WO2005090980A1 - Procédé de détection d'une substance biologique avec une sensibilité élevée - Google Patents

Procédé de détection d'une substance biologique avec une sensibilité élevée Download PDF

Info

Publication number
WO2005090980A1
WO2005090980A1 PCT/JP2005/005125 JP2005005125W WO2005090980A1 WO 2005090980 A1 WO2005090980 A1 WO 2005090980A1 JP 2005005125 W JP2005005125 W JP 2005005125W WO 2005090980 A1 WO2005090980 A1 WO 2005090980A1
Authority
WO
WIPO (PCT)
Prior art keywords
substance
carrier
substrate
detecting
solution
Prior art date
Application number
PCT/JP2005/005125
Other languages
English (en)
Japanese (ja)
Inventor
Hiroko Sakamoto
Hiromi Sanuki
Original Assignee
Olympus Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Olympus Corporation filed Critical Olympus Corporation
Publication of WO2005090980A1 publication Critical patent/WO2005090980A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Definitions

  • the present invention relates to a method for detecting a biologically relevant substance (analyte) such as a nucleic acid with high sensitivity.
  • a small amount of the mRNA thus obtained is amplified by a method that is applied to the T7- & RNA amplification method several times, and is detected by nucleic acid hybridization.
  • this has problems such as the necessity of expensive reagents for preparing RNA samples used for hybridization, and the necessity of two to four days of complicated work.
  • Patent Document 1 Japanese Patent No. 2948904
  • the enzyme is activated by activating a detectably modified substrate using an enzyme that has been modified to specifically bind to a label attached to the analyte.
  • This is a method in which the activating substance is deposited everywhere where the receptor for the substrate is immobilized, and the detection sensitivity of the analyte in the sample is improved by a signal from the deposited substance modifier.
  • a tyramide compound is radicalized in the presence of hydrogen peroxide by the catalytic action of horseradish peroxidase. Is covalently linked to aromatic amino acids in the vicinity.
  • tyramide signal amplification kits use this technique, and by adding a detectable modifying substance (eg, fluorescent substance) to a tyramide compound, highly sensitive detection can be performed.
  • Non-Patent Document 1 the hybridization reaction step of the nucleic acid as the analyte, the washing step of the carrier on which the probe is immobilized, and the detection step of the analyte are described.
  • each step is performed by flowing the solution at a constant speed (500 / L / min) in a certain direction.
  • a constant speed 500 / L / min
  • the solution is continuously supplied to the carrier by flowing in a certain direction, the flow of the solution becomes laminar in the microchannel of the carrier.
  • the laminar flow occurs, the substantial flow velocity near the inner wall surface of the microchannel becomes slow, and there is a problem in that the efficiency of enzyme and substrate substitution decreases, and the efficiency of increasing chemiluminescence deteriorates.
  • Patent Document 1 Japanese Patent No. 2948904
  • Patent Document 2 Japanese Patent No. 3208390
  • Patent Document 3 Patent No. 3130316
  • Non-Patent Document 1 Cheek et. Al., Analytical 'Analytical Chemistry, (USA), 2001, Vol. 73, pp. 5777-5783
  • Non-Patent Document 2 Van Belkum, A. et al., BioTechniques, 1994, Vol. 16, pp. 148-153 Disclosure of the invention
  • an object of the present invention is to provide a method that enables highly sensitive detection of a biologically related substance, particularly a substance that can be obtained only in a trace amount at a medical site where diagnosis is performed.
  • the present inventors succeeded in finding that an analyte can be detected with high sensitivity by changing the spectroscopic properties of the analyte by an enzymatic reaction and insolubilizing the analyte.
  • the present invention has been completed.
  • the present invention has the following configuration.
  • a conjugate B composed of a substance and an enzyme that specifically binds to one substance is reacted with a conjugate A bound to a probe immobilized on a carrier in the step a), and the conjugate A and the conjugate B are reacted.
  • the spectroscopic property of the enzyme reaction product is a fluorescence spectrum having no substrate before the enzyme reaction or having a different wavelength from that of the substrate before the enzyme reaction.
  • the spectroscopic properties of the enzymatic reaction product have no substrate before the enzymatic reaction, or have a wavelength significantly different from that of the substrate before the enzymatic reaction, a visible region, an ultraviolet region, Or the absorption or emission spectrum in the infrared region.
  • the probe-immobilized region on the carrier is washed with a solution that does not dissociate the conjugate formed in each step.
  • the amount of the solution used for the washing is larger than the amount of the solution supplied to the carrier in each step of the step a) and / or the step b) ( Method for detecting the target substance described in 4).
  • the sample solution, the conjugate B or the substrate is driven into and out of the porous carrier.
  • step c) after holding the substrate in the carrier for a certain time, the substrate is moved out of the carrier;
  • the method for detecting a substance to be analyzed according to (6) or (7) comprising:
  • step c) the substrate is held in the carrier for a certain period of time
  • the method for detecting a substance to be analyzed according to (6) or (7) which comprises: (11) The method for detecting a substance to be analyzed according to (8) or (9), wherein in the step d), quantification is performed using the signal intensity when the change rate of the acquired signal intensity is positive. .
  • the enzyme is mainly selected from the group consisting of a phosphatase system, an oxidase system, a galactosidase system, a dalicosidase system, and a sulfatase system, and the substrate is an endogenous substrate or a synthetic enzyme of the enzyme.
  • the analyte is composed of one or more types, and one or more types of probes for each analyte are respectively provided on a porous carrier in a single reaction vessel. And the solid phase is immobilized in separate spots. How to detect substances.
  • the diameter or the length of the diagonal line of the spot or the area where the probe can be immobilized in the spot is three-dimensional.
  • the diameter or diagonal length of the cross section of the spot, parallel to the base of the carrier is in the range of 50 to 500 ⁇ m;
  • the number of the spots on a single porous carrier is 3 or more
  • the signal intensity of a detection spot is significantly improved, and a low-expression bio-related substance that could not be detected by the conventional method can be detected. Furthermore, it is possible to detect with high accuracy regardless of the level of expression, from low-expression bio-related substances to high-expression bio-related substances.
