemiluminescent substance. In a further preferred embodiment of the first aspect of the present invention, the stimulable phosphor is a barium fluorohalide-based phosphor, and the sensitizer is poly [vinylbenzyl (benzyldimethyl) represented by the following general formula. Ammonium chloride)] as a main component.

[0009]

Embedded image

In a further preferred embodiment of the first aspect of the present invention, the sensitizer further contains a trace amount of a fluorescent dye and a surfactant. A second object of the present invention is, according to the second invention, that the chemiluminescent substance can be brought into contact with a polymer that is selectively labeled with a labeling substance that contacts the chemiluminescent substance to generate chemiluminescence. And a stimulable phosphor that is capable of accumulating radiation energy, is excited by light in the visible wavelength range, and emits the accumulated radiation energy as light. Pre-exposure means for uniformly irradiating low-energy radiation to the stimulable phosphor sheet having the stimulable phosphor layer formed thereon to uniformly accumulate the energy of the radiation, the labeling substance, and the chemiluminescent substance. Exciting light irradiating means for irradiating exciting light to the stimulable phosphor sheet that has released the energy of the accumulated radiation as light by chemiluminescence generated by contact with It is accomplished by chemiluminescent detection apparatus having a detection means for photoelectrically detecting light emitted from the stimulable phosphor sheet. In a preferred embodiment of the second aspect of the present invention, as radiation,
X-rays with energies below 20 KV have been chosen.

In another preferred embodiment of the second aspect of the present invention, an electron beam having an energy of 100 KV or less is selected as the radiation. In a further preferred aspect of the second aspect of the present invention, the excitation light irradiating means is configured to scan the stimulable phosphor sheet with an electromagnetic wave to excite it. In a further preferred aspect of the second invention of the present application, the chemiluminescent substance contains a sensitizer capable of converting the emission wavelength of the chemiluminescent substance. In a further preferred embodiment of the second aspect of the present invention, the stimulable phosphor is a barium fluorohalide-based phosphor, and the sensitizer is poly [vinylbenzyl (benzyldimethylammonium) represented by the following general formula. Chloride)] as a main component.

[0012]

[Chemical 4]

In a further preferred embodiment of the second aspect of the present invention, the sensitizer further contains a trace amount of a fluorescent dye and a surfactant. The stimulable phosphor contained in the stimulable phosphor layer formed in the stimulable phosphor sheet that can be used in the present invention is capable of accumulating energy of radiation and is excited by light in the visible light wavelength range, It is not particularly limited as long as it can release the energy of the accumulated radiation as light. Specifically, for example, the alkaline earth metal fluorohalide-based phosphor (Ba _1-x, M disclosed in JP-A-55-12145).
²⁺ _x ) FX: yA (where M ²⁺ is Mg, Ca, Sr,
At least one alkaline earth metal element selected from the group consisting of Zn and Cd, X is at least one halogen selected from the group consisting of Cl, Br and I, and A is E
u, Tb, Ce, Tm, Dy, Pr, Ho, Nd, Yb
And at least one 3 selected from the group consisting of
Valent metal element, x is 0 ≦ x ≦ 0.6, y is 0 ≦ y ≦ 0.2
Is. ), An alkaline earth metal fluorohalide-based phosphor SrF disclosed in JP-A-2-276997.
X: Z (where X is at least one halogen selected from the group consisting of Cl, Br and I, and Z is Eu or Ce), and europium activation disclosed in JP-A-59-56479. Composite halogen-based phosphor BaF
X · xNaX ′: aEu ²⁺ (wherein X and X ′ are at least one halogen selected from the group consisting of Cl, Br, and I, x is 0 <x ≦ 2, and a is 0 < a ≦ 0.2), MOX: xCe (where M is Pr, and M is Pr, which is a cerium-activated trivalent metal oxyhalogen-based phosphor disclosed in JP-A-58-69281).
Nd, Pm, Sm, Eu, Tb, Dy, He, Er, T
At least one trivalent metal element selected from the group consisting of m, Yb and Bi, X is one or both of Br and I, and x is 0 <x <0.1. ), LnOX: xCe, which is a cerium-activated rare earth oxyhalogen-based phosphor disclosed in JP-A-60-101179 and JP-A-60-90288, where Ln is Y,
At least one rare earth element selected from the group consisting of La, Gd and Lu, X is at least one halogen selected from the group consisting of Cl, Br and I, and x is 0 <
x ≦ 0.1. ) And europium-activated disclosed in JP-59-75200 JP complex halide phosphor ^{M II FX · aM I X '} · bM' II X '' 2 · cM III
X ^''' ₃ xA: yEu ²⁺ (where M ^II is Ba, Sr
And at least one alkaline earth metal selected from the group consisting of Ca, M ^I is Li, Na, K, of at least one alkali metal element selected from the group consisting of Rb and Cs, M ^'II is from Be and Mg at least one divalent metal element selected from the group consisting of, M ^III is Al,
At least one trivalent metal element selected from the group consisting of Ga, In and Tl, A is a metal oxide, X is Cl, B
at least one halogen selected from the group consisting of r and I, X ′, X ^″ and X ^{′ ″} are at least one halogen selected from the group consisting of F, Cl, Br and I, and a represents 0 ≤a≤2, b is 0≤b≤10 ^-2 , c
Is 0 ≦ c ≦ 10 ⁻² and a + b + c ≧ 10 ⁻² , x is 0 <x ≦ 0.5, and y is 0 <y ≦ 0.2. ) Can be preferably used.

In the present invention, the chemiluminescent substance is
If the chemiluminescent substance used in the chemiluminescent detection method,
It is not particularly limited and includes, for example, luminol, lucigenin, acridinium ester, AMPPD
(Adamantyl-1,2-dioxetane), TROP
IX "CDP-Star" (registered trademark), "CD
Dioxycetanes such as P "(registered trademark) and the like can be preferably used. In the present invention, the labeling substance that causes chemiluminescence upon contact with the chemiluminescent substance is not particularly limited as long as it is a chemiluminescent substance used in the chemiluminescence detection method, but it is not limited to a catalyst or an enzyme. Is preferably used. Examples of catalysts include, for example, various metals and adenosine triphosphate, and examples of enzymes include phosphatases such as lactate dehydrogenase, luciferase, peroxidase, and alkaline phosphatase. Among these, enzymes, particularly phosphatase, are preferred, and alkaline phosphatase is most preferred. In the present invention, determination of the base sequence of nucleic acids such as DNA and RNA by electrophoresis, screening of genes by electrophoresis and colony hybridization, generation of reference data of genes by dot blotting, determination of gene amount and expression amount For simple detection, a labeling substance for labeling DNA or RNA complementary to the target DNA or RNA can be used. Examples of such labeling substance include digoxigenin (DIG) and avidin. , Streptavidin, biotin, fluorescein and the like.

