JP6075610B2 - Protein-immobilized gel microarray - Google Patents

Description

  The present invention relates to a microarray for detecting a protein.

  In recent years, in the life science research field, a microarray method has been developed as an analysis method for collectively analyzing a large amount of biological information.

  A microarray is an array of a plurality of substances that bind to a substance (target substance) to be examined as a capture probe on a small substrate such as a microchip.

  The specimen is brought into contact with the probe placed on the microarray substrate, the target substance reacts with the probe, and then the bound substance is detected, so that the presence / absence of the target substance present in the specimen or its abundance can be collectively measured. Can be examined.

  Various types of microarrays have already been developed, and microarrays (Patent Document 1) in which capture probes (DNA (deoxyribonucleic acid), peptides, etc.) are sequentially synthesized on the two-dimensional surface of the substrate by photolithography are synthesized in advance. There is a microarray (Patent Document 2) in which a capture probe that has been prepared is spotted on a two-dimensional surface of a substrate.

  As another example, there has been proposed a microarray for detecting a bio-related substance, which has a plurality of compartments, each compartment is formed of holes or hollow fibers, and a gel-like material containing a capture probe is held in each compartment. (See Patent Document 3 and Patent Document 4). This manufacturing method has the advantage that stable quality microarrays can be produced in large quantities without the need for expensive manufacturing equipment as compared with the photolithography method and spotting method.

  In addition, since the microarray manufactured by this method holds the gel-like material containing the capture probe in the hollow portion of the hollow fiber, the microarray is larger in one section than the microarray that simply holds the capture probe on the flat substrate. Can hold the capture probe. Furthermore, since the hybridization reaction by electrophoresis can be performed, the inspection time can be shortened.

  Among such microarrays, those in which DNA is arranged as a probe are particularly called DNA microarrays. In addition to DNA, various microarrays for the detection of biologically related substances in which proteins, peptides, sugar chains, other compounds, etc. are used as target substances and the substances to which they bind are arranged are manufactured and used mainly in basic research fields. ing.

  An attempt has been made to apply such a microarray method to the clinical diagnosis field, and to perform an existing clinical diagnosis using a microarray. For example, a method for detecting a cancer marker using a microarray has been reported. A method is disclosed in which a pancreatic cancer marker protein, which is a blood protein, is reacted with a capture probe disposed on a microarray substrate, and then the pancreatic cancer marker protein bound to the probe is analyzed by mass spectrometry (Patent Document 5). . A method for detecting a lung cancer marker using a microarray in which peptides serving as epitopes of autoantibodies present in a specimen of a lung cancer patient are arranged is disclosed (Patent Document 6).

  In addition to cancer marker detection, a method for performing an allergy test using a microarray has also been reported. Specifically, a method is disclosed in which a microarray in which allergen proteins that cause an allergic reaction are arranged is prepared and the amount of IgE specific for allergy present in serum is examined (Patent Document 7).

  The test for determining allergy is a test for examining the amount of specific IgE against various allergens, ie, an antibody that specifically binds to an allergen protein present in blood. In contrast, only one type of allergy item can be examined.

US Pat. No. 5,405,783 US Pat. No. 5,601,980 International Publication No. 00/40942 Pamphlet International Publication No. 01/98781 Pamphlet International Publication No. 06/98087 Pamphlet JP 2010-507069 gazette JP 2010-266448 A JP 2001-133453 A JP-A-11-108928 JP 2010-204130 A

"Proteins for protein purification and handling" edited by Tatsuya Moriyama H. Tanaka, et al., Macromolecular Rapid Communication, 31 (14), 1267-1271 (2010) U.K.Laemmli.Nature, 1970,227,680

  However, biological samples used for clinical diagnosis contain many contaminants in addition to the target substance, and it is not easy with existing microarray technology to accurately detect a very small amount of target substance from among them. Absent. Therefore, in practice, the microarray method is not used in the clinical diagnosis, and the actual situation is that single item inspection (CAP_RAST method) using a conventional biochemical method is still mainstream.

  Therefore, a main object of the present invention is to provide a microarray for use in diagnosis that simultaneously and stably detects several hundred types of target substances from a very small amount of biological sample.

  As a result of intensive studies to solve the above problems, the present inventors have immobilized a substance that captures a target substance (target protein) on a gel held in each through hole of a substrate having a through hole. As a result, the inventors have found that the above-mentioned problems can be achieved and have completed the present invention.

  That is, the present invention relates to a microarray having a plurality of through-holes and a gel in which a protein or an epitope of the protein is immobilized in the through-hole.

  According to the present invention, a microarray that detects a plurality of minute target substances (target proteins) present in a specimen with high sensitivity is provided.

It is a conceptual diagram of an arrangement | sequence fixing device. In the Example of this invention, it is a figure which shows the result at the time of using various sample liquids, washing | cleaning liquids, etc.

  The microarray of the present invention is a microarray having a plurality of through-holes and a gel in which a protein or an epitope of the protein is immobilized in the through-hole.

  The microarray can be obtained by forming a through-hole in a thin plate, but is obtained by cutting a block including a plurality of linear bodies or through-holes in a direction crossing the longitudinal direction of the linear body or through-holes. Is preferred. This is because stable quality microarrays can be mass-produced.

  Each configuration will be described in detail below.

(1) Formation of a block including a plurality of linear bodies or through holes A linear body or a block including through holes is a block having linear bodies or through holes oriented in the coaxial direction. The method for forming the through hole is not particularly limited. For example, a method of arranging hollow fibers in the coaxial direction as described in Patent Document 8 and then hardening with resin can be used.

(1-1) Hollow fiber Although various materials can be used for the hollow fiber, an organic material is preferable.