  • RNA 10 ng—
  • the application range of a microarray is wide from clinical research to diagnosis. Can be expected.
  • the signal strength is greatly improved by the method of the present invention, that is, signal amplification by an enzyme reaction
  • the S / N ratio signal Z noise ratio
  • stable signal detection is possible even with a small hybridization amount per probe spot. Therefore, it is possible to significantly reduce the amount of a substance to be analyzed (for example, the amount of a nucleic acid in gene expression analysis) added to a carrier.
  • the amount of a substance to be analyzed for example, the amount of a nucleic acid in gene expression analysis
  • 5 zg of a fluorescently labeled nucleic acid was required, but when this method is used, it can be reduced to 10 ng (lZ500 amount).
  • RNA for example, about 0.1 ⁇ g of RNA does not require amplification by T7 RNA polymerase, and analysis can be performed by, for example, performing a single labeling by reverse transcription reaction.
  • trace amounts of nucleic acids and proteins to be analyzed are determined by a chemical reaction such as that used in the Universal Linkage System (Non-Patent Document 2). It is also possible to add labels to biomolecules and analyze them, which can simplify and speed up the process of labeling analytes, which was conventionally time-consuming and labor-intensive. It is possible.
  • the label means a step of adding the binding pair according to claim 1 of the present invention to the substance to be analyzed.
  • FIG. 1 This figure shows a time-dependent change in a detection signal when an enzyme amplification reaction is carried out by the method of the present invention when a gene as a substance to be detected is detected (FIG. 1).
  • FIG. 3 shows the relationship between the enzymatic reaction time in the signal amplification reaction using alkaline phosphatase using ELF as a substrate and the transition of signal luminance.
  • FIG. 4 This figure shows the relationship between the amount of added sampnole and the transition of signal luminance in a signal amplification reaction using alkaline phosphatase using ELF as a substrate.
  • a probe Prior to carrying out the method of the present invention, a probe is immobilized on a carrier.
  • the method for immobilization is not particularly limited as long as the combination of the probe and the carrier is known in the art, but the reaction between the probe and the analyte in step a) is inhibited.
  • a method is employed in which the probe is immobilized on a carrier in a form that is not performed.
  • the probe to be used a probe capable of specifically capturing the analyte is used.
  • the analyte is a nucleic acid
  • a nucleic acid molecule having a sequence complementary to at least a part of the nucleic acid is used.
  • the substance to be analyzed is a protein or peptide, an antibody capable of specifically recognizing and binding them, a substrate for an enzyme reaction catalyzed by the protein, a protein or peptide, etc. Examples include cells that bind, and phage that express the antibody in the outer shell.
  • the substance to be analyzed is any substance having a known ligand-receptor relationship, the substance can be a receptor or a ligand.
  • nucleic acids, sugars, proteins, peptides, or substances in which these biological substances are phosphorylated can be mentioned. .
  • the analyte is a nucleic acid
  • its amount should be in the range of 0.01-1.
  • detection can be performed more suitably, and the amount of a substance other than nucleic acid is preferably within the same range.
  • the combination of probe / analyte may be DNA / DNA, DNA / RNA or
  • RNA / RNA Antibodies / proteins, peptides / proteins, proteins (ligands) / proteins (receptors), sugars / proteins, small molecules / proteins, cells / peptides, Cell / protein combinations are also included. Any of the above combinations may be a probe or a substance to be analyzed.
  • the method for detecting an analyte of the present invention comprises:
  • Forming a conjugate C consisting of: c) reacting a substrate for the enzyme in the conjugate B with the conjugate C to obtain a spectroscopic reaction product (hereinafter referred to as an enzyme reaction product). Making the properties significantly different from the spectroscopic properties of the substrate before the reaction, and insolubilizing the enzymatic reaction product in the reaction system;
  • a conjugate is formed by binding one of the substances of the binding pair to the substance to be analyzed in advance.
  • binding pair refers to a combination of substances that specifically bind to each other biologically, such as, for example, biotin-avidin, biotin-streptavidin, antigen-antibody, and ligand-receptor. "One member of a binding pair” means one member of these combinations.
  • the binding method between the analyte and one of the substances in the binding pair can be performed by a method known in the art under the conditions and the like. There is no particular limitation as long as the form does not inhibit the conjugate formation reaction formed with the other substance.
  • the sample solution containing the conjugate A is transferred to a carrier on which the probe is immobilized.
  • a specific bond is formed between the probe and the analyte in the conjugate A.
  • the formation of a specific bond is performed under conditions considered to be optimal for the combination of the analyte and the probe.
  • the analyte and the probe are nucleic acids having complementary sequences, Determine the reaction temperature, the composition of the reaction solution, etc., taking into account the Tm value of the complementary double strand formed between the substance and the probe.
  • a step for removing unreacted analytes and substances not captured by the probe from the reaction system can be appropriately performed. This removal is usually carried out by a washing step using a solution that does not dissociate the conjugate formed in step a), and preferably by removing a larger amount of the solution supplied to the carrier in step a). Do better.
  • the conjugate B is reacted with the conjugate A.
  • the conjugate B is a substance in which an enzyme is previously bound to a substance that specifically binds to one substance of the above-mentioned binding pair, ie, “the other substance of the binding pair”.
  • the binding between the other substance of the binding pair and the enzyme is not particularly limited as long as it is performed by a method known in the technical field as long as the enzyme activity is not inhibited.
  • a conjugate C is formed on the carrier.
  • This conjugate C is composed of a conjugate A composed of (analyte-one substance of the binding pair) and a conjugate B composed of (the other substance of the binding pair-enzyme).