In the present invention, the stimulable phosphor sheet comprises
When X-rays are used as the uniform radiation, the energy is 20 KV or less, preferably 1
It is preferable to use X-rays of 2.5 KV. The energy of X-rays that are uniformly applied to the stimulable phosphor sheet is 20
When the voltage exceeds KV, the energy of X-rays is uniformly accumulated even in the deep part of the stimulable phosphor layer of the stimulable phosphor sheet, so that the stimulable phosphor sheet is exposed by weak chemiluminescence. However, the energy of the X-rays accumulated in the deep portion of the stimulable phosphor layer cannot be released, and the chemiluminescence detection sensitivity decreases, which is not preferable. In the present invention, when an electron beam is used as the radiation for uniformly irradiating the stimulable phosphor sheet, the energy is preferably 100 KV or less. The preferable electron beam energy varies depending on the thickness of the stimulable phosphor layer provided on the stimulable phosphor sheet and the presence or absence of the protective layer, but it has a thickness of about 50 μm and no protective layer is provided. In the case of a stimulable phosphor layer, if an electron beam with an energy of 150 to 200 KV is used for uniform irradiation, the electron beam will pass through the stimulable phosphor layer and the stimulable phosphor sheet The energy of the electron beam is evenly accumulated up to the deep part of the stimulable phosphor layer, and even if the stimulable phosphor sheet is exposed by the weak chemiluminescence, it is accumulated in the deep part of the stimulable phosphor layer. In the case of a stimulable phosphor layer that cannot release the energy of the electron beam and has a thickness of about 50 to 150 microns and is not provided with a protective layer, the stimulable phosphor sheet is Energy of electron beam , 100
When it is KV or less, the energy of the electron beam is uniformly accumulated only in the vicinity of the surface of the stimulable phosphor layer, and when the stimulable phosphor sheet is exposed by weak chemiluminescence, the stimulable fluorescence is exposed. It is preferable since the energy of the electron beam accumulated in the body layer can be released and the chemiluminescence detection sensitivity can be improved. 50 to 150 provided with a protective layer
In the case of a stimulable phosphor sheet with a thickness of about micron,
The energy of the electron beam uniformly irradiated is preferably 60 KV or more.

The sensitizer used in the preferred embodiment of the present invention is capable of converting the emission wavelength of the chemiluminescent substance into a wavelength capable of efficiently erasing the radiation energy accumulated in the stimulable phosphor. If a barium fluorohalide-based phosphor is used as the stimulable phosphor, the poly [vinylbenzyl (represented by the following general formula (1) A sensitizer containing benzyldimethylammonium chloride)] as a main component can be preferably used.

[0017]

Embedded image

In the present invention, the sensitizer preferably contains a trace amount of a fluorescent dye, and when a sensitizer containing poly [vinylbenzyl (benzyldimethylammonium chloride)] as a main component is used, , The following equation (2)
Fluorescein (CI-45350 Acid Y
Ellow 73, Dye No. 54101: Dye Handbook (Kodansha)), or sulforodamine represented by the following formula (3) (Dye No. 54012) is preferably contained.

[0019]

[Chemical 6]

[0020]

[Chemical 7]

In the present invention, the sensitizer preferably contains a small amount of a surfactant, and when a sensitizer containing poly [vinylbenzyl (benzyldimethylammonium chloride)] as a main component is used, As a surfactant, anionic dodecyloxypolyoxyethylene sulfate sodium salt, that is, C ₁₂ H ₂₅ O (CH
₂ CH ₂ O) _n SO ₃ Na is preferable. In the present invention, the sensitizer preferably contains a small amount of an inorganic substance, and when a sensitizer containing poly [vinylbenzyl (benzyldimethylammonium chloride)] as a main component is used, the inorganic substance is NaC
1 or an inorganic cation such as Na, Ca, Al, K or Ti is preferably contained. When it contains an inorganic cation, Na, Ca, A, compared with K, Ti
The amount of 1 is preferably Oikata. In the present invention, when a sensitizer containing poly [vinylbenzyl (benzyldimethylammonium chloride)] as a main component is used, the sensitizer preferably contains a small amount of sodium acetate. Specifically, for example, "EM manufactured by TROPIX Co., Ltd.
"ERALD" or "RUBY" may be preferably used. TROPIX's "EMERALD", "RUB"
“Y” contains poly [vinylbenzyl (benzyldimethylammonium chloride)] represented by the general formula (1) as a main component, and “EMERALD” manufactured by TROPIX is represented by the formula (2). Trace amount of fluorescein (CI-45350 Acid Yellow 73, pigment
No. 54101: Dye Handbook (Kodansha)), a small amount of anionic dodecyloxypolyoxyethylene sulfate sodium salt, that is, C ₁₂ H ₂₅ O (CH ₂ CH _2).
O) _n SO ₃ Na, a small amount of NaCl and a small amount of sodium acetate. In addition, TROPIX's "R
UBY "is a trace amount of sulforhodamine (dye No. 54012) represented by the formula (3), and a small amount of anionic dodecyloxypolyoxyethylene sulfate sodium salt, that is, C ₁₂ H ₂₅ O (CH ₂ CH ₂ O). _It contains _n SO ₃ Na, a small amount of inorganic cations such as Na, Ca, Al, K and Ti and a small amount of sodium acetate.

