JP2004109081A - Assay method using unit for biochemical analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve solution flowability in an adsorptive region to reduce a background. <P>SOLUTION: A ligand or a receptor is joined to the adsorptive region of a unit for biochemical analysis having the porous adsorptive region. A hybridization buffer with a label receptor or a label ligand labeled by a label substance added and without containing a Denhardt's solution or a Salmon Sperm DNA is forcibly made to flow so as to cross the adsorptive region to specifically join the label receptor or the label ligand to the ligand or the receptor. Next, an enzyme label antibody is specifically joined to the label receptor or the label ligand, and a chemical luminescent substance is brought into contact with the enzyme label antibody specifically joined to the label receptor or the label ligand to photoelectrically detect chemical light emission emitted from the adsorptive region of the unit for the biochemical analysis. <P>COPYRIGHT: (C)2004,JPO

Description

【００６３】
表１から明らかなように、デンハーツソリューションとサーモンスパームＤＮＡを含まないハイブリダイゼーションバッファを用いた実施例１〜４では、設定の流速と、１８時間循環させた後の系内のハイブリダイゼーションバッファの流速が殆ど変化せず、流動性が維持されているため、その後の洗浄工程で、吸着性領域に非特異的に結合している標識レセプタまたは標識リガンドを充分に洗い落とすことができた。この結果、吸着性領域のバックグラウンドの発光量を低減させることができた。一方で、デンハーツソリューションとサーモンスパームＤＮＡを含むハイブリダイゼーションバッファを用いた比較例１および比較例４では、１８時間循環させた後の系内のハイブリダイゼーションバッファの流速が設定の流速の半分以下となり、バックグラウンドは、デンハーツソリューションとサーモンスパームＤＮＡを含まないハイブリダイゼーションバッファを用いた実施例１〜４の約５倍も高いものとなった。
【００６４】
以上のように、本発明の生化学解析用ユニットを利用した化学発光法は、標識物質により標識された標識レセプタまたは標識リガンドが添加されたハイブリダイゼーションバッファを吸着性領域を横切るように強制的に流動させて標識レセプタまたは標識リガンドを特異的に結合させる場合に、ハイブリダイゼーションバッファとしてデンハーツソリューションとサーモンスパームＤＮＡを含まないハイブリダイゼーションバッファを用いることとしたので吸着性領域が詰まることがなく、その後の洗浄工程で、吸着性領域に非特異的に結合している標識レセプタまたは標識リガンドを充分に洗い落とすことができ、吸着性領域のバックグラウンドの発光量を低減させることができた。従って、これにより、微量の標識レセプタまたは標識リガンドの検出を可能なものとすることができる。
【図面の簡単な説明】
【図１】本発明の化学発光法に用いられる生化学解析用ユニットの概略斜視図
【図２】本発明の生化学解析用ユニットを利用した化学発光法に用いられるリアクタの実施の形態を示す概略断面図
【図３】本発明の生化学解析用ユニットを利用した化学発光法に用いられるリアクタの別の実施の形態を示す概略断面図
【符号の説明】
１　　生化学解析用ユニット
２　　基板
３　　孔
４　　吸着性領域
５　　リガンドまたはレセプタ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an assay method for detecting a receptor or a ligand, and more particularly to an assay method for detecting a receptor or a ligand using a biochemical analysis unit having a porous adsorptive region.
[0002]
[Prior art]
In recent years, for example, hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, at different positions on the carrier surface of glass arrays or membrane arrays using glass slides or membrane filters After dropping a ligand or receptor that can specifically bind to a substance derived from a living body such as DNA or RNA and has a known base sequence, base length, composition, characteristics, etc. using a spotter device, Fixed and then collected from the body by extraction, isolation, etc. of hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, DNA, mRNA, etc. Treated substance that is labeled with a fluorescent labeling substance such as a fluorescent substance or fluorescent dye The septa or ligand is specifically bound to the ligand or receptor immobilized on the adsorptive region by hybridization or the like, followed by irradiation with excitation light to photoelectrically emit fluorescence emitted from a labeling substance such as a fluorescent substance or a dye. Analyzing systems that detect and analyze biologically derived substances have been developed.