  • a conjugate C which is a larger composite as a whole, is formed.
  • the conjugate C is bound to the probe immobilized on the carrier via the substance to be analyzed therein.
  • a step of removing unreacted substances and the like that have not been incorporated into the conjugate C can be appropriately performed. This removal is usually performed by a washing step using a solution that does not dissociate the conjugate formed in steps a) and b), and is preferably larger than the amount of the solution supplied to the carrier in step b). Perform with the volume of solution.
  • the substrate of the enzyme in the conjugate B is reacted with the conjugate C formed in the steps a) and b).
  • the spectroscopic properties of the enzyme reaction product are significantly different from the spectroscopic properties of the enzyme before the reaction, and the enzyme reaction product is insolubilized in the reaction system.
  • the change in the spectroscopic properties in the enzymatic reaction needs to be significantly different before and after the reaction. More specifically, the substrate does not have a substrate before the enzymatic reaction or the substrate before the enzymatic reaction. It is preferable to obtain a fluorescence spectrum having a different wavelength or an absorption or emission spectrum in a visible region, an ultraviolet region, or an infrared region.
  • Specific examples include a case where a substrate that did not emit a fluorescence spectrum before the reaction acquires a fluorescent spectrum due to the enzyme reaction, or a substrate that was colorless before the reaction, A case where the compound is converted into a colored substance by a reaction is exemplified.
  • the change in the spectroscopic properties more specifically, the change in the wavelength of the fluorescence spectrum is significantly different before and after the enzymatic reaction, the difference between the substrate before the reaction and the enzymatic reaction product after the reaction is obtained. Since the spectroscopic discrimination between them is easy, it is possible to spectroscopically discriminate them in a situation where the substrate before the reaction and the enzyme reaction product after the reaction are mixed. Therefore, depending on the degree of change in spectroscopic properties, it is possible to measure the substance to be detected (enzyme reaction product) without performing a washing step for unreacted substances after the reaction, etc. It is possible to reduce labor.
  • step c) it is necessary to select an enzyme-substrate combination such that the solubility of the enzyme reaction product in the reaction system is sufficiently low and substantially insoluble in the reaction system.
  • the enzymatic reaction product is insolubilized, the equilibrium of the enzymatic reaction is inclined toward the reaction product, so that more enzymatic reaction product, which is a substance to be detected, can be created on the carrier. Thereby, the detection sensitivity can be increased.
  • the degree of insolubilization is large, and if the change in the spectroscopic properties is an increase in the spectral intensity, it is possible to increase the sensitivity as a result without a significant change in wavelength, It is preferable to obtain a fluorescence spectrum or the like having a different wavelength from the substrate.
  • Examples of enzyme-substrate combinations in which the substrate acquires new spectroscopic properties and insolubility include those described in Patent Document 3. More specifically, examples of the enzyme include those selected from the group consisting of phosphatase enzymes, oxidase enzymes, glycosidase enzymes, and sulfatases enzymes. Among the endogenous substrates and synthetic substrates of this enzyme, those that can be selected from the group of those that generate insoluble substances in aqueous systems such as organic alcohols and phenols by enzymatic reaction can be mentioned.
  • BCIP 5, -bromo-4-chloro-3-3-indorisgalactoside
  • X_Gal -bromo-4-chloro-3-3-indorisgalactoside
  • the force generated by the insolubilized enzyme reaction product is determined indirectly by quantifying the spectroscopic properties acquired by the enzyme reaction product.
  • the acquired spectroscopic property is a fluorescence spectrum
  • each spot where the probe is immobilized on the carrier is measured by a fluorescence measurement system, while the acquired spectroscopic property is measured in the visible region
  • each spot is measured by a visible, ultraviolet or infrared measurement system, respectively.
  • FD10 manufactured by Olympus Corporation or the like can be used.
  • the carrier used in the present invention can substantially hold a liquid, and moves the liquid in the carrier. Any material that can be used and has a large surface area can be used. For example, there are hollow fibers, hollow fibers + gel, aluminum oxide film, silicon wafer etching, glass fiber, multiple glass and resin beads held in a container, and more preferably three-dimensional porous Is good.
  • the three-dimensional porous carrier include a filter made of nylon, nitrocellulose, or resin, a gel matrix, and a metal oxide film.
  • the three-dimensional porous carrier used in Examples of the present invention is a metal oxide film produced electrochemically described in Patent Document 2.
  • the porous carrier is provided with a spot for immobilizing one or more types of probes for each analyte with the porous carrier.
  • the carrier used in the detection method of the present invention generally has a plurality of probe-immobilized spots so that a plurality of samples can be simultaneously measured, and each spot contains a separate analyte. Probes for capture are immobilized.
  • the carrier used in the detection method of the present invention is such that only one type of probe for one type of analyte is immobilized on one spot and one type of analyte.
  • One type of probe for a substance should be immobilized on multiple spots, and two or more types of probes for one type of analyte should be immobilized on separate spots for each type. Alternatively, two or more types of probes for one type of analyte can be immobilized on a plurality of spots for each type.
  • This carrier can be used as a dedicated carrier for a specific purpose.
  • the diameter (diameter or diagonal length) where the probe is spotted is in the range of 50 to 500 zm.
  • the diameter (diameter or diagonal length) of the cross section on which the probe is spotted, which is parallel to the base of the carrier is 50 to 500 zm.
  • the number of spots on a single porous carrier can be three or more.
  • the diameter or the diagonal length of the spot (in the case of two dimensions) of the probe is
  • the diameter or diagonal length of the cross section (in the case of three dimensions) is in the range of 50 to 500 ⁇ m
  • about 20 to 400 types are available in the area of about 4 mm in diameter or diagonal length on the carrier.