  Examples of hollow fibers made of organic materials include polyamide hollow fibers such as nylon 6, nylon 66 and aromatic polyamide, polyethylene hollow fibers such as polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and polycarbonate, poly Acrylic hollow fibers such as acrylonitrile, polyolefin hollow fibers such as polyethylene and polypropylene, polymethacrylate hollow fibers such as polymethyl methacrylate, polyvinyl alcohol hollow fibers, polyvinylidene chloride hollow fibers, polyvinyl chloride hollow fibers, Examples include polyurethane-based hollow fibers, phenol-based hollow fibers, fluorine-based hollow fibers made of polyvinylidene fluoride and polytetrafluoroethylene, and polyalkylene paraoxybenzoate-based hollow fibers.

  The manufacturing method of the said hollow fiber is not limited, It can manufacture by the well-known method as described in patent document 9. FIG. For example, a melt spinning method is preferable, and a horseshoe type, a C type nozzle, a double tube nozzle, or the like can be used as the nozzle. In the present invention, it is preferable to use a double tube nozzle in that a continuous and uniform hollow portion can be formed.

  The hollow fiber used in the present invention may be porous, and can be obtained by combining a known spinning technique such as a drawing method, a microphase separation method, and an extraction method with a melt spinning method or a solution spinning method. The porosity is not particularly limited, but from the viewpoint of increasing the density of the biopolymer immobilized around the fiber material unit length, the porosity is desirably high so that the specific surface area is increased.

  The inner diameter of the hollow fiber can be arbitrarily set. Preferably it is 10-2000 micrometers, More preferably, it can be 150-1000 micrometers.

  If necessary, hollow fibers containing a suitable amount of black pigment such as carbon black can also be used. By containing a black pigment, optical noise derived from foreign substances such as dust can be reduced during detection, and the strength of the resin can be increased. The content of the pigment is not limited and can be appropriately selected according to the size of the hollow fiber, the purpose of use of the microarray, and the like. For example, it can be 0.1 to 10% by mass, preferably 0.5 to 5% by mass, and more preferably 1 to 3% by mass.

(1-2) In order to form a through hole in a block block, a plurality of the hollow fibers are arranged three-dimensionally so that the fiber axes of the hollow fibers are in the same direction, so that the arrangement is not disturbed. A method of fixing with a resin such as an adhesive can be used.

  For example, there is a method (Patent Document 9) in which a plurality of hollow fibers are arranged in parallel at a predetermined interval on a sheet-like material such as a pressure-sensitive adhesive sheet to form a sheet, and then the sheet is wound in a spiral shape (Patent Document 9).

  Further, two porous plates each having a plurality of holes provided at a predetermined interval are overlapped so that the hole portions coincide with each other, the hollow fibers are passed through the hole portions, and the interval between the two porous plates is opened. A method (Patent Document 8) in which the periphery of the hollow fiber between two porous plates is filled with a curable resin raw material and cured.

  As a curable resin raw material, what consists of organic materials, such as a polyurethane resin and an epoxy resin, is preferable. Specifically, those formed from one or more kinds of materials composed of an organic polymer or the like are preferable.

  Organic polymers include rubber materials such as polyurethane, silicone resin, epoxy resin, polyamide resins such as nylon 6, nylon 66, aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid, polycarbonate, etc. Polyester resins, acrylic resins such as polyacrylonitrile, polyolefin resins such as polyethylene and polypropylene, polymethacrylate resins such as polymethyl methacrylate, polyvinyl alcohol resins, polyvinylidene chloride resins, polyvinyl chloride resins, Examples thereof include phenol resins, fluorine resins made of polyvinylidene fluoride and polytetrafluoroethylene, polyalkylene paraoxybenzoate resins, and the like.

  An appropriate amount of a black pigment such as carbon black can be contained in the organic polymer. By adding a black pigment, optical noise derived from impurities such as dust can be reduced during detection, and the strength of the resin can be increased. The content of the pigment is not limited and can be appropriately selected according to the size of the hollow fiber, the purpose of use of the microarray, and the like. For example, it can be 0.1 to 10% by mass, preferably 0.5 to 5% by mass, and more preferably 1 to 3% by mass.

In addition, an organic block can be used, and a plurality of through holes can be formed in the organic block with a laser or the like. The through holes are preferably arranged in parallel to each other in the organic block and formed with a predetermined interval. The shape of the through hole is a square, a rectangle, a circle, or the like. In the case of a circle, the diameter is about 10 to 2000 μm. The arrangement of the through holes is concentric, spiral or the like. The through holes are formed in the block at a density of 10 to 10,000 holes / cm ² .

  Furthermore, a hollow fiber bundle can also be manufactured by fusing the outer surfaces of adjacent hollow fibers to each other.

  The arrangement is fixed to the entire length or a predetermined portion of the arranged hollow fibers. A portion having an appropriate length from at least one end portion of the arranged hollow fibers may be not fixed.

  The number of hollow fibers arranged in the present invention, that is, the number of spots is not limited, and can be appropriately selected according to the intended experiment. Therefore, the distance between the hollow fibers can be appropriately selected according to the area of the microarray and the number of hollow fibers arranged.

(2) Thinning of block In each through-hole of a block having a through-hole obtained by the above method, a protein that binds to a target substance is held via a gel, and then the direction in which the through-hole is oriented By cutting in the vertical direction, a microarray in which through holes are arranged in the substrate can be obtained.

  The cutting method is not limited as long as it can be sliced. For example, it can be performed by a microtome, a laser, or the like. The thickness of the obtained flake is not limited and can be appropriately selected depending on the purpose of the experiment. For example, it can be 5 mm or less, preferably 0.1 to 1 mm.

(3) Immobilization of protein or epitope of the protein
(3-1) Protein or epitope of the protein In the microarray of the present invention, a protein that binds to a target substance (for capturing the target substance) or an epitope of the protein is held in a through-hole through a gel.