[0022]

According to the present invention, stimulable fluorescent light containing a stimulable phosphor capable of accumulating radiation energy, being excited by light in the visible light wavelength range, and emitting the energy of the accumulated radiation as light. A labeling substance that uniformly irradiates a stimulable phosphor sheet having a body layer with low-energy radiation to evenly accumulate the energy of the radiation and contact with a chemiluminescent substance to generate chemiluminescence. By selectively labeling the polymer with the chemiluminescent substance by contacting the polymer labeled with the labeling substance with the chemiluminescent substance, and accumulating by chemiluminescence generated by the contact between the polymer labeled with the labeling substance and the chemiluminescent substance. Exciting the stimulable phosphor by exposing the stimulable phosphor sheet, the stimulable phosphor releases the energy of low-energy radiation that has been accumulated in advance as light, and accumulates the accumulated energy. By erasing, the chemiluminescence is recorded in the stimulable phosphor, so it is not necessary to shield the stimulable phosphor sheet from visible light, and the stimulable phosphor sheet can be handled easily. Therefore, it becomes possible to efficiently and accurately obtain information on macromolecules such as genetic information. Further, since the stimulable phosphor can store the energy of radiation, the stimulable phosphor sheet is used not only for the chemiluminescence detection method but also for the autoradiographic measurement method used for the same purpose. Therefore, the user can obtain information on macromolecules such as gene information by a chemiluminescence detection method or an autoradiography measurement method, depending on the object, and DNA, RNA, etc. by the electrophoresis method. Determination of nucleotide sequence of nucleic acid, screening of gene by electrophoresis method and colony hybridization, generation of reference data of gene by dot blotting,
It becomes possible to efficiently perform simple detection of gene amount and expression amount, separation and identification of proteins, or evaluation of molecular weight and characteristics.

Furthermore, since low-energy radiation is uniformly applied to the stimulable phosphor sheet, the energy of the radiation is uniformly accumulated.
The stimulable phosphor was accumulated in advance only in the vicinity of the surface of the stimulable phosphor layer. Therefore, even when the stimulable phosphor sheet was exposed by weak chemiluminescence, the stimulable phosphor was accumulated in advance. Since most of the energy of radiation can be emitted as light and the stored energy can be erased, it becomes possible to improve the detection sensitivity of chemiluminescence. Furthermore, according to a preferred embodiment of the present invention, the chemiluminescent substance contains a sensitizer, and the wavelength of light capable of effectively erasing the radiation energy accumulated in the stimulable phosphor is a chemiluminescent substance. Even when the wavelength of the emitted light is deviated, the stimulable phosphor efficiently emits the energy of the radiation accumulated in advance as light by chemiluminescence, and the accumulated energy is erased. The emitted light can be recorded in the stimulable phosphor. According to a further preferred embodiment of the present invention, the stimulable phosphor sheet exposed by chemiluminescence is further scanned by electromagnetic waves, excited, and photoelectrically converted from light emitted from the stimulable phosphor sheet, Since the image data is obtained, it is possible to easily analyze the image data by subjecting it to digital image processing, and it is possible to accurately obtain information about the polymer such as gene information.

According to a further preferred embodiment of the present invention, since the chemiluminescent substance contains a sensitizer containing poly [vinylbenzyl (benzyldimethylammonium chloride)] as a main component, the wavelength of chemiluminescence is Radiation energy accumulated in barium fluorohalide-based phosphors, which are widely used as a detection material for autoradiography, can be matched with the wavelength of light that can be efficiently erased, and the stimulable fluorescence can be efficiently used. The body emits the energy of radiation that has been previously accumulated as light, and the accumulated energy is erased, whereby chemiluminescence can be recorded in the stimulable phosphor.

[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. FIG.
And FIG. 2 is a flow sheet showing the procedure of the chemiluminescence detection method according to the embodiment of the present invention. 1 and 2
Shows an example of a chemiluminescence detection method in the case where the gene amount is detected by dot blotting to generate gene reference data. In FIGS. 1 and 2, first, a dilution series 1 of 7 unlabeled DNAs is prepared. Each dilution series 1 is, for example, 10 ng
/ Microliter, 1ng / microliter, 100
pg / microliter, 10 pg / microliter, 1
It contains pg / microliter, 100 fg / microliter and 10 fg / microliter of DNA.
Then, using a pipette, each diluted solution of DNA is dropped on the nylon membrane filter 2 to form spots 3.

After the membrane filter 2 is dried, it is irradiated with ultraviolet rays to cross-link the DNA and the membrane filter 2 (UV crosslink), and the DNA is fixed on the membrane filter 2. After that, the membrane filter 2 on which the DNA is fixed is capable of transmitting visible light and
It is housed in a sealable plastic hybridization bag 4. Next, a hybridization solution is prepared and poured into the hybridization bag 4 for prehybridization. Meanwhile, a complementary DNA probe labeled with digoxigenin (DIG) -dNTP was prepared and added to the hybridization solution, and the hybridization solution for prehybridization was discharged from the hybridization bag 4, 4 and hybridize with the DNA immobilized on the membrane filter 2. In FIG. 1, 5
Indicates DNA hybridized with complementary DNA. In this embodiment, since the gene amount is detected and the reference data of the gene is generated, all the DNA dropped on the membrane filter 2 is hybridized with the complementary DNA.

After completion of the hybridization, the membrane filter 2 is washed, and an anti-digoxigenin alkaline phosphatase-labeled antibody solution is prepared and added to the hybridization bag 4. As a result, the complementary D labeled with digoxigenin (DIG)
DNA binding to NA and alkaline phosphatase
Are labeled. In FIG. 2, 6 indicates a complementary DNA labeled with alkaline phosphatase. In this embodiment, the membrane filter 2
The complementary DNA hybridized with all the DNA dropped above is labeled with alkaline phosphatase. Next, after discharging the anti-digoxigenin alkaline phosphatase-labeled antibody solution from the hybridization bag 4, the AMPPD (adamantyl-1,2-dioxetane) solution is added into the hybridization bag 4. For AMPPD solution,
"RUBY", which is a sensitizer manufactured by TROPIX, is contained. “RUBY” manufactured by TROPIX is mainly composed of poly [vinylbenzyl (benzyldimethylammonium chloride)] represented by the general formula (1), and a small amount of a fluorescent dye sulforodamine (dye No. represented by the formula (3)). . 54012) and, dodecane siloxy polyoxyethylene sulfate sodium salt anionic is a small amount of a surfactant, _{i.e., C 12 H 25 O (CH} 2 CH 2 O)
_It contains _n SO ₃ Na, a small amount of inorganic cations such as Na, Ca, Al, K, and Ti, and a small amount of sodium acetate.