  • Probe can be immobilized, and the volume of the sample to be supplied to each spot can be extremely small. Therefore, even when only a very small amount of the substance to be analyzed is available, detection using different probes can be performed in the same place and efficiently.
  • a sample solution, a solution containing the conjugate B, or a solution containing the substrate can be reciprocated by driving (bombing or pitting) into and out of the porous carrier.
  • the method of proceeding an enzyme reaction and the method of quantifying a substance to be analyzed are determined. It is possible to set more flexibly.
  • the conjugate formation reaction can be performed over a larger surface area by holding the sample solution in the carrier for a certain period of time, and the sample solution is driven into and out of the carrier,
  • the system is capable of increasing the frequency of contact between reactants and making the reaction more efficient.
  • the sample solution introduced into the carrier is driven out of the carrier after reacting for a certain period of time, and unreacted substances and the like are excluded outside the carrier, and the substance to be detected (insoluble enzyme reaction product Sample) and then re-introduce the sample solution, which had been driven out of the carrier, into the carrier, or drive a new substrate, etc., into the carrier to allow the unreacted substance to react. It can be excluded to the outside and measurement of the target substance can be repeated again. Therefore, in the method of the present invention,
  • step c) after holding the substrate in the carrier for a certain time, the substrate is moved out of the carrier;
  • step C) a step of quantifying the spectroscopic properties of the enzyme reaction product insolubilized in steps c) and e) above. Can be included. Further, in the method of the present invention, in the step C), the substrate is kept in the carrier for a certain time;
  • the combination of the above steps e) and f) can be performed a plurality of times. Even if it is not possible to measure a signal of sufficient intensity in the measurement in step d), by performing the combination of steps e) and f) multiple times, the insolubilized enzyme-reactive It accumulates so much that a detectable level of signal can be obtained.
  • the substrate-containing solution used in the step contains a sufficient amount of the substrate. In step e), the substrate-containing solution is used in step c), which is not a new substrate. It is also possible to use a solution that has been driven.
  • the signal intensity eg, the fluorescence intensity
  • the signal intensity becomes saturated. If the signal intensity is saturated, quantitative comparison between different spots on the carrier cannot be performed accurately. Therefore, in the method of the present invention, in particular, in the method of amplifying the signal intensity by performing the combination of the above steps e) and f) a plurality of times, when the change rate of the signal intensity is positive, more preferably the signal intensity is increased. It is desirable to perform quantification using the signal intensity in the process of increasing linearly. More specifically, the signal intensity increases linearly, more specifically, the signal is measured in a range where the signal intensity changes substantially linearly, that is, a differential value of the rate of change is in a range of 0 to 0.5. Is more preferable.
  • the step a) d) or the step a) -f) is carried out in a substantially constant range of 20 ° C. to 70 ° C. It is preferably performed at a temperature. More preferably, it is a substantially constant temperature range within the range of 30 ° C-50 ° C, 30 ° C and 37 ° C.
  • the analyte is a nucleic acid
  • hybridization with the probe is usually performed at about 50-65 ° C
  • enzymatic reaction is preferably performed at 20-45 ° C. Les ,.
  • the optimal temperature for hybridization is 50.
  • the optimal temperature for hybridization was 42 ° C, which is within the temperature range in which the enzymatic reaction can be effectively performed.
  • the steps a) to d) or the steps a) to f) can be performed at substantially the same temperature. . This eliminates the need for temperature changes, thus improving the temperature control system and reducing time.
  • the steps a) to d) or the steps a) to f) are performed at substantially the same temperature.
  • the steps a) and b) are performed at substantially the same temperature X
  • the steps c), d) and f) are performed at substantially the same temperature
  • the steps a) and It can be performed at a temperature lower than the temperature X in b).
  • Biotin-labeled aRNA was prepared from human mammary gland-derived total RNAl ⁇ g using messageAMP aRNAkit (manufactured by Ambion). First, using an oligo d (T) primer containing the promoter sequence of T7 RNA polymerase, 42 ⁇ g of total RNA in the presence of dNTPs and reverse transcriptase 42. C. for 2 hours to synthesize single-stranded cDNA. Subsequently, the mixture was incubated at 16 ° C for 2 hours in the presence of dNTPs, RNaseH, and DNA polymerase to synthesize double-stranded cDNA.
  • T oligo d
  • the double-stranded cDNA was purified to form type III, in the presence of ATP, CTP, GTP, UTP, biotin_UTP and T7 RNA polymerase37. After incubation at C for 12 hours, an in vitro transcription reaction (IVT reaction) was performed to synthesize aRNA. After the IVT reaction, DNasel was added to the IVT reaction solution, and the mixture was incubated at 37 ° C for 30 minutes to decompose the cDNA used as type I. After degradation, the product of the IVT reaction was purified.
  • IVTT reaction in vitro transcription reaction
  • 1 ⁇ g of the purified aRNA sample was subjected to fragmentation using 10 ⁇ Fragmentation Reagent manufactured by Ambion.
  • 10 ⁇ Fragmentation Reagent manufactured by Ambion.
  • the aRNA solution adjusted to 37.5 ⁇ l with 1 ⁇ 1 ⁇ 236-6 ⁇ water was incubated at 95 ° C for 5 minutes, quenched in ice, and then placed on ice for 5 minutes or more.
  • the solution was then preheated to 42 ° C, the temperature at which hybridization was performed, and the 20X SSPE melt was added to 7.5 ⁇ l and 10 ° C immediately before adding to the array.
  • 5 ⁇ l of the% SDS solution was added to the mixture, and 50 ⁇ l of an aRNA solution (1 ⁇ gaRNA / 3 ⁇ SSPE / 1% SDS) was finally prepared.
  • the DNA microarray is a flow-through type three-dimensional porous membrane substrate that has an aggregated structure of many branched cyclic structures with openings at the top and bottom.
  • a microarray having a solid phase immobilized thereon was used.
  • This substrate has a structure that allows the liquid to pass through the structure and retains it, so that the reaction can be performed by applying force without driving the solution into and out of the carrier.