  The target substance means a biological substance collected from a living body to be detected in the microarray of the present invention contained in a specimen, and is a biopolymer such as DNA, RNA, peptide, protein, polysaccharide, nucleotide, nucleoside , Sugars, amino acids, vitamins, lipids, and other low molecular organic compounds, inorganic compounds, and the like that exist in the living body. The protein referred to here includes not only enzymes, antibodies and antigens but also glycoproteins to which sugar chains are bound. Among these, in the present invention, it is preferable to detect a biological substance involved in allergic reaction as a target substance.

  The type of protein or epitope of the protein to be retained in the through-hole of the microarray of the present invention is not particularly limited, and can be appropriately selected according to the type of target substance. In addition to proteins having a recognition site that binds to a target substance, there are antibody proteins such as polyclonal antibodies and monoclonal antibodies, antigen proteins that specifically bind to antibodies, and lectin proteins. Further, it may be a specific recognition site that binds to the target substance, that is, an epitope.

  As a method for preparing a protein or an epitope of the protein, a general method as described in Non-Patent Document 1 can be used. For example, one extracted from a natural product may be purified, or one produced from insects such as moths or E. coli may be used. Moreover, you may synthesize | combine artificially with an automatic synthesizer.

  As an example, a method for extracting and purifying protein from soybean is given below. First, the soybean is immersed in ultrapure water for 3 hours. Next, this soybean is ground and ground, then wrapped with gauze and compressed, and this compressed solution is treated with a column (PD10) to obtain a protein extract. A protein extract with a known concentration can be prepared by quantifying the amount of protein in the obtained extract by the Bradford method.

  In the present invention, it is preferable to detect a biological substance involved in an allergic reaction as a target substance, and therefore, the protein to be immobilized on the gel or the epitope of the protein is preferably an allergen protein or an epitope of the allergen protein. The type of allergen protein is not particularly limited and can be appropriately selected depending on the detection purpose and the like. For example, house dust, mite mite, mushroom mite, mushroom mite, mite, mite, mite, cedar, cypress, juniper (genus), alder (genus), hippopotamus (birch genus), oak (genus), beech (genus), Pine (genus), elm (genus), willow (genus), maple (genus), walnut (genus), mulberry (genus), acacia (genus), olives, anemonefish, blue whale, hargaya, gyosiba, nagahagusa, osuzumenoteppou, Sebum sorghum, barley, hirohashinokegusa, reed, wheat (genus), giant grass, genus, euglena, mugwort, ragweed, ragweed, dandelion (genus), sagebrush, redwood, canola Nettle (genus), psyllium, Ndida, Aspergillus, Penicillium, Mucor, Himesiva, Hermintosporium, Malassezia (genus), Alternaaria, Staphylococcus aureus enterotoxin A, Pityrosporum (malassezia), Cladosporium, cat (epithelium), cat (dandruff) ), Guinea pig (epithelium), S. aureus enterotoxin B, trichophyton, hamster (epithelium), dog (epithelium), dog (dandruff), rabbit (epithelium), goat (epithelium), mouse, rat, pig (epithelium) Budgerigar dung, horse (dandruff), cow (dandruff), budgerigar (feather), goose (feather), sheep (epithelium), duck (feather), chironomid (adult), chicken (feather), cockroach, wasp , Aedes (genus), pigeons, bees, roundworms, wasps, moths, cotton, worms, ani Kiss, plantain seed, isocyanate TDI, isocyanate MDI, isocyanate HDI, phthalic anhydride, ethylene oxide, formalin, silk, latex, egg yolk, milk, egg white, buckwheat, ovomucoid (heat-resistant egg protein), rice, barley, wheat, ω -5, millet, oat, rye, millet, peanut, walnut, almond, millet, corn, green beans, cashew nut, coconut, pea, hazel, strawberry, soybean, orange, peach, brazil nut, tomato, mango, apple, onion , Celery, Banana, Melon, Yam, Watermelon, Grapefruit, Garlic, Pumpkin, Kiwi, Cheese, Sesame, Avocado, Pear, β-Lactoglobulin, Malt, Parsley, Beef, Molded Cheese, Carrot, Lamb, Casein, Gluten, Potato, crab, pork, sweet potato, abalone, oyster (oyster), shrimp, spinach, bamboo shoot, octopus, scallop, mustard, sardine, mackerel, brewer's yeast, cacao, salmon, salmon roe, tuna, α-lactalbumin, taraco, chicken Lobster, clams, blue mussels, squid, horse mackerel, cod, flounder, matsutake, human insulin, gelatin can be used. Among these, shrimp, crab, wheat, buckwheat, egg yolk, white egg, milk, peanut, abalone, squid, salmon roe, orange, kiwi, beef, walnut, salmon, mackerel, soy, chicken, banana, pork, matsutake, peach , Yam, apple and gelatin are preferred.

  The amount of protein immobilized on the gel and the epitope of the protein can be appropriately selected according to the type of the protein. For example, it is preferable to maintain the concentration in the concentration range of 0.25 to 250 pmol / μL from the viewpoint that it can be stably fixed to the gel while maintaining the function of capturing the target protein. The concentration range is more preferably 1 to 150 pmol / μL, further preferably 2.5 to 100 pmol / μL, and particularly preferably 5 to 50 pmol / μL.

  As a method of immobilizing proteins and epitopes of the proteins on the gel, in addition to the method of embedding in the gel as it is, the protein is dispersed in the gel and then heated to utilize the structural change caused by thermal denaturation of the protein. A method in which the gel is entangled and fixed in the gel may be used. Moreover, you may make a polymer bond using the some functional group in protein (nonpatent literature 2). In addition, a functional group capable of binding to the gel may be introduced into the protein and covalently bonded to the gel. For example, a method in which a vinyl group is introduced into a protein and copolymerized with a gel-forming polymerizable monomer can be used (see the examples on the following page).

(3-2) Gel The type of gel used in the present invention is not particularly limited, and if it is a gel obtained from a natural product, in addition to polysaccharides such as agarose and sodium alginate, proteins such as gelatin and polylysine are used. it can.