[0003]
According to this analysis system, a large number of spots of ligands or receptors are formed at different positions on the surface of a carrier such as a membrane filter, and the receptors or ligands labeled with a fluorescent labeling substance are hybridized. Therefore, there is an advantage that it becomes possible to analyze a substance derived from a living body in a short time.
[0004]
These analysis systems are also required to have improved detection limits and reproducibility as well as being able to detect with sufficient accuracy. However, the analysis system using the fluorescent labeling substance described above requires a large amount of labeled receptor or labeled ligand necessary for expression analysis because of its low detection sensitivity. In addition, there is a problem that the amount of ligand or receptor that can be immobilized on the glass array is small, and the ligand or receptor immobilized on the array is peeled off in the analysis operation step.
[0005]
[Patent Document 1]
Japanese Patent No. 2837276 [0006]
[Non-Patent Document 1]
"Nature genetics" Vol. 21, p. 25-p. 32 (1999)
[0007]
[Non-Patent Document 2]
“Bioindustry” Vol. 18, p. 13-p. 19 (2001)
[0008]
[Problems to be solved by the invention]
Conventionally, in the above analysis system, when performing hybridization or the like, an experimenter manually places an array on which a ligand or receptor is fixed in a hybridization bag, and puts a labeled receptor or labeled ligand in the hybridization bag. It is carried out by a so-called shaking method in which the reaction solution is added, the labeled bag or labeled ligand is moved by convection or diffusion by shaking the hybridization bag, and the labeled receptor or labeled ligand is specifically bound to the ligand or receptor. It was general.
[0009]
However, in the case of the shaking method, it is difficult to efficiently diffuse the labeled receptor or labeled ligand in the hybridization reaction solution to a large number of spot-like regions containing the ligand or receptor. The ligand or receptor and the labeled receptor or labeled ligand are difficult to diffuse. Cannot be efficiently hybridized. If the labeled receptor or labeled ligand in the hybridization reaction solution cannot be efficiently diffused into a large number of spot-like regions containing the ligand or receptor, the amount of light emitted from the adsorptive region to which the labeled receptor or labeled ligand is not bound ( When the ratio (S / N ratio) of light emission (signal) corresponding to the amount of labeled receptor or labeled ligand bound to noise or background is small, the amount of labeled receptor or labeled ligand binding to the adsorptive region becomes very small. There is a problem that it is difficult to detect.
[0010]
In order to sufficiently permeate the labeled receptor or the labeled ligand to the inside of the adsorptive region, it is considered that the reaction solution is forced to circulate inside the adsorptive region. When the containing hybridization buffer is forced to flow into the adsorptive region, there is a problem that the circulation property of the hybridization buffer containing the labeled receptor or the labeled ligand circulating in the adsorbing region is lowered. When the circulation property of the adsorptive region is reduced, the labeled receptor or the ligand that is non-specifically bound to the adsorptive region cannot be sufficiently washed out in the subsequent washing step. The amount of light emitted from the ground increases, making it difficult to detect a small amount of labeled receptor or labeled ligand.
[0011]
The present invention has been made in view of the above circumstances, and an object thereof is to provide an assay method using a biochemical analysis unit capable of improving the fluidity of an adsorptive region and reducing the background. It is what.
[0012]
[Means for Solving the Problems]
The assay method using the biochemical analysis unit of the present invention comprises a labeled receptor labeled with a labeling substance on the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which a ligand or receptor is bound. Alternatively, a hybridization buffer to which a labeled ligand is added is forced to flow across the adsorptive region to specifically bind the labeled receptor or labeled ligand to the ligand or receptor, and the labeled receptor or labeled ligand In the assay method for detection, a hybridization buffer that does not contain Denhardt's solution and Salmon Palm DNA is used as the hybridization buffer. It is the method characterized by these.
[0013]
The assay method using the biochemical analysis unit of the present invention is a high-level assay system in which a receptor or ligand is added to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound. A binding buffer is forced to flow across the adsorptive region to specifically bind the receptor or ligand, the receptor or ligand is specifically bound to a label labeled with a labeling substance, and the receptor Alternatively, in the assay method for detecting a ligand, a hybridization buffer that does not contain Denharz Solution and Salmon Palm DNA is used as the hybridization buffer.