Since AMPPD has the property of decomposing and emitting chemiluminescence when contacted with alkaline phosphatase, chemiluminescence is generated from DNA labeled with alkaline phosphatase. On the other hand, the surface of the stimulable phosphor sheet 7 having a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor capable of accumulating radiation energy is uniformly irradiated with low energy X-rays. Then, low-energy X-ray energy is uniformly accumulated in the stimulable phosphor layer. Next, after discharging a part of the AMPPD solution from the hybridization bag 4, the hybridization bag 4 is heated in order to increase the decomposition rate of AMPPD, and thus the energy of low-energy X-rays is uniformly accumulated. The stimulable phosphor sheet 7 on which the exhaustible phosphor layer is formed is brought into surface contact with the hybridization bag 4 in a dark room.
Here, in the present embodiment, a barium fluorobromide-based stimulable phosphor capable of accumulating radiation energy and excitable by an electromagnetic wave in the visible light wavelength region is used. Therefore, it is generated by decomposition of AMPPD. When the stimulable phosphor layer formed on the stimulable phosphor sheet 7 is exposed by chemiluminescence, the accumulated X-ray energy is released to the exposed part of the stimulable phosphor layer, Erased. The energy of X-rays accumulated in the barium fluorobromide stimulable phosphor can be efficiently
The wavelength of the light to be erased is about 580 nm,
The wavelength of chemiluminescence generated by decomposition of
However, since the AMPPD solution contains "RUBY", which is a sensitizer manufactured by TROPIX,
The wavelength of chemiluminescence generated by the decomposition of PD is converted to 620 nm, and therefore the energy of X-rays accumulated in the stimulable phosphor can be efficiently erased.

In this embodiment, the surface of the stimulable phosphor sheet 7 on which the stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor is formed is uniformly irradiated with low energy X-rays. The reason is that X-ray energy is uniformly accumulated only in the stimulable phosphor near the surface of the stimulable phosphor layer, and the stimulable phosphor sheet 7 is formed even by weak chemiluminescence. This is because most of the energy of the X-ray accumulated in the stimulable phosphor layer can be released and erased, and the chemiluminescence detection sensitivity is improved. That is, when irradiating the surface of the stimulable phosphor sheet 7 with high-energy X-rays, not only the stimulable phosphor near the surface of the stimulable phosphor layer but also the stimulable fluorescence in the deep part Even the body will accumulate X-ray energy, and even if it is exposed to weak chemiluminescence after that, the stimulable phosphor layer is made to sufficiently release the accumulated X-ray energy. However, since it becomes difficult to erase, the sensitivity of detecting chemiluminescence decreases, but when uniformly irradiating with low-energy X-rays, the energy of the X-rays is the same as that of the stimulable phosphor layer. Only for the stimulable phosphor near the surface,
Most of the X-ray energy accumulated in the stimulable phosphor layer can be released and erased when it is stored and exposed by weak chemiluminescence, and the chemiluminescence image is
The stimulable phosphor sheet 7 can be recorded with high sensitivity. The energy of X-rays uniformly applied to the surface of the stimulable phosphor sheet 7 is preferably 20 KV or less.

After a lapse of a predetermined time, the stimulable phosphor sheet 7
Is separated from the hybridization bag 4. Thus, in the stimulable phosphor layer of the stimulable phosphor sheet 7 exposed by chemiluminescence, the energy of X-rays is uniformly accumulated only in the part which is not exposed by chemiluminescence. In the exposed portion, the X-ray energy accumulated in advance is released and erased according to the intensity of chemiluminescence. In this embodiment, an image corresponding to the DNA concentration of each spot 2 is accumulated and recorded at the position of the stimulable phosphor layer corresponding to each spot 2. FIG. 3 is a schematic perspective view showing an example of an image data reading device and an image processing device for reading image data from the stimulable phosphor sheet 7 on which the stimulable phosphor layer on which the image is accumulated and recorded is formed in this way. It is a figure. The image data reading device is configured to scan and excite the stimulable phosphor sheet 7 with the laser beam 10 to generate stimulated emission. The laser light 10 is generated by the laser light source 11 and passes through the filter 12 to cut a portion of the wavelength region corresponding to the wavelength region of the photostimulable light generated from the stimulable phosphor sheet 7 by the excitation by the laser light 10. To be done. Next, the beam diameter of the laser beam 10 is accurately adjusted by the beam expander 13, and the laser beam 10 enters the optical deflector 14 such as a galvanometer mirror. The laser beam 2 deflected by the optical deflector 14 is f
The light is reflected by the plane reflecting mirror 16 via the θ lens 15 and is one-dimensionally incident on the stimulable phosphor sheet 7.
The fθ lens 15 ensures that when the stimulable phosphor sheet 7 is scanned by the laser light 10, the scanning is always performed at a uniform beam velocity.

In synchronization with the scanning by the laser light 10 as described above, the stimulable phosphor sheet 7 is indicated by an arrow A in FIG.
Is moved in the direction of, and the entire surface is scanned by the laser beam 10. Accumulative phosphor sheet 7
When irradiated with the laser beam 10, emits stimulated light of a light amount proportional to the energy of accumulated X-rays, and the emitted stimulated light enters the light guide sheet 17. The light guide sheet 17 has a light receiving end formed in a linear shape, and is arranged in proximity to the scanning line on the stimulable phosphor sheet 7 so as to face the scanning line.
The exit end thereof has an annular shape and is connected to the light receiving surface of a photoelectric conversion type photodetector 18 such as a photomultiplier. The light guide sheet 17 is made by processing a transparent thermoplastic resin sheet such as an acrylic synthetic resin, and the light incident from the light receiving end repeats total reflection on the inner surface of the light receiving end while the light exiting end is emitted. The shape is determined so as to be transmitted to the light receiving surface of the photodetector 18 through the section. Therefore, the photostimulable light emitted from the stimulable phosphor sheet 7 in response to the irradiation of the laser light 10 is emitted from the light guide sheet 1.
The light is incident on the light source 7, and the light is received by the photodetector 18 through the exit end while repeating total reflection inside the light.