  • Hybridization was performed using Olympus FD10. Add 50 samples of aRNA solution to the array, and apply this solution at 42 ° C at a constant rate of 5 ⁇ l / sec to the probe-immobilized 3D porous carrier 150 times. A hybridization reaction was performed while repeatedly passing through.
  • Hybridization was detected using ELF97kit manufactured by Molecular Probe. After the above hybridization reaction, the hybridization sample solution is driven out of the carrier and removed, and the 70 ⁇ l (7) 3 X SSPE solution is removed at a constant speed of 5 ⁇ l / sec. Washing was performed by repeating the operation of driving one to three times into and out of the device three times. After washing, add 50 ⁇ of blocking solution (1% BSA), and similarly make the probe permeate the solid-phased three-dimensional porous carrier 5 times repeatedly at a constant rate of 5 ⁇ // sec. , A blocking treatment was performed. After blocking, remove the blocking solution and add 50 ⁇ l of freshly prepared streptavidin- ⁇ solution.
  • blocking solution 1% BSA
  • the signal analysis of the array image was performed by the image analysis software attached to the FD10 system.
  • the brightness of each signal spot increases in proportion to the reaction time in the enzyme amplification reaction in signal detection.
  • Stage images were selected and used to quantify signal brightness. Specifically, for genes that are expected to have high signal brightness due to high gene expression levels, images acquired at a relatively early stage of the enzyme amplification reaction can be used for signal analysis. On the other hand, for genes that are expected to have low signal brightness due to low expression levels, images acquired at a later stage of the enzyme amplification Used for signal analysis.
  • Example 1 hybridization was performed using 1 ⁇ g of aRNA directly fluorescently labeled using FITC-UTP instead of the aRNA used in Example 1 above, and image analysis was performed.
  • aRNA directly fluorescently labeled using FITC-UTP instead of the aRNA used in Example 1 above
  • image analysis was performed.
  • Fig. 2 As shown in FIG. 2, it was found that when the method of the present invention was performed, the signal quantification was possible in about three to four times as many spots as in the conventional method using direct labeling.
  • nucleic acids, sugars, proteins, peptides, and substances obtained by modifying these substances can be detected.
  • Various modifications to these substances include phosphorylation, methylation, acetylation, and deacetylation.
  • measuring protein kinase activity is one of them. The activity of this protein kinase can be determined by measuring whether or not a specific amino acid in the peptide has been phosphorylated. In this case, detection is carried out by using a peptide serving as a substrate for proteinase as a probe.
  • a 30-mer run consisting of 30 random sequences of four types of bases, adenine, thymine, guanine, and cytosine, with 5 'end labeled with one molecule of biotin and 3' end labeled with one molecule of FITC.
  • the dam sequence oligo DNA was chemically synthesized and dissolved in a TE solution to prepare a 1 ⁇ concentration random oligo stock solution.
  • 25 ⁇ l of the above 1 ⁇ random oligo stock solution was adjusted to 37.5 ⁇ l with RNase-free water, incubated at 95 ° C for 5 minutes, quenched in ice, and then kept in ice for 5 minutes or more. I smelled.
  • This solution is then pre-heated to 42 ° C, the temperature at which hybridization is performed, and immediately before addition to the array, 20X SSPE solution is added to 7.5 ⁇ ⁇ and 5% lauroyl sarcosine. 5 ⁇ l of the solution was added to the mixture and finally 50 ⁇ l of a random oligo target nucleic acid solution (500 ⁇ random oligo / 3 ⁇ SSPE / 0.5% lauroyl sarcosine) was prepared.
  • DNA microarrays consist of a flow-through type three-dimensional porous membrane substrate that has an aggregate structure of a number of branched circular structures with openings at the top and bottom. A microarray having a solid phase immobilized thereon was used. This substrate has a structure that allows the liquid to pass through and holds the structure, and has a structure that can perform the reaction while driving the solution into and out of the unit.
  • Hybridization was performed using Olympus FD10. 50 ⁇ l of the sample random oligo solution was added to the array, and the solution was applied at 42 ° C at a constant rate of 5 / il / s to the 3D porous carrier on which the probe was immobilized. The hybridization reaction was carried out repeatedly while passing through 150 times.
  • the hybridization sample solution is driven out of the carrier and removed, and 70 ⁇ l of the 3X SSPE solution is moved into and out of the carrier at a constant rate of 5 ⁇ l / sec. 1 The operation of driving three times was repeated three times to perform washing.
  • 50 ⁇ l of 3X SSPE solution was added to the carrier, and the entire amount of this solution was transmitted through the lower surface of the array substrate (behind the array surface to be imaged), and an array image was acquired using an optical filter for FITC detection. Then, the amount of target nucleic acid hybridized to each probe spot was detected from the signal luminance using FITC fluorescence as a label.
  • Amplification and detection of the hybridization signal were performed using ELF97 mRNA In Situ Hybridization Kit # 2 (Cat #: E-6605) manufactured by Molecular Probe. After the above signal detection by FITC, remove the 3X SSPE solution by driving it out of the carrier, add 50 ⁇ l of blocking solution (1% BSA solution), and similarly probe at a constant speed of 5 ⁇ l ⁇ second. Blocking treatment was performed by repeatedly permeating the solid-phased three-dimensional porous carrier five times. After blocking treatment, remove the blocking solution, add 50 ⁇ of freshly prepared streptavidin 'alkaline phosphatase solution (hereinafter referred to as streptavidin_AP solution), and similarly add 3D pores at a rate of 5 ⁇ seconds.
  • streptavidin_AP solution freshly prepared streptavidin 'alkaline phosphatase solution
  • the membrane was repeatedly permeated 15 times through a porous carrier, and a binding reaction between biotin and streptavidin was carried out. After the binding reaction, the streptavidin- ⁇ solution is removed, 70 ⁇ l of 3X SSPE solution is added, and the probe is immobilized at a speed of 5 ⁇ lZ seconds on the three-dimensional porous support on which the probe is immobilized. Washing was repeated four times through the process of repeating permeation three times. After washing, add 50 ⁇ l of the ELF solution prepared according to the protocol attached to the kit, and allow it to pass through the porous carrier at a rate of 5 ⁇ l / s by 25 ⁇ l by volume.
  • the ELF was insolubilized by alkaline phosphatase.
  • the ELF solution was further passed through at 25 / il, and in this state, the first array image acquisition was performed using a CCD camera.