[0014]
The assay method using the biochemical analysis unit of the present invention includes an auxiliary substance in which an auxiliary substance is bound to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound. A hybridization buffer to which a binding receptor or an auxiliary substance-binding ligand is added is forced to flow across the adsorptive region to specifically bind to the auxiliary substance-binding receptor or the auxiliary substance-binding ligand, and bind to the auxiliary substance. A binding substance capable of binding specifically can be forced to flow across the adsorptive region to specifically bind to the auxiliary substance binding receptor or auxiliary substance binding ligand, and the auxiliary substance binding receptor or auxiliary substance In an assay for detecting bound ligand, the hybridization buffer As a method which comprises using a hybridization buffer containing no den Hearts solutions and salmon Sperm DNA.
[0015]
【The invention's effect】
The assay method using the biochemical analysis unit of the present invention comprises a hybridization receptor to which a labeled receptor or a labeled ligand labeled with a labeling substance is added, a hybridization buffer to which a receptor or a ligand is added, or a labeling substance. In the case where the hybridization buffer to which the labeled label is added is forced to flow across the adsorptive region, a hybridization buffer not containing Denhart's solution and salmon palm DNA is used as the hybridization buffer. Therefore, the adsorptive region is not clogged, and the labeled receptor or the labeled ligand non-specifically bound to the adsorptive region can be sufficiently washed away in the subsequent washing step. Therefore, it is possible to reduce the amount of light emitted from the background of the adsorptive region and detect a small amount of labeled receptor or labeled ligand.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic perspective view of a biochemical analysis unit used in the assay method of the present invention. The biochemical analysis unit 1 shown in FIG. 1 includes a substrate 2 provided with a plurality of holes 3 and an adsorptive region 4 filled in the holes 3 and having a porous material bonded to the substrate 2. A ligand or receptor 5 having a known structure or characteristic is dropped on the adsorptive region 4 and immobilized by subsequent processing.
[0017]
The material of the substrate 2 is preferably a material that does not transmit or attenuate light in order to prevent light scattering inside the biochemical analysis unit, and is preferably a metal or ceramic. In addition, when a plastic that can be easily processed to form holes is used as the substrate, it is preferable to disperse the particles inside the plastic in order to further attenuate the light.
[0018]
Preferred examples of the metal include copper, silver, gold, zinc, lead, aluminum, titanium, tin, chromium, iron, nickel, cobalt, tantalum, and alloys such as stainless steel and brass. Preferred examples of the ceramic include alumina, zirconia, magnesia, and quartz. Plastics include polyolefins such as polyethylene and polypropylene, acrylic resins such as polystyrene and polymethyl methacrylate, polyvinyl chloride, polyvinylidene chloride, polyvinylidene fluoride, polytetrafluoroethylene, polychlorotrifluoroethylene, polycarbonate, polyethylene naphthalate, Polyesters such as polyethylene terephthalate, aliphatic polyamides such as nylon-6 and nylon-6,6, silicon resins such as polyimide, polysulfone, polyphenylene sulfide, polydiphenylsiloxane, phenolic resins such as novolac, epoxy resins, polyurethane, cellulose acetate And celluloses such as nitrocellulose, copolymers such as butadiene-styrene copolymers, and plastics. Such as those obtained by blending is preferably raised.
[0019]
Area of the opening of the hole 3 opening in the substrate 2 (size), in order to increase the density of the holes 3, typically less than 5 mm ^2, preferably less than 1 mm ^2, more preferably less than 0.3 mm ^2, more preferably less than 0.01 mm ^2. And it is more preferable that it is 0.001 mm ² or more.
[0020]
The pitch of the holes 3 (distance from the center of two adjacent holes to the center) is preferably in the range of 0.05 to 3 mm, and the interval between the holes 3 (from the end to the end of the two adjacent holes) The shortest distance) is preferably in the range of 0.01 to 1.5 mm. The number (density) of the holes 3 is generally 10 / cm ² or more, preferably 100 / cm ² or more, more preferably 500 / cm ² or more, and further 1000 / cm ² or more. Is preferred. And it is preferable that it is 100000 piece / cm < ² > or less further, Furthermore, it is 10,000 pieces / cm < ² > or less. Note that the holes 3 do not necessarily have to be provided at regular intervals as shown in FIG. 1, and a plurality of holes may be provided for each block divided into several blocks (units).