  As a preferable synthetic polymer, for example, a gel obtained by reacting a polymer having a reactive functional group such as polyacryloylsuccinimide and a crosslinking agent having reactivity can be used. In addition, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid And a synthetic polymer gel obtained by copolymerization with a polyfunctional monomer such as methylene bis (meth) acrylamide, polyethylene glycol di (meth) acrylate, etc. using a polymerizable monomer such as allyl dextrin as a monomer .

  The density | concentration of the gel used for the microarray of this invention is not specifically limited, According to the kind and quantity of protein etc. to be used, the kind of target substance, etc., it can select suitably. For example, it is preferably 2% by mass or more, more preferably 2 to 10% by mass, even more preferably 3 to 7% by mass, and particularly preferably 3.5 to 5% by mass in terms of the concentration of the monomer component. It is. At 2% by mass or more, proteins and the like can be reliably immobilized, and target substances can be detected efficiently. Moreover, even if the concentration is 10% by mass or more, it is difficult to obtain a dramatic effect.

  When the synthetic polymer gel is held on the microarray of the through-hole substrate, the block can be filled with a synthetic polymer gel precursor solution and then gelled and held in the block. Thereafter, the microarray in which the gel is held on the through-hole substrate can be obtained by slicing in a direction perpendicular to the orientation direction of the through-holes of the block. Here, the gel precursor solution refers to a solution containing a chemical substance that forms a crosslinked structure and causes gelation. For example, it can be defined as a solution containing a monomer, a polyfunctional monomer, a polymerization initiator and water.

  The method of filling the gel precursor solution into the through-hole of the block can be introduced, for example, by sucking the solution into a syringe having a fine needle and inserting the needle into the hollow portion of each hollow fiber.

  In addition, when using a block produced by solidifying a hollow fiber bundle with a resin, if one end of the hollow fiber bundle is not fixed, the gel precursor solution is introduced into the hollow fiber by the following method. You can also. First, the hollow portion of the end portion where the hollow fiber bundle is fixed is sealed, and the other non-fixed end portion of the hollow portion is opened. Next, a gel precursor solution containing the protein that binds to a target substance or an amino acid sequence fragment of the protein that becomes an epitope is prepared, the gel precursor solution and the hollow fiber bundle are placed in a decigarator, and then hollow fibers The end of the bundle where the hollow fibers are not fixed is immersed in this solution, the inside of the desiccator is decompressed, and then returned to normal pressure, so that the solution is hollowed out from the end immersed in the hollow fiber solution. It can introduce | transduce into a fiber hollow part.

  By polymerizing (reacting) the gel precursor solution introduced into the hollow portion of the hollow fiber, the gel or the gel-like material containing the epitope of the protein is held in the hollow portion of the hollow fiber. The polymerization conditions are not particularly limited, and can be appropriately selected depending on the type of gel precursor used. For example, an acrylamide-based monomer can be polymerized using a radical initiator, and can be preferably polymerized by a thermal polymerization reaction using an azo-based initiator.

(4) Use of the microarray of the present invention The method of using the microarray of the present invention is not particularly limited as long as the target substance in the sample can be detected.

(4-1) Contact between microarray and specimen (reaction)
For example, first, the microarray and the specimen are brought into contact with each other. At this time, the specimen may be brought into contact with the microarray by dropping the specimen onto the microarray, or the specimen may be brought into contact with the microarray by immersing the microarray in the specimen.

  The method for preparing the sample solution is not particularly limited, and a generally used method (known method) can be used. A person skilled in the art can prepare a sample solution suitable for a microarray. The sample solution can contain an additive such as a salt or a surfactant as required. The type of the salt is not limited and can be appropriately selected according to the purpose of the experiment.

  As the salt, for example, sodium chloride, sodium citrate, sodium acetate, sodium phosphate and the like are preferable. The salt concentration in the sample solution is preferably 0.001 to 0.1M, more preferably 0.005 to 0.08M, and still more preferably 0.01 to 0.05M. By setting the salt concentration in the sample solution to 0.001 M or more, non-specific interaction between the microarray and the biological substance is hardly formed. Moreover, by setting it to 0.1 M or less, a bond due to a specific interaction between the biological substance and the probe is likely to be formed.

  The type of surfactant is selected in consideration of foaming properties and solubility of the standard substance in the sample solution. However, since the sample solution contains various buffer solutions, either a cationic surfactant or a nonionic surfactant is used. It is preferred to select from surfactants. The surfactant concentration is preferably 0.01 to 0.04% by mass, more preferably 0.015 to 0.038% by mass, and still more preferably 0.02 to 0.035% by mass.

  The temperature of the specimen at the time of contact is not limited and can be about 4 to 35 ° C, preferably about 20 to 30 ° C. Further, the contact time is not limited and can be appropriately selected according to the type of the target substance. For example, it may be 60 seconds to 18 hours, preferably 1 to 8 hours, more preferably 2 to 4 hours.

(4-2) Washing of microarray After that, the microarray may be washed if necessary. By washing, substances other than the target substance in the sample non-specifically bound to the gel or protein in the microarray are washed away, and only the target substance specifically bound to the probe can be detected.

  The type of cleaning liquid used for the cleaning is not particularly limited as long as it can efficiently wash away ecologically related substances that are non-specifically bound in the microarray or through-hole. For example, it is preferable to include salts such as sodium chloride, sodium citrate, sodium acetate, and sodium phosphate. The salt concentration in the cleaning liquid is preferably 0.12 to 0.4M, more preferably 0.13 to 0.3M, and even more preferably 0.15 to 0.25M. By setting the concentration to 0.1 M or more, non-specifically bound substances can be efficiently removed. Moreover, it can avoid that the biologically relevant substance couple | bonded specifically is removed by setting it as 0.4 M or less.