A filter is attached to the light-receiving surface of the photodetector 18 for transmitting only the light in the wavelength region of the photostimulable light emitted from the stimulable phosphor sheet 7 and cutting the light in the wavelength region of the laser light 10. The photodetector 18 is attached so as to photoelectrically detect only the stimulated emission emitted from the stimulable phosphor sheet 7. The photostimulable light photoelectrically detected by the photodetector 18 is converted into an electric signal, amplified by an amplifier 19 having a predetermined amplification factor into an electric signal of a predetermined level, and then the A / D converter 20. Entered in. The electric signal is converted into a digital signal by the A / D converter 20 with a scale factor suitable for the signal fluctuation width, input to the image processing device 21, stored in an image data memory (not shown), and required. The image is displayed as an image on the screen of the CRT 22 after predetermined digital image processing is performed according to the above. According to this embodiment, a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor that does not accumulate visible light and can be used in an autoradiography measurement method is uniformly dispersed by low-energy X-rays. The X-ray energy accumulated in the stimulable phosphor layer is released and erased by the chemiluminescence generated by the decomposition of AMPPD by irradiating the substrate with X-ray energy.
An image corresponding to the DNA concentration of each spot 2 is accumulated in the stimulable phosphor layer, and then the stimulable phosphor layer of the stimulable phosphor 7 is scanned by the laser beam 2 to generate the stimulable phosphor layer. Which is photoelectrically detected by the photodetector 18,
It is converted into a digital signal by the A / D converter 20, stored in the image data memory, subjected to predetermined digital image processing if necessary, and reproduced on the screen of the CRT 22. The body 7 does not always need to be shielded from visible light and is convenient to handle. Also, the stimulable phosphor 7 is shared with an autoradiography measurement method used for the same purpose as the chemiluminescence detection method. Moreover, since it can be easily converted into a digital signal, it becomes possible to detect chemiluminescence very efficiently and accurately. In addition, in the present embodiment, a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor is provided with a low energy X-ray.
Since the X-ray energy is uniformly irradiated with the X-ray to uniformly accumulate the X-ray energy, the X-ray energy is accumulated only in the vicinity of the surface of the stimulable phosphor layer. Even in this case, most of the X-ray energy accumulated in the stimulable phosphor layer can be released and erased, and a chemiluminescence image can be recorded with high sensitivity. It becomes possible to improve the sensitivity. Further, in the present embodiment, the chemiluminescent substance AMPPD is added to TRUPIX “RUB”.
Y ”is contained and the wavelength of the chemiluminescence generated by the decomposition of AMPPD is about 470 nm, and the energy of the X-ray accumulated in the stimulable phosphor including the barium fluorobromide-based stimulable phosphor is efficiently erased. Can be 6
Since the wavelength is converted to 20 nm, chemiluminescence can be detected more efficiently by the stimulable phosphor.

FIG. 4 and FIG. 5 are flow sheets showing the procedure of the chemiluminescence detection method according to another embodiment of the present invention. 4 and 5 show a method for determining a DNA sequence by the Southern blotting method. In FIGS. 4 and 5, first, a DNA containing a target gene
The fragments 30 are electrophoresed on the agarose gel support medium 31 to be separated and developed. Then
As is well known, the DNA fragment 30 separated and developed on the agarose gel support medium 31 is denatured by alkali treatment, and the agarose gel support medium 31 and the nylon membrane filter 32 are allowed to stand for a predetermined time. Membrane contact 3
The denatured DNA fragment is transferred onto 2. Then, the membrane filter 32 is dried and irradiated with ultraviolet rays to cross-link the DNA and the membrane filter 32 (UV cross-link), and the DNA is fixed on the membrane filter 32.

Thereafter, the membrane filter 32, on which the DNA is fixed, is housed in a plastic hybridization bag 34 which is transparent to visible light and can be sealed. Next, a hybridization solution is prepared and poured into the hybridization bag 34 for prehybridization. On the other hand, a DNA probe complementary to the DNA containing the gene of interest labeled with digoxigenin (DIG) -dNTP is prepared and added to the hybridization solution, and the hybridization solution for prehybridization is added to the hybridization bag 34. After being discharged from the
Hybridize with the DNA fixed to the membrane filter 32. In FIG. 4, 35 is a complementary DN
DN containing a target gene hybridized with A
A is shown. After the hybridization is completed in this way, the membrane filter 32 is washed, and an anti-digoxigenin alkaline phosphatase-labeled antibody solution is prepared, and the hybridization bag 34 is prepared.
Add in As a result, the complementary DNA labeled with digoxigenin (DIG) and alkaline phosphatase bind to each other to label the DNA. In FIG. 1, 36 indicates a complementary DNA labeled with alkaline phosphatase.

Then, after the anti-digoxigenin alkaline phosphatase-labeled antibody solution is discharged from the hybridization bag 34, the AMPPD solution is added into the hybridization bag 34. As a result, chemiluminescence is generated from the position where the DNA labeled with alkaline phosphatase is present. Where AMP
For the PD solution, the sensitizer “EM” manufactured by TROPIX is used.
ERALD "has been added. "EMERALD" manufactured by TROPIX is mainly composed of poly [vinylbenzyl (benzyldimethylammonium chloride)] represented by the general formula (1), and a small amount of a fluorescent dye represented by the formula (2), fluorescein (CI-). 45350 Acid
Yellow 73, Dye No. 54101: Dye Handbook (Kodansha)), and a small amount of anionic dodecyloxypolyoxyethylene sulfate sodium salt, which is a surfactant.
That is, C ₁₂ H ₂₅ O (CH ₂ CH ₂ O) _n SO ₃ Na
And a small amount of NaCl and a small amount of sodium acetate. On the other hand, on the surface of the stimulable phosphor sheet 37 formed with a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor capable of accumulating radiation energy,
A low-energy electron beam is uniformly irradiated to accumulate the energy of the electron beam in the stimulable phosphor layer.

Next, the hybridization bag 3
4, after discharging a part of the AMPPD solution, the hybridization bag 34 was heated to increase the decomposition rate of AMPPD, thus forming a stimulable phosphor layer in which the energy of ultraviolet rays was uniformly accumulated. The stimulable phosphor sheet 37 and the hybridization bag 34 are
Make surface contact in a dark room. As a result, the energy of ultraviolet rays accumulated in the portion of the stimulable phosphor layer exposed by the chemiluminescence generated by the decomposition of AMPPD is released and erased, and the image of DNA is transferred to the stimulable phosphor layer. Will be recorded. In this embodiment, the surface of the stimulable phosphor sheet 7 on which the stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor is formed is uniformly irradiated with a low-energy electron beam. Is the electron beam energy only in the stimulable phosphor near the surface of the stimulable phosphor layer,
The energy of the electron beam accumulated in the stimulable phosphor layer formed on the stimulable phosphor sheet 7 can be released and erased by evenly accumulating it even by weak chemiluminescence. This is to improve the detection sensitivity of chemiluminescence. That is, when irradiating the surface of the stimulable phosphor sheet 7 with a high-energy electron beam, not only the stimulable phosphor near the surface of the stimulable phosphor layer but also the stimulable fluorescence in the deep part Even the body will accumulate the energy of the electron beam, and even if it is exposed to weak chemiluminescence after that, the stimulable phosphor layer is made to sufficiently release the energy of the electron beam accumulated. , It becomes difficult to erase, so that the sensitivity of detection of chemiluminescence decreases, but when uniformly irradiated with a low-energy electron beam, the energy of the electron beam is equal to that of the stimulable phosphor layer. Most of the energy of the electron beam accumulated in the stimulable phosphor layer can be released and erased when it is accumulated only in the stimulable phosphor near the surface and exposed by weak chemiluminescence. , Chemiluminescent images, accumulative fluorescence The sheet 7, it is possible to record with high sensitivity. Accumulative phosphor sheet 7
The energy of the electron beam that is uniformly irradiated on the surface of the is different depending on the thickness of the stimulable phosphor layer and the presence or absence of the protective layer, but the thickness of the stimulable phosphor layer is about 50 to 150 μm. In this case, it is preferably 100 KV or less.