[0021]
As the porous material forming the adsorptive region, a porous material or a fiber material is preferably used. Further, the adsorptive region can be formed by using a porous material and a fiber material together. The porous material used to form the adsorptive region may be either an organic material or an inorganic material, or an organic / inorganic composite.
[0022]
The organic porous material used for forming the adsorptive region is not particularly limited, but a carbon porous material such as activated carbon or a porous material capable of forming a membrane filter is preferably used. As a porous material capable of forming a membrane filter, a polymer that is soluble in a solvent is preferably used. Examples of the polymer that can be dissolved in the solvent include cellulose derivatives (for example, nitrocellulose, regenerated cellulose, cellulose acetate, cellulose acetate, cellulose butyrate acetate, etc.), aliphatic polyamides (for example, nylon 6, nylon 6,6, nylon 4, 10), polyolefins (eg, polyethylene, polypropylene, etc.), chlorine-containing polymers (eg, polyvinyl chloride, polyvinylidene chloride, etc.), fluororesins (eg, polyvinylidene fluoride, polytetrafluoride, etc.), polycarbonate , Polysulfone, alginic acid and derivatives thereof (for example, alginic acid, calcium alginate, alginic acid / polylysine polyion complex, etc.), collagen, etc., and copolymers and composites (mixtures) of these polymers It can be used.
[0023]
In addition, the fiber material for forming the adsorptive region is not particularly limited, but preferred examples include the cellulose derivatives and aliphatic polyamides described above.
[0024]
The inorganic porous material used to form the adsorptive region is not particularly limited, but is preferably a metal (for example, platinum, gold, iron, silver, nickel, aluminum, etc.), a metal or the like Examples thereof include oxides (for example, alumina, silica, titania, zeolite, etc.), metal salts (for example, hydroxyapatite, calcium sulfate, etc.), and composites thereof.
[0025]
Examples of the method for forming the plurality of holes 3 in the substrate 2 include punching by punching with pins, electric discharge machining for applying a high voltage to the electrodes in a pulsed manner to volatilize the substrate, etching, and laser irradiation. When the substrate material is a metal material or a plastic material, a corona discharge or plasma discharge is applied to the surface of the substrate and an adhesive is applied, and then a porous material for forming the adsorptive region is pressed. The unit for biochemical analysis is produced by pasting together. When the porous material for forming the adsorptive region is heated and softened at the time of bonding, the adsorptive region is easily formed inside the hole. In addition, when pressing a porous material for forming an adsorptive region on a substrate, the material for forming the substrate and the adsorptive region is divided in advance and then intermittently pressed. Alternatively, the material for forming the substrate and the adsorptive region may be continuously conveyed between the two rolls in the form of a long band.
[0026]
In addition, in the chemiluminescence method using the biochemical analysis unit of the present invention, it is possible to use a biochemical analysis unit produced by the above-mentioned materials and methods. A unit may be used, and a biochemical analysis unit in which a ligand or a receptor is already bound to a porous adsorptive region may be used.
[0027]
FIG. 2 is a schematic cross-sectional view showing an embodiment of a reactor used in the chemiluminescence method using the biochemical analysis unit of the present invention. The reactor includes a reaction vessel 8, a solution circulation pipe 14, and a pump 15. The reaction vessel 8 holds the biochemical analysis unit 1 and has a sealing function for preventing liquid leakage and a biochemical analysis unit holding unit 7. The reaction vessel main body 9 comprises a reaction vessel upper half 10 and a reaction vessel lower half 11, and the reaction vessel upper half 10 is detachably provided on the reaction vessel main body 9. The chemical analysis unit 1 is configured to be set by removing the upper half 10 of the reaction vessel.
[0028]
Further, as shown in FIG. 2, a solution inlet 12 through which the solution can flow is formed on the bottom wall of the lower half portion 11 of the reaction vessel, and the solution flows similarly on the top wall of the upper half portion 10 of the reaction vessel. A possible solution outlet 13 is formed. Further, a solution circulation pipe 14 is detachably attached to the inlet 12 and the outlet 13, respectively. The reactor is configured such that the solution enters the reaction vessel main body 9 from the inlet 12 by the pump 15, passes through the biochemical analysis unit 1, exits from the outlet 13, and circulates through