  The method of washing is not limited. For example, an operation of immersing in a 0.12 M Tris-HCl, 0.12 M sodium chloride solution containing 0.05% Tween-20 and shaking at room temperature of 20 to 30 ° C. for 10 to 20 minutes is performed by 1 to 3 It can be washed by a method such as repeating about several times.

(4-3) After the detection washing, the target substances bound to the microarray are labeled with various labeling agents. For example, labeling can be performed by preparing an aqueous solution of a labeling agent and immersing the washed microarray in the labeling agent solution for 10 to 30 minutes. The concentration of the labeling agent solution can be arbitrarily determined. The labeling agent is not limited, and for example, a luminescent labeling agent such as horseradish peroxidase which is generally commercially available, or a fluorescent labeling agent such as Cy3, Cy5 or Alexa680 can be used. An apparatus capable of detecting fluorescence or luminescence, such as a fluorescence microscope or a fluorescence scanner, can be used. However, in the case of detection, it is preferable to shield light such as by making the detection environment a dark room. This is because when light from the outside is irradiated onto the detection environment, it becomes noise and the light amount of the labeling agent may not be detected accurately.

<Example 1: BSA-immobilized microarray>
In this example, a microarray was prepared by immobilizing BSA (bovine serum albumin, molecular weight of about 66 kDa) as a protein on a gel.

(1) Production of vinylated BSA In order to fix BSA to the gel, vinyl groups were introduced into BSA by the following method.

Preparation of vinylating agent We measure Wako Pure Chemical's special grade reagent dimethyl sulfoxide (998.5 μL) with a micropipette into a 1 cc capacity microtube and add Aldrich's methacrylic anhydride (methacrylic anhydride) with a micropipette. 1.5 μL was added to prepare a dimethyl sulfoxide solution (10 mM) of methaic anhydride (vinylating agent).

BSA Vinylation Next, 0.01035 g of Aldrich BSA crystals were weighed, placed in a microtube, and dissolved in 1.5 mL of ultrapure water (MilliQ water).

  The BSA solution (20 μL) was put into a reaction volume 100 μL microtube with a micropipette, and then a carbonate buffer (10 μL) manufactured by Nacalai tex and the vinylating agent (10 μL) prepared above were sequentially added. After stirring with a vortex mixer so that the mixture was uniform, the liquid adhering to the tube wall was collected at the bottom of the tube with a centrifuge. Thereafter, the reaction was allowed to stand at room temperature for 30 minutes.

Isolation and purification of vinylated BSA 40 μL of the reaction solution thus obtained was transferred to a spin column (MICROCON (registered trademark), YM-30 manufactured by Millipore), and 60 μL of ultrapure water was added to make a total volume of 100 μL. did. Next, the spin column containing the reaction solution was set in a centrifuge and treated at 10,000 G for 10 minutes. Thereafter, the process of adding 100 μL of ultrapure water to the concentrated part of the spin column and further centrifuging at 10,000 G for 10 minutes was repeated 5 times.

  The spin column was taken out, and the vinylated BSA solution concentrated in the concentration part of the spin column was diluted with 20 μL of ultrapure water. The concentrating part of the spin column was once removed from the tube body, turned upside down and attached to the body again, and centrifuged at 10000 G for 10 minutes. The vinylated BSA solution collected in the tube was dried at room temperature with a centrifugal evaporator to obtain about 16 μg of vinylated BSA.

(2) Production of Hollow Fiber Bundle A hollow fiber bundle was produced using the array fixture shown in FIG. In the figure, x, y, and z are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber.

  First, two perforated plates with a thickness of 0.1 mm were prepared in which a total of 256 holes each having a diameter of 0.32 mm were provided in a total of 256 rows in 16 rows each with a distance between the centers of the holes being 0.12 mm. These perforated plates were overlapped, and one polycarbonate hollow fiber having an outer diameter of 280 μm, an inner diameter of 180 μm, and a length of 150 mm was passed through all of the holes.

  The position of the two perforated plates was moved in a state in which a tension of 0.1 N was applied to each fiber in the X-axis direction, and was fixed at two positions of 20 mm and 100 mm from one end of the hollow fiber. . That is, the interval between the two perforated plates was 80 mm. Next, the three surrounding surfaces of the space between the perforated plates were surrounded by a plate-like object. In this way, a container having an open top only was obtained.

  Next, the resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the gross mass of a polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd., Nipponran 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate and the plate-like material were removed to obtain a hollow fiber bundle. The obtained hollow fiber bundle was put in a desiccator and the inside was purged with nitrogen, and then allowed to stand for 16 hours.

(3) Immobilization of protein As shown in Table 1, four types of gel precursor solutions were prepared according to the concentration level of vinylated BSA, 30 sets were prepared for each concentration level, and 80 μL was added to each well of the well plate. Noted. The well plate was placed in a desiccator, and the gel precursor solution dispensed into each well from the end was sucked and introduced into the hollow part of the hollow fiber.

次いで、前記の中空繊維束を前記デシケーター内に設置し、デシケーター内を窒素雰囲気下、５５℃まで昇温して、５５℃で３時間、重合反応を実施した。

Next, the hollow fiber bundle was placed in the desiccator, the temperature inside the desiccator was raised to 55 ° C. under a nitrogen atmosphere, and a polymerization reaction was carried out at 55 ° C. for 3 hours.

  After completion of the polymerization reaction, a hollow fiber bundle was sliced into a thickness of 250 μm in a direction perpendicular to the longitudinal direction of the hollow fiber using a microtome. In this way, 100 microarrays having a thickness of 250 μm on which gel spots including 256 capture probes were mounted were produced.

(4) Evaluation of microarray Using the prepared BSA-fixed microarray, detection of anti-BSA antibody was attempted.