  • an optical filter matched to the fluorescence characteristics of the insolubilized ELF was used.
  • the ELF solution was passed through the ELF solution at 25 / l, this time in the reverse direction, and the substrate was again immersed in the ELF solution for 5 minutes.
  • 25 ⁇ l of the ELF solution was transmitted again, and a second array image acquisition was performed.
  • a series of operations such as:
  • a powerful cycle is repeated several times (preferably about 210 times), Images were acquired at each point in the home during the elapsed time of the null amplification reaction, and a graph was created in which individual signal intensities were plotted against the elapsed time of the enzyme reaction.
  • one molecule was detected on the random oligo as the target nucleic acid sample without signal amplification by the enzyme, and the hybridization detected using the directly labeled FITC as an indicator was also plotted, and the signal from the enzyme reaction using ELF as a substrate was plotted. The degree of amplification was observed.
  • the step of signal amplification by the enzyme was performed at 37 ° C.
  • the signal analysis of the array image was performed by the image analysis software attached to the FD10 system.
  • a short image acquisition time (2 milliseconds) to a long image acquisition time Acquisition of multiple array images at different imaging times up to 2560 milliseconds
  • select the signal luminance was used for quantification.
  • images acquired with a short imaging time are used for signal analysis
  • images acquired with a long imaging time are used for signal analysis.
  • the signal brightness for the uniform signal acquisition time was calculated for each probe spot and used as the signonole brightness of the probe spot.
  • the signal brightness of each probe spot increased in proportion to the reaction time 30 minutes after the disclosure of the enzyme amplification reaction.
  • the signal luminance after the enzyme amplification reaction is amplified up to about 300 times the signal luminance detected by FITC before the amplification reaction, and by performing signal amplification by the enzyme reaction, a small amount of hybridization can be achieved.
  • FITC signal luminance detected by FITC
  • a small amount of hybridization can be achieved.
  • Fig. 3 it was suggested that stable signal detection would be possible even for genes with low expression levels (see Fig. 3).
  • a biotin-labeled aRNA was prepared from 1 ⁇ m of Universal Human Reference RNA (Stratagene Ca 40000) using the messageAMP aRNA kit (Ambion). First, using oligo d (T) primer containing the promoter sequence of T7 RNA polymerase, 1 ⁇ g of totalRNA was incubated at 42 ° C for 2 hours in the presence of dNTPs and reverse transcriptase to synthesize single-stranded cDNA. . Subsequently, the mixture was incubated at 16 ° C for 2 hours in the presence of dNTPs, RNaseH, and DNA polymerase to synthesize double-stranded cDNA.
  • 1 ⁇ g of the purified aRNA sample was subjected to fragmentation using 10 ⁇ Fragmentation Reagent manufactured by Ambion.
  • 10 ⁇ Fragmentation Reagent manufactured by Ambion.
  • RNA solution adjusted to 25 ⁇ l with RNase-free water was incubated at 95 ° C for 5 minutes, quenched on ice, and then kept on ice for 5 minutes or more. The solution is then preheated to 37 ° C, the temperature at which the hybridization is performed, and immediately prior to addition to the array, 2X hybridization buffer (20% formamide / 6X SSPE / 1% lauroyl zanorecosin). ) Dissolve f night with 25 ⁇ l of calorie, and finally 50 ⁇ l of aRNA night (10, 20, 50, and 100 ng of aRNA / 10% formamide ⁇ 3 X SSPE / 0.5% lauroyl sarcosine) Prepare did.
  • DNA microarrays consist of a flow-through type three-dimensional porous membrane substrate that has an aggregate structure of a number of branched circular structures with openings at the top and bottom. A microarray having a solid phase immobilized thereon was used. This substrate has a structure that allows the liquid to pass through and holds the structure, and has a structure that can perform the reaction while driving the solution into and out of the unit.
  • Hybridization was performed using Olympus FD10. Add 50 ⁇ l of the sample random oligo solution to the array, and apply this solution at 37 ° C at a constant speed of 5 ⁇ s 150 times to the 3D porous carrier on which the probe is immobilized. The hybridization reaction was carried out while repeatedly passing through.
  • Amplification and detection of the hybridization signal were performed using ELF97 mRNA In Situ Hybridization Kit # 2 (Cat #: E-6605) manufactured by Molecular Probe.
  • the hybridization sample solution is driven off the carrier and removed, and 70 ⁇ l of the 3X SSPE solution is injected into and out of the carrier at a constant speed of 5 ⁇ l / sec.
  • the operation of driving one to three times was repeated three times to perform washing.
  • the blocking solution was removed, 50 ⁇ l of freshly prepared streptavidin- ⁇ solution was added, and permeation was repeated 15 times through the three-dimensional porous carrier at a rate of 5 ⁇ l ⁇ second.
  • 'A binding reaction between streptavidin was performed.
  • the streptavidin-AP solution is removed, 70 ⁇ l of 3X SSPE solution is added, and the probe is immobilized at a speed of 5 ⁇ lZ seconds on the three-dimensional porous support on which the probe is immobilized. Washing was repeated four times through the process of repeating permeation three times.
  • the ELF solution prepared according to the protocol attached to the kit, and add 25 ⁇ 1 of the volume at a rate of 5 ⁇ 1 / sec to the porous carrier.
  • the ELF was insolubilized with alkaline phosphatase by holding the substrate in this state for 15 minutes and holding the substrate immersed in the ELF solution. After 15 minutes, the ELF solution was further transmitted at 25 / il, and in this state, the first array image acquisition was performed using a CCD camera.
  • an optical filter matched to the fluorescence characteristics of the insolubilized ELF was used. All the above steps were performed at 37 ° C.
  • the signal analysis of the array image was performed by the image analysis software attached to the FD10 system.
  • a short image acquisition time (2 milliseconds) to a long image acquisition time Acquisition of multiple array images at different imaging times up to 2560 milliseconds
  • select the signal luminance was used for quantification.
  • images acquired with a short imaging time are used for signal analysis
  • images acquired with a long imaging time are used for signal analysis.
  • the signal brightness for the uniform signal acquisition time was calculated for each probe spot and used as the signonole brightness of the probe spot.
  • the signal brightness of each probe spot increased in proportion to the increase in the amount of biotin-labeled aRNA to be analyzed from 20 ng to 100 ng.