Here, the wavelength of the light for efficiently erasing the energy of the X-rays accumulated in the barium fluorobromide stimulable phosphor is about 580 nm, and the AMP
The wavelength of chemiluminescence generated by the decomposition of PD is about 470.
However, since the AMPPD solution contains the sensitizer "EMERALD" manufactured by TROPIX, the wavelength of chemiluminescence generated by the decomposition of AMPPD is 5 nm.
The energy of the X-rays converted to 40 nm and thus accumulated in the stimulable phosphor can be efficiently erased. After a predetermined time has passed, the stimulable phosphor sheet 37 is separated from the hybridization bag 34. Thus
In the stimulable phosphor layer of the stimulable phosphor sheet 37 exposed by chemiluminescence, the energy of ultraviolet rays is uniformly accumulated only in the part which is not exposed by chemiluminescence, while it is exposed by chemiluminescence. According to the intensity of chemiluminescence, the portion releases and erases the energy of ultraviolet rays that has been accumulated in advance, and a DNA image is recorded. In this way, the image of the DNA recorded on the stimulable phosphor layer of the stimulable phosphor sheet 37 is read as a digital signal by the image data reading device shown in FIG. 3 and sent to the image processing device 21, It is stored in the image data memory, subjected to digital image processing as necessary, and displayed on the screen of the CRT 22. CRT
It is possible to easily determine the DNA sequence based on the image displayed on the screen of 22.

Also in this embodiment, similarly to the above-described embodiment, it becomes possible to detect chemiluminescence extremely efficiently and accurately. Further, in this embodiment, a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor is uniformly irradiated with a low-energy electron beam to uniformly accumulate electron beam energy. Therefore, the electron beam energy is accumulated only in the vicinity of the surface of the stimulable phosphor layer, and therefore, it is uniformly accumulated in the stimulable phosphor layer even when exposed by the weak chemiluminescence. Releases most of the energy of the electron beam
Since the chemiluminescence image can be recorded with high sensitivity after erasing, the chemiluminescence detection sensitivity can be improved. Further, in the present embodiment, AMPPD, which is a chemiluminescent substance, and “E”, which is a sensitizer manufactured by TROPIX, are used.
"MERALD" is contained, and the chemiluminescence wavelength generated by the decomposition of AMPPD is about 470 nm, and the energy of X-rays accumulated in the stimulable phosphor including the barium fluorobromide-based stimulable phosphor is efficiently erased. The stimulable phosphor allows the chemiluminescence to be detected more efficiently since it has been converted to a wavelength of 540 nm, which is possible.

[0039]

EXAMPLES Examples and comparative examples will be given below in order to further clarify the effects of the present invention. Example First, a dilution series of 7 unlabeled DNAs was prepared.
10 ng / microliter, 1 ng / microliter, 100 pg / microliter, 10 pg /
Microliter, 1pg / microliter, 100fg
/ Microliter and 10 fg / microliter of DNA. Then, using a pipette, each diluted solution of DNA was dropped onto a 15 cm × 10 cm nylon membrane filter having a pore diameter of 0.45 μm to form spots. After drying the membrane filter, irradiate it with UV light with a wavelength of about 265 nm, and
The NA and the membrane filter were cross-linked (UV cross-linked), and the DNA was immobilized on the membrane filter. Then, the membrane filter having the DNA immobilized thereon was moistened with sterilized water and housed in a plastic hybridization bag which was permeable to visible light and sealable.

After that, 30 ml of a hybridization solution consisting of 500 mM sodium phosphate buffer (pH 7.2), 7% SDS and 1 mM EDTA was added to a membrane containing a membrane filter to which DNA was immobilized. Pre-hybridization was performed by pouring into a hybridization bag. On the other hand, a DNA probe was prepared as follows. 1 microgram of the DNA fragment was dissolved in 15 microliters of distilled water, placed in a microtube, sealed, put in boiling water, heated at 100 ° C. for 5 minutes, and denaturated. , The DNA fragment was single-stranded. The single-stranded DNA solution was rapidly cooled in ice water for 5 minutes, precipitated by a small centrifuge, and added with 2 microliters of the hexanucleotide mixture and 2 microliters of the dNTP labeling mixture,
Mixed well. Here, the dNTP labeling mixture contains digoxigenin-dNTP. In this mixture,
Add 1 microliter of Klenow enzyme and mix gently by pipetting at 37 ° C
The reaction was allowed to proceed for 2 hours. Then 2 moles of EDT
The reaction was stopped by adding 2 microliters of A.

Further, 4 mol of lithium chloride (2.5 microliters) and 100% ethanol (75 microliters) were added, and the mixture was left at -70 ° C. for 1 hour to precipitate the precipitate. Then, with a microcentrifuge, at 4 ℃, 10
Centrifuge at 15,000 rpm for 1 minute and collect the precipitate at the bottom of the tube. After gently aspirating the supernatant with a pipette, 500 microliters of 70% ethanol,
In addition, after vigorously stirring with a test tube mixer and confirming that the precipitate had peeled off from the bottom of the tube, it was centrifuged with a microcentrifuge at 4 ° C. for 5 minutes at 15,000 rpm to precipitate the precipitate. Collect again at the bottom of the tube. The supernatant was then gently pipetted off and air dried. Then add 50 microliters of TE buffer and add DN
A probe was prepared and stored at -20 ° C. A predetermined amount of the thus prepared DNA probe is put in a microtube, sealed, put in boiling water, and heated to 100 ° C.
Heat for 5 minutes to denature (denaturatio
n) Then, the DNA probe was made single-stranded.

The denatured DNA probe was subjected to 5 in ice water.
Quenched for a minute and allowed to settle. Then, cut off the corner of the hybridization bag and discard the pre-hybridization solution in the hybridization bag,
A solution prepared by adding a DNA probe to a concentration of 20 ng / ml in 10 ml of the hybridization solution which had been heated to 65 ° C. in advance,
Pour into a hybridization bag containing a membrane filter with DNA fixed, and seal.
Hybridization was carried out at 5 ° C. for 15 hours. After the hybridization was completed, the hybridization solution was discharged from the hybridization bag, and 40 mmol of sodium phosphate buffer solution (pH 7.2) that had been preheated and 1% S were added.
The washing solution consisting of DS was poured into the hybridization bag, the hybridization bag was sealed, and the membrane filter was washed while shaking vigorously. For cleaning, change the cleaning solution to a new cleaning solution and do 3
It was divided into two times and carried out for 20 minutes. After the washing is completed, the washing solution is discharged from the hybridization bag, and an anti-digoxigenin alkaline phosphatase-labeled antibody solution is prepared. Combine,
The DNA was labeled.

Then, the anti-digoxigenin alkaline phosphatase-labeled antibody solution was discharged from the hybridization bag, and T was placed in the hybridization bag.
An AMPPD solution added with "EMERALD", which is a sensitizer manufactured by ROPIX, was added. On the other hand, it was placed on the surface of the stimulable phosphor sheet on which the stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor was formed, at a position 50 cm away from the surface of the stimulable phosphor sheet. The low energy X-rays are uniformly irradiated by the X-ray source of 8 kilovolts and 150 microamperes, and the low energy X-rays are emitted.
Lines were deposited on the stimulable phosphor layer. On the other hand, after discharging a part of the AMPPD solution from the hybridization bag, the hybridization bag was heated to 37 ° C. in order to enhance the decomposition rate of AMPPD, and the low energy X-rays were uniformly accumulated to be stimulable. The stimulable phosphor sheet having the phosphor layer formed thereon and the hybridization bag were brought into surface contact with each other in a dark room for 3 hours. After that, using the image data reading device shown in FIG. 3, the surface of the stimulable phosphor sheet is scanned with helium-neon laser light, photoelectrically detecting photostimulable light, and read as a digital signal. When the dilution series was reproduced on the CRT screen, 100 fg / microliter DN
Up to A were detected. Comparative Example Placed on the surface of a stimulable phosphor sheet on which a stimulable phosphor layer containing a barium fluorobromide-based stimulable phosphor was formed, at a position separated by 50 cm from the surface of the stimulable phosphor sheet. Exactly the same as the example except that X-rays were uniformly irradiated by an X-ray source of 80 kV and 150 microamperes to accumulate X-ray energy in the stimulable phosphor layer. , DNA dilution series, CR
When it was reproduced on the T screen, only 1 pg / microliter of DNA was detected.

According to the above Examples and Comparative Examples, low-energy X-rays were applied to the surface of the stimulable phosphor sheet on which the stimulable phosphor layer containing the barium fluorobromide stimulable phosphor was formed. When the X-ray energy is uniformly accumulated in the photostimulable phosphor layer by irradiation, the X-ray energy is irradiated to the photostimulable phosphor layer by irradiation with high-energy X-rays. It was found that the chemiluminescence detection sensitivity is significantly improved as compared with the case of uniform accumulation. The present invention
Without being limited to the above-described embodiments and examples, various modifications can be made within the scope of the invention described in the claims, and they are also included in the scope of the present invention. Needless to say. For example, in the above-mentioned embodiment, an example of detecting the gene amount and determining the nucleotide sequence of DNA by dot-blotting and electrophoretic method for generating reference data of the gene is shown. It is not limited to these, and can be used for the determination of nucleotide sequences of nucleic acids other than DNA by electrophoresis, screening of genes by electrophoresis or colony hybridization, separation and identification of proteins, or evaluation of molecular weight and characteristics. Can also be used. Therefore, the labeling substance and the chemiluminescent substance are not limited to those described in the above embodiment, and those suitable for each chemiluminescence detection method can be used.

Further, in the above-mentioned embodiment, an example of dot-blotting for detecting gene amount and generating gene reference data is shown, but the present invention is not limited to this method. Can also be used for Furthermore, in the above-mentioned embodiments and examples,
As the stimulable phosphor, a barium fluorobromide-based phosphor is used, but as the stimulable phosphor, it is possible to store energy of light having a wavelength other than the visible light wavelength range, and light in the visible light wavelength range. As long as it can be excited by barium fluorobromide-based phosphors, barium fluorohalide-based phosphors other than barium fluorobromide-based phosphors, and other alkaline earth metal fluoride halide-based phosphors or europium-activated composite halogens -Based phosphors, cerium-activated trivalent metal oxyhalogen-based phosphors, cerium-activated rare earth oxyhalogen-based phosphors, europium-activated composite halogen-based phosphors and the like can also be used. Further, in the above-mentioned embodiment, AMPPD is used as the chemiluminescent substance.
However, a chemiluminescent substance other than AMPPD may be used. Further, in the embodiment shown in FIG. 1 to FIG. 3, the TROM is added to the chemiluminescent substance AMPPD.
The sensitizer “RUBY” manufactured by PIX was added, and FIG.
In addition, in the embodiment shown in FIG. 5 and the above-mentioned examples, TROPIX is added to AMPPD which is a chemiluminescent substance.
Although the sensitizer “EMERALD” manufactured by the company is added, it is not always necessary to add the sensitizer.
In the embodiment shown in Figures 3 to 3, TROPIX
Instead of "RUBY" which is a sensitizer manufactured by the company, "EMER
ALD ”is added, but in the embodiment shown in FIGS. 4 and 5 and the above-mentioned examples,“ RUBY ”is used instead of“ EMERALD ”which is a sensitizer manufactured by TROPIX.
May be added, and further, if the wavelength of chemiluminescence can be converted to a wavelength capable of efficiently erasing the energy of radiation accumulated in the stimulable phosphor, T
"RUBY" and "EME" which are sensitizers manufactured by ROPIX
Any sensitizer other than "RALD" may be used.

In the embodiment shown in FIGS. 1 to 3 and the above-mentioned embodiment, low-energy X-rays are used.
In the stimulable phosphor layer formed on the stimulable phosphor sheet 7,
Irradiate uniformly to accumulate X-ray energy, while
In the embodiment shown in FIGS. 4 and 5, low-energy electron beams are uniformly applied to the stimulable phosphor layer to accumulate the electron beam energy. In the embodiment shown in 3 and the above examples,
Even if the stimulable phosphor layer formed on the stimulable phosphor sheet 7 is uniformly irradiated with a low-energy electron beam to accumulate the energy of the electron beam, FIG. 4 and FIG. In the embodiment shown in FIG. 3, it is of course possible to irradiate the stimulable phosphor layer with low energy X-rays uniformly to accumulate X-ray energy. The stimulable phosphor layer may be uniformly irradiated with radiation to accumulate the energy of the radiation. Further, in the above embodiment, the chemiluminescence information detected and converted into a digital signal is displayed as an image on the screen of the CRT 22, but it may be displayed on other display means, or It may be reproduced on a photo film or other recording medium.

[0047]

EFFECTS OF THE INVENTION According to the present invention, a stimulable phosphor sheet which is easy to handle and can be used in both chemiluminescence detection methods and autoradiography measurement methods can be used efficiently and accurately. It is possible to provide a chemiluminescence detection method and device capable of obtaining information on macromolecules such as genetic information.

[Brief description of drawings]

FIG. 1 is a flow sheet showing a first half of a procedure of a chemiluminescence detection method according to an embodiment of the present invention.

FIG. 2 is a flow sheet showing the latter half of the procedure of the chemiluminescence detection method according to the embodiment of the present invention.

FIG. 3 is a schematic perspective view showing an example of an image data reading device and an image processing device.

FIG. 4 is a flow sheet showing the first half of the procedure of the chemiluminescence detection method according to another embodiment of the present invention.

FIG. 5 is a flow sheet showing the latter half of the procedure of the chemiluminescence detection method according to another embodiment of the present invention.

[Explanation of symbols]

1 Dilution series of DNA 2 Membrane filter 3 Spot 4 Hybridization bag 5 DNA 6 hybridized with complementary DNA 6 Complementary DNA labeled with alkaline phosphatase 7 Accumulative phosphor sheet 10 Laser light 11 Laser light source 12 Filter 13 Beam expander 14 Optical deflector 15 fθ lens 16 Planar reflecting mirror 17 Light guide sheet 18 Photodetector 19 Amplifier 20 A / D converter 21 Image processing device 22 CRT 30 DNA 31 Agarose gel support medium 32 Membrane Filter 34 Hybridization bag 35 DNA hybridized with complementary DNA 36 Complementary DNA 37 labeled with alkaline phosphatase 37 Accumulative phosphor sheet

Claims (16)

[Claims] 1. A stimulable phosphor layer containing a stimulable phosphor capable of accumulating radiation energy, being excited by light in the visible light wavelength range, and releasing the accumulated radiation energy as light. The stimulable phosphor sheet thus formed is uniformly irradiated with low-energy radiation to evenly accumulate the energy of the radiation, and the chemiluminescent substance is brought into contact with the chemiluminescent substance to generate chemiluminescence. By selectively contacting the polymer labeled with the labeling substance and the chemiluminescent substance, and chemiluminescence generated by contacting the polymer labeled with the labeling substance with the chemiluminescent substance A method for detecting chemiluminescence, which comprises exposing the stimulable phosphor sheet. 2. The method for detecting chemiluminescence according to claim 1, wherein the radiation is selected from the group consisting of X-rays, α-rays, β-rays, γ-rays and electron beams. 3. The chemiluminescence detection method according to claim 2, wherein the radiation is an X-ray having an energy of 20 KV or less. 4. The method for detecting chemiluminescence according to claim 2, wherein the radiation is an electron beam having an energy of 100 KV or less. 5. Further, by scanning the stimulable phosphor sheet exposed by chemiluminescence with an electromagnetic wave,
5. The chemiluminescence detection method according to claim 1, wherein the image data is obtained by photoelectrically converting light that is excited and emitted from the stimulable phosphor sheet. 6. The chemiluminescent detection method according to claim 1, wherein the chemiluminescent substance contains a sensitizer capable of converting the emission wavelength of the chemiluminescent substance. . 7. The photostimulable phosphor is a barium fluorohalide-based phosphor, and the sensitizer is mainly composed of poly [vinylbenzyl (benzyldimethylammonium chloride)] represented by the following general formula. The method for detecting chemiluminescence according to claim 6, characterized in that Embedded image 8. The chemiluminescence detection method according to claim 7, wherein the sensitizer further contains a trace amount of a fluorescent dye and a surfactant. 9. A visible-light-transmissive housing member that accommodates the chemiluminescent substance and a polymer selectively labeled with a labeling substance that comes into contact with the chemiluminescent substance to generate chemiluminescence. A stimulable phosphor layer containing a stimulable phosphor that is capable of accumulating radiation energy and that is excited by light in the visible light wavelength range and emits the accumulated radiation energy as light. Pre-exposure means for uniformly irradiating the phosphor sheet with low-energy radiation to evenly accumulate the energy of the radiation, and the chemiluminescence generated by the contact between the labeling substance and the chemiluminescent substance cause accumulation. The stimulable phosphor sheet that has emitted the energy of the radiation as light was emitted from the stimulable phosphor sheet by excitation light irradiation means for irradiating excitation light, and excitation light. A chemiluminescence detection device comprising a detection means for photoelectrically detecting the emitted light. 10. The radiation uniformly irradiated by the pre-exposure means is selected from the group consisting of X-rays, α-rays, β-rays, γ-rays and electron beams. The chemiluminescence detection device described. 11. The chemiluminescence detection device according to claim 10, wherein the pre-exposure means is capable of irradiating an X-ray having an energy of 20 KV or less. 12. The chemiluminescence detection device according to claim 10, wherein the pre-exposure means is capable of irradiating an electron beam having an energy of 100 KV or less. 13. The exciting light irradiating means is configured to scan the stimulable phosphor sheet with electromagnetic waves to excite the stimulable phosphor sheet, according to any one of claims 9 to 12. Chemiluminescence detector. 14. The chemiluminescent detection method according to claim 9, wherein the chemiluminescent substance contains a sensitizer capable of converting the emission wavelength of the chemiluminescent substance. . 15. The photostimulable phosphor is a barium fluorohalide-based phosphor, and the sensitizer is mainly composed of poly [vinylbenzyl (benzyldimethylammonium chloride)] represented by the following general formula. 15. The chemiluminescence detection device according to claim 14, wherein the chemiluminescence detection device comprises: Embedded image 16. The sensitizer further contains trace amounts of a fluorescent dye and a surfactant.
The chemiluminescence detection device described in.