Sample Preparation and Reaction Add 180 μL of 0.012 M TNT buffer (0.012 M Tris.HCl / 0.012 M, NaCl / 0.03%, Tween-20) to 20 μL of biotin-labeled anti-BSA antibody manufactured by ABCAM. The sample solution was mixed so as to be uniform by pipetting to prepare a sample solution having a total amount of 200 μL.

  This sample solution is injected into the Genopearl (registered trademark) hybrid chamber manufactured by Mitsubishi Rayon Co., Ltd., and the BSA-fixed microarray prepared earlier is slowly inserted, the chamber lid is closed, and the reaction is allowed to stand at room temperature for about 2 hours. I let you.

6 mL of 0.12M Tris.HCl / 0.12M NaCl / 0.05% Tween-20 solution was injected into a tube (made by SARSTATED) having a washing volume of 8 mL after the reaction, and the BSA-fixed microarray taken out from the chamber after the reaction. Was inserted so as to be completely immersed in the liquid.

  Next, the lid of the tube was closed and rotated at room temperature using a rotator for 20 minutes (10 rpm). Thereafter, the microarray was removed from the tube, replaced with a tube containing a 0.12M Tris.HCl / 0.12M NaCl solution, and rotated using a rotator for 10 minutes at room temperature (10 rpm).

Labeled after reaction Jackson Immunoresearch Laboratories, Inc. 1 mL of Nuclease-free water was added and dissolved in Streptavidin-Cy5 (1 mg). After centrifuging at 10,000 G for 3 minutes to remove the precipitate, 12 μL of Streptavidin-Cy5 solution was prepared.

  12 μL of Streptavidin-Cy5 solution was diluted with 6 mL of 0.12M Tris · HCl / 0.12M NaCl solution to prepare a labeling solution.

  The previously washed microarray was immersed in the labeling solution and allowed to stand at room temperature for 30 minutes. The labeled microarray was immersed in a 0.12 M Tris.HCl / 0.12 M NaCl / 0.05% Tween-20 solution and rotated with a rotator at room temperature for 20 minutes (10 rpm). Thereafter, the microarray was immersed in a 0.12M Tris.HCl / 0.12M NaCl solution.

The labeled microarray was placed on the sample stage of a detection laser type fluorescence microscope (Yokogawa's MB-03) and exposed to excitation light having a wavelength of 633 nm, and the fluorescence signal of each spot of the protein microarray was detected. The exposure time was measured under four conditions of 0.1 second, 1 second, 4 seconds, and 40 seconds, and the fluorescence signal intensity per unit second was adopted as fluorescence signal intensity data. The measured fluorescence signal intensity of each spot of the microarray is shown in Table 2.

表２に示すとおり、プローブ濃度の水準毎に蛍光シグナルの変動係数を算出すると、５ｐｍｏｌ／μＬから２５ｐｍｏｌ／μＬまで水準を上げるに従い、バラツキが小さくなっていることが確認できた。

As shown in Table 2, when the variation coefficient of the fluorescence signal was calculated for each probe concentration level, it was confirmed that the variation was reduced as the level was increased from 5 pmol / μL to 25 pmol / μL.

<Example 2: Allergen protein-immobilized microarray>
Preparation of allergen protein Buckwheat, wheat, peanut, soybean, rice, shrimp, crab, milk and egg white were selected as allergen proteins to be immobilized on the gel, and each protein was extracted by the following method.

  <Soba> 50 mL of distilled water was added to 20 g of buckwheat flour and mixed with a spatula until uniform.

  <Wheat> Distilled water (50 mL) was added to 20 g of wheat flour (Nisshin Flour Milling Co., Ltd.) and mixed with a spatula until uniform.

  <Peanuts> 50 mL of distilled water was added to 20 g of peanuts, and after milling, they were wrapped in gauze and pressed.

  <Soybean> 50 mL of distilled water was added to 5 g of soybean, soaked for 3.5 hours, milled, wrapped in gauze and pressed.

  <Rice> 75 g of distilled water was added to 75 g of rice, soaked for 3.5 hours, milled, wrapped in gauze and pressed.

  <Shrimp> 50 mL of distilled water was added to 40 g of edible portion of shrimp (black tiger), and it was put on a mill.

  <Crab> 50 mL of distilled water was added to 30 g of the edible portion of the crab, and it was put on a mill.

  <Milk> Commercial milk was used.

  <Egg white> 8 mL of distilled water was added to 4 mL of egg white and stirred well.

  About each liquid, after calculating | requiring a density | concentration by Bradford method, adjusting to 20 mg / mL with distilled water, mixing with the extract of the composition shown in Table 3 by 1: 1 mass ratio, and centrifuging for 10 minutes at 3000 rpm The obtained supernatant was used as each allergen protein extract. The amount of protein (concentration) in these extracts was determined using 2D Quant Kit (GE Healthcare).

アレルゲンタンパク質へのビニル基導入
アレルゲンタンパク質のビニル化反応を行うにあたり、反応させるアレルゲンタンパク質とビニル化剤の反応量比をモル比で換算するために、まずアレルゲンタンパク質の分子量を以下の手順で決定した。

In order to convert the reaction ratio of allergen protein and vinylating agent to be reacted in the molar ratio, the molecular weight of the allergen protein was first determined by the following procedure. .

  The molecular weight of the protein was determined by electrophoresis (SDS-PAGE) using a polyacrylamide gel as a support, which is the most common technique. Colored molecular weight markers (manufactured by GE; Full-Range Rainbow Molecular Markers) were separated by electrophoresis according to the Laemmli method (Non-patent Document 3), and a correlation curve between the migration distance and molecular weight of each marker protein was prepared. .

  Next, the constituent proteins were separated from each prepared allergen protein extract by electrophoresis in the same manner as described above, and each band of the obtained constituent proteins was stained with SYPRO-ORANGE. The fluorescence intensity of each band was measured with a fluorescence microscope, and the quantitative ratio of the constituent proteins was determined from the intensity ratio.

  After determining the molecular weight of each band using the molecular weight-migration distance correlation curve of the marker created earlier, extract the value obtained by adding the obtained molecular weight value multiplied by the amount ratio of the constituent proteins. The average molecular weight of the protein contained in the product was used.

About 100 μg of vinylated protein was obtained in the same manner as in Example 1 except that each allergen protein was used instead of the vinylation reaction BSA.

Preparation of microarray 100 microarrays were prepared by adjusting the polymerization solution composition as shown in Table 4 in the same manner as in Example 1 except that each allergen protein was used instead of BSA.

（４）マイクロアレイの評価
作製したマイクロアレイを使って、アレルゲン特異的ＩｇＥ抗体の検出を試みた。
検体の調製及び反応
ＭＡＳＴＩＩ検査により、表５に示すアレルギー判定結果が得られたＩｎｔｅｒｎａｔｉｏｎａｌ ｅｎｚｙｍｅｓ社製のヒト血清を三菱レイヨン社製ジェノパール（登録商標）用ハイブリチャンバーに２００μＬ注入し、先に作製したマイクロアレイを１枚挿入し、チャンバーの蓋を閉め、室温で２時間ほど静置反応させた。

(4) Evaluation of microarray Using the prepared microarray, detection of an allergen-specific IgE antibody was attempted.
Preparation of sample and reaction MASTII test gave 200 μL of human serum manufactured by International enzymes, which had obtained the allergy determination results shown in Table 5, into a hybrid chamber for Genopearl (registered trademark) manufactured by Mitsubishi Rayon Co., Ltd. One microarray was inserted, the lid of the chamber was closed, and the reaction was allowed to stand at room temperature for about 2 hours.

反応後の洗浄
容量８ｍＬのチューブ（ＳＡＲＳＴＥＤＴ社製）に０．１２Ｍ Ｔｒｉｓ・ＨＣｌ／０．１２Ｍ ＮａＣｌ／０．０５％ Ｔｗｅｅｎ−２０溶液を６ｍＬ注入し、そこに反応後にチャンバーから取り出したマイクロアレイを完全に液中に浸漬するように挿入した。

6 mL of 0.12M Tris.HCl / 0.12M NaCl / 0.05% Tween-20 solution was injected into a tube (made by SARSTATED) having a washing volume of 8 mL after the reaction, and the microarray taken out of the chamber after the reaction was completely completed. It was inserted so as to be immersed in the liquid.

Next, the lid of the tube was closed and rotated at room temperature using a rotator for 20 minutes (10 rpm). Thereafter, the microarray was removed from the tube, replaced with a tube containing a 0.12M Tris.HCl / 0.12M NaCl solution, and rotated using a rotator for 10 minutes at room temperature (10 rpm).
Reaction with biotin-labeled antibody Biotin-labeled anti-human IgE antibody manufactured by KPL was diluted 100-fold by adding 0.012M TNT buffer, mixed uniformly by pipetting, and a total amount of 200 μL of reaction solution was prepared. Prepared.
This reaction solution was poured into a hybrid chamber for Genopearl (registered trademark) manufactured by Mitsubishi Rayon Co., Ltd., one microarray prepared earlier was slowly inserted, the chamber lid was closed, and the reaction was allowed to stand at room temperature for 2 hours. .
6 mL of 0.12M Tris.HCl / 0.12M NaCl / 0.05% Tween-20 solution was injected into a tube (made by SARSTATED) having a washing volume of 8 mL after the reaction, and the microarray taken out of the chamber after the reaction was completely completed. It was inserted so as to be immersed in the liquid.

Next, the lid of the tube was closed and rotated at room temperature using a rotator for 20 minutes (10 rpm). Thereafter, the microarray was removed from the tube, replaced with a tube containing a 0.12M Tris.HCl / 0.12M NaCl solution, and rotated using a rotator for 10 minutes at room temperature (10 rpm).
Labeled after reaction Jackson Immunoresearch Laboratories, Inc. 1 mL of Nuclease-free water was added and dissolved in Streptavidin-Cy5 (1 mg). After centrifuging at 10,000 G for 3 minutes to remove the precipitate, 12 μL of Streptavidin-Cy5 solution was prepared.

  12 μL of Streptavidin-Cy5 solution was diluted with 6 mL of 0.12M Tris · HCl / 0.12M NaCl solution to prepare a labeling solution.

  The previously washed microarray was immersed in the labeling solution and allowed to stand at room temperature for 30 minutes. The labeled microarray was immersed in a 0.12 M Tris.HCl / 0.12 M NaCl / 0.05% Tween-20 solution and rotated with a rotator at room temperature for 20 minutes (10 rpm). Thereafter, the microarray was immersed in a 0.12M Tris.HCl / 0.12M NaCl solution.

A labeled microarray was placed on a sample stage of a detection laser type fluorescence microscope (Yokogawa Electric Corporation MB-03), and an excitation light with a wavelength of 633 nm was exposed to detect a fluorescent signal at each spot of the microarray. The exposure time was measured under four conditions of 0.1 second, 1 second, 4 seconds, and 40 seconds, and the fluorescence signal intensity per unit second was adopted as fluorescence signal intensity data. Table 6 shows the measured fluorescence signal intensity of each spot of the microarray.

アレルギー判定結果で抗体反応が高かったアレルゲン項目について、本マイクロアレイにおいても、表６に示すとおり、抗体特異的なシグナルを確認することができた。

As shown in Table 6, antibody-specific signals could be confirmed for allergen items that showed a high antibody response as a result of allergy determination.

<Example 3: Synthetic peptide-immobilized microarray>
Preparation of microarray A microarray was prepared in the same manner as in Example 1 except that a synthetic peptide (a peptide consisting of an amino acid sequence vinylated on the C-terminal side (sequence (N-terminal → C-terminal): DYKDDDDK)) was used instead of BSA. Manufactured. As shown in Table 7, 16 types of gel precursor solutions were prepared according to the concentration level of the probe.

検体溶液の調製
ビオチン標識モノクローナル抗ＦＬＡＧ抗体（アルドリッチ社製：本発明で使用するプローブに特異的に結合する）、及びビオチン標識モノクローナル抗ＨＡ抗体（アルドリッチ社製）を表８に示す量を含む検体溶液（溶媒はＴＮＴ溶液）が、それぞれ２００μＬになるように調製した。また、表９に示すように、当該検体中に種々の濃度のＮａＣｌ（Ｍ）及びＴｗｅｅｎ−２０（質量％）を含むように調製し、９種類準備した。

Preparation of sample solution Sample containing biotin-labeled monoclonal anti-FLAG antibody (manufactured by Aldrich: specifically binds to the probe used in the present invention) and biotin-labeled monoclonal anti-HA antibody (manufactured by Aldrich) as shown in Table 8 Each solution (the solvent was a TNT solution) was prepared to be 200 μL. Moreover, as shown in Table 9, it prepared so that various density | concentrations of NaCl (M) and Tween-20 (mass%) might be included in the said sample, and nine types were prepared.

検体溶液とマイクロアレイの接触
調製した検体溶液に対し、マイクロアレイを２時間浸漬した。浸漬させる際の検体溶液の温度は、表８に示される番号１、３、５は３０℃、番号２、４、６は室温とした。

The microarray was immersed in the sample solution prepared by contacting the sample solution and the microarray for 2 hours. The temperature of the specimen solution at the time of immersion was 30 ° C. for numbers 1, 3, and 5 shown in Table 8, and room temperature for numbers 2, 4, and 6 were shown.

Washing First, the microarray after being immersed in the specimen solution was subjected to nine kinds of washing liquids shown in Table 9 (NaCl, Tween-20 and 0.12M Tris-HCl aqueous solutions described in the column of "Sample solution" in Table 9). And washed for 20 minutes at room temperature. Next, the chip was immersed in nine types of cleaning solutions (9 types of NaCl and 0.12 M Tris-HCl aqueous solution) shown in the right column (cleaning solution) of Table 9 and washed at room temperature for 10 minutes.

Staining was carried out using Cy5 Streptavidin (manufactured by GE Healthcare). Specifically, 1 mg of streptavidin-Cy5 was dissolved in 1 mL of sterile water, 10 μL of which was mixed with 5 mL of 0.12 M TNT solution (0.12 M Tris-HCl, 0.12 M NaCl, 0.5% Tween 20 solution), A staining solution was prepared. The microarray was immersed in the prepared staining solution at room temperature for 30 minutes.

The washing microarray was taken out from the staining solution, and the chip was immersed in a washing solution containing salts and surfactants prepared based on the concentration levels of Nos. 1 to 9 shown in Table 9 and washed at room temperature for 20 minutes.

A labeled microarray was placed on a sample stage of a detection laser type fluorescence microscope (Yokogawa Electric Corporation MB-03), and an excitation light with a wavelength of 633 nm was exposed to detect a fluorescent signal at each spot of the microarray. The exposure time was measured under five conditions of 50 msec, 100 msec, 1 sec, 4 sec, and 40 sec, and the fluorescence signal intensity per unit second was adopted as fluorescence signal intensity data.

(3) Results The experiment under the conditions of Nos. 1 to 9 shown in Table 9 was performed at n = 3 with respect to the specimen concentration levels of Nos. 1 to 6 shown in Table 8, and the fluorescence signal intensity at each spot of the microarray From the above, an S / N ratio serving as a quantitative index was calculated. The S / N ratio was calculated with reference to “Introductory Parameter Design”.

  Although not shown in the chart, the SN ratio of the microarray in which the concentration of the probe fixed in the through hole was less than 15 pmol / μL was lower than that of 15 pmol / μL or more.

  The results when using various specimen solutions and cleaning solutions are shown in FIG. This is a graph showing the influence of the level of each control factor on the SN ratio when the concentration of the probe fixed in the through hole is 40 pmol / μL. The obtained S / N ratio decreases depending on the salt concentration of the sample solution, tends to decrease when the surfactant concentration of the sample solution exceeds 0.03% by mass, and the salt concentration of the cleaning solution is 0.11M. There was a tendency for it to become higher by making it higher.

DESCRIPTION OF SYMBOLS 1 Array fixing tool 11 Hole 21 Perforated plate 31 Hollow fiber 41 Plate-shaped object

Claims (3)

A microarray having a plurality of through-holes and having a gel with a monomer concentration of 2 % by mass or more, in which the allergen protein or an epitope of the allergen protein is immobilized in the through-hole at a concentration of 20 to 50 pmol / μL. A method for producing a microarray, comprising the following steps.
(1) Step of producing a hollow fiber bundle by arranging a plurality of hollow fibers in a three-dimensional manner so that the fiber axes of the hollow fibers are in the same direction, and fixing the arrangement with a resin (2) Allergen protein Alternatively, a step of introducing a gel precursor solution having a monomer concentration containing an epitope of the allergen protein of 2% by mass or more into the hollow portion of each hollow fiber bundle (3) The monomer concentration introduced into the hollow portion of the hollow fiber bundle is 2 mass% or more gel precursor solution is reacted, step epitopes of allergen protein or the allergen protein retains the immobilized gel into the hollow portion of the hollow fibers (4) crossing the hollow fiber bundle in the longitudinal direction of the fibers Cutting in the direction of cutting A step of bringing the microarray according to claim 1 into contact with the specimen to bind the allergen protein immobilized on the microarray or an epitope of the allergen protein to a target substance in the specimen; and
Comprising the step of quantifying the amount of target substance bound to an epitope of the allergen protein or the allergen protein, method for detecting a biological substance.

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