  • the amount of aRNA added was increased from 10 ng to 20 ng, the signal luminance increased but the luminance did not reach twice.
  • the minimum required amount of aRNA sample with aRNA sample is about 20 ng, and the required sample amount is 1/100 1/500 compared to the conventional method without this method. It was found that it could be reduced.
  • the amount of addition was increased to 100ng, the relationship between the amount of sample added and the signal brightness was maintained in a proportional relationship, suggesting that the signal could be detected with high quantitativeness (see Fig. 4).
  • Example 5 In the case of gene expression analysis, a conventional method in which a signal amplification reaction by an enzyme is performed and a method in which a fluorescent signal is detected by hybridizing directly labeled sampnoles without signal amplification are performed. In order to examine the correlation of the obtained expression change data when the above was performed, the following examination was performed.
  • the messageAMP aRNA kit (Ambion ), And a biotin-labeled aRNA (drug administration group, control group) and FITC-labeled aRNA (drug administration group, control group) were prepared.
  • oligo d (T) primer containing the promoter sequence of T7 RNA polymerase, 1 ⁇ g of total RNA was incubated at 42 ° C for 2 hours in the presence of dNTPs and reverse transcriptase, and the single-stranded cDNA was recovered. Synthesized.
  • the mixture was incubated at 16 ° C for 2 hours in the presence of dNTPs, RNaseH, and DNA polymerase to synthesize double-stranded cDNA.
  • the double-stranded cDNA was purified to form ⁇ , and ATP, CTP, GTP, UTP, and biotin-labeled biotin-UTP, FITC-labeled FITC-UTP, and T7RNA polymerase in the presence of T7 RNA polymerase.
  • an in vitro transcription reaction IVT reaction
  • DNasel was added to the IVT reaction solution, and the mixture was incubated at 37 ° C for 30 minutes to decompose the cDNA used as type I. After degradation, the product of the IVT reaction was purified.
  • Fragmentation treatment was performed on 10 ⁇ g of each purified aRNA sample using 10 ⁇ Fragmentation Reagent manufactured by Ambion.
  • RNA solution adjusted to 37.5 ⁇ l with RNase-free water was incubated at 95 ° C for 5 minutes, quenched in ice, and then kept in ice for 5 minutes or more. The solution is then pre-heated to 37 ° C, the temperature at which hybridization is performed, and immediately before addition to the array, 7.5 ⁇ l of 20X SSPE solution and 5 ⁇ l of 5% lauroyl sarcosine solution are added.
  • RNA solution 5 ⁇ g (FITC-labeled) or 50 ng (biotin-labeled) of aRNA / 3 ⁇ SSPE / 0.5% lauroyl sarcosine was prepared.
  • DNA microarrays consist of a flow-through type three-dimensional porous membrane substrate that has an aggregate structure of a number of branched circular structures with openings at the top and bottom. A microarray having a solid phase immobilized thereon was used. This substrate has a structure that allows the liquid to pass through and holds the structure, and has a structure that can perform the reaction while driving the solution into and out of the unit.
  • Hybridization was performed using Olympus FD10. 50 ⁇ l of the sample random oligo solution was added to the array, and this solution was applied at 37 ° C at a constant rate of 5 / il / s to a 3D porous support on which the probe was immobilized. The hybridization reaction was performed while allowing the permeation to be repeated repeatedly.
  • the hybridization sample solution is driven out of the carrier and removed, and 70 ⁇ l of the 3X SSPE solution is moved into and out of the carrier at a constant rate of 5 ⁇ l / sec. 1
  • the operation of driving three times was repeated three times to perform washing.
  • a 50/11 1 333 solution was added to the carrier, and the entire amount of this solution was allowed to penetrate the lower surface of the array substrate (behind the array surface to be imaged). And an array image was obtained for the FITC-labeled sample.
  • Hybridization signal using Hybridization Kit # 2 (Cat #: E-6605) Amplification and detection were performed.
  • hybrida I See Chillon above reaction hybrida the I See Chillon sample solution was removed by driving out the carrier, and out of the carrier 3 X SSPE solution 70/1 at a constant speed of 5 beta 1 / sec The operation of driving one to three times was repeated three times to perform washing. After washing, add 50 ⁇ l of blocking solution (1% BSA), and repeat 5 times permeation through the 3D porous carrier on which the probe is immobilized at a constant speed of 5 ⁇ l / sec. By doing so, a blocking process was performed.
  • blocking solution 1% BSA
  • the signal analysis of the array image was performed by the image analysis software attached to the FD10 system.
  • a short image acquisition time (2 milliseconds) to a long image acquisition time Acquisition of multiple array images at different imaging times up to 2560 milliseconds
  • select the signal luminance was used for quantification.
  • images acquired with a short imaging time are used for signal analysis
  • images acquired with a long imaging time are used for signal analysis.
  • the spot brightness was calculated with respect to the uniform signal acquisition time, and the signal brightness was defined as the signal intensity of the probe spot.
  • the luminance ratio of the signal detected at each probe spot was calculated for the drug administration group and the control group.
  • FITC-labeled samples and biotin-labeled samples were compared, respectively.
  • the signal brightness ratio the sum of signal brightness obtained from one array image was calculated. Calculations were made after making corrections to make them the same.
  • the probe spots where a change in gene expression was detected and the signal luminance ratio of each probe spot are shown below.
  • the FITC-labeled sample is used in the conventional method and when the biotin-labeled sample is used according to the present invention, the gene whose expression change is detected is the same, and the value of the change (signal luminance ratio) is also the same. Was confirmed.
  • the above table shows that, for the same RNA sample, the conventional method of direct fluorescence-labeled aRNA sample was used. Detection of changes in expression when a pull is prepared and signal detection is performed without signal amplification by an enzyme reaction, or when a signal is detected by preparing a biotin-labeled aRNA sample according to the present invention and amplifying the signal by an enzyme reaction. This figure shows the expressed genes and the expression ratio in each case.
  • a bio-related substance contained in a small amount in a sample available at a clinical site can be detected without complicated preparatory operations or expensive reagents. It is expected to be used at medical sites where diagnosis is performed.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

L'intention est de fournir un procédé de détection d'une substance biologique avec une sensibilité élevée. A savoir, procédé de détection d'un sujet devant être analysé caractérisé en ce qu'il comprend de faire réagir une enzyme, laquelle est indirectement liée à la substance biologique immobilisée sur un support via une sonde, avec un substrat, lequel est insolubilisé du système de réaction après la fin de la réaction avec l'enzyme et présente un grand changement en termes de propriétés spectroscopiques, et de quantifier ensuite les propriétés spectroscopiques du produit de la réaction avec l'enzyme au cours et/ou après la réaction avec l'enzyme.
PCT/JP2005/005125 2004-03-22 2005-03-22 Procédé de détection d'une substance biologique avec une sensibilité élevée WO2005090980A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2004082400 2004-03-22
JP2004-082400 2004-03-22

Publications (1)

Publication Number Publication Date
WO2005090980A1 true WO2005090980A1 (fr) 2005-09-29

Family

ID=34993834

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2005/005125 WO2005090980A1 (fr) 2004-03-22 2005-03-22 Procédé de détection d'une substance biologique avec une sensibilité élevée

Country Status (1)

Country Link
WO (1) WO2005090980A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110286221A (zh) * 2019-06-11 2019-09-27 遵义医学院附属医院 一种用于监测hpv杂交捕获的远程观察系统

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5830667A (ja) * 1981-08-05 1983-02-23 エフ・ホフマン−ラ・ロシユ・ウント・コンパニ−・アクチエンゲゼルシヤフト 標識化免疫学的活性物質
JPH06109734A (ja) * 1992-09-30 1994-04-22 S R L:Kk 抗原の測定方法
JPH08501628A (ja) * 1992-09-11 1996-02-20 ベーリンガー マンハイム コーポレーション 免疫診断検査系およびその利用
JPH10239314A (ja) * 1997-02-24 1998-09-11 Eiken Chem Co Ltd 結合分析の高感度化方法
WO2002034950A2 (fr) * 2000-10-23 2002-05-02 Beckman Coulter, Inc. Immobilisation de biopolymeres sur des substrats amines par adsorption directe
JP2004061431A (ja) * 2002-07-31 2004-02-26 Yanaihara Kenkyusho:Kk 長期間保存可能なヒト・クロモグラニンａ測定キット

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5830667A (ja) * 1981-08-05 1983-02-23 エフ・ホフマン−ラ・ロシユ・ウント・コンパニ−・アクチエンゲゼルシヤフト 標識化免疫学的活性物質
JPH08501628A (ja) * 1992-09-11 1996-02-20 ベーリンガー マンハイム コーポレーション 免疫診断検査系およびその利用
JPH06109734A (ja) * 1992-09-30 1994-04-22 S R L:Kk 抗原の測定方法
JPH10239314A (ja) * 1997-02-24 1998-09-11 Eiken Chem Co Ltd 結合分析の高感度化方法
WO2002034950A2 (fr) * 2000-10-23 2002-05-02 Beckman Coulter, Inc. Immobilisation de biopolymeres sur des substrats amines par adsorption directe
JP2004061431A (ja) * 2002-07-31 2004-02-26 Yanaihara Kenkyusho:Kk 長期間保存可能なヒト・クロモグラニンａ測定キット

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110286221A (zh) * 2019-06-11 2019-09-27 遵义医学院附属医院 一种用于监测hpv杂交捕获的远程观察系统

Similar Documents

Publication Publication Date Title
US11377689B2 (en) Chemical compositions and uses thereof
US20220026433A1 (en) Cleavable fluorescent tyramide for sensitive and multiplexed analysis of biological samples
US20030013091A1 (en) Methods for detection and quantification of analytes in complex mixtures
US20230175047A1 (en) Array and method for detecting spatial information of nucleic acids
EA004271B1 (ru) Способы амплификации и секвенирования нуклеиновых кислот
JP3872762B2 (ja) Ｄｎａチップの品質管理方法
JPWO2006095550A1 (ja) Ｐｃｒプライマー、それを利用したｐｃｒ法及びｐｃｒ増幅産物、並びにｐｃｒ増幅産物を利用するデバイス及びｄｎａ−タンパク複合体
KR20100075524A (ko) 표적 ｒｎａ의 검출·정량방법 및 키트
Wei et al. A highly sensitive SPRi biosensing strategy for simultaneous detection of multiplex miRNAs based on strand displacement amplification and AuNP signal enhancement
US20070015223A1 (en) Method of detecting substance to be analyzed
Joo et al. Highly selective FRET-aided single-molecule counting of microRNAs labeled by splinted ligation
US20100029492A1 (en) Nucleic acid chip for obtaining binding profile of single strand nucleic acid and unknown biomolecule, manufacturing method thereof and analysis method of unknown biomolecule using nucleic acid chip
JP2002357604A (ja) 反応容器及びこれを用いる生物学的に活性な物質の分析方法
US20170175177A1 (en) Method for detecting nucleic acid, capture probe, detection probe set, microarray, nucleic acid detection kit, nucleic-acid-immobilized solid phase, and fluid device
CN111718985A (zh) 一种microRNAs检测试剂盒及检测方法和应用
WO2005090980A1 (fr) Procédé de détection d'une substance biologique avec une sensibilité élevée
KR100923048B1 (ko) 미지의 생체분자와 단일가닥핵산의 결합 프로파일을생성하기 위한 핵산칩, 핵산칩의 제조방법, 및 핵산칩을이용한 미지의 생체분자 분석방법
CN107058576B (zh) 基因芯片杂交组件、包含该杂交组件的试剂盒、其应用及杂交检测方法
JP6288650B2 (ja) 核酸配列決定用のフローセル
US20220025430A1 (en) Sequence based imaging
WO2010133849A1 (fr) Détection d'un analyte cible par amplification de signal
WO2005106030A1 (fr) Méthode de détection d'acide nucléique
Aviñó Andrés et al. Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)
WO2019229792A1 (fr) Procédé de détection d'un fragment d'acide nucléique cible
JP2006029953A (ja) 生体関連物質検出用固相化担体及びプローブの固相化方法、並びに生体関連物質解析方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SM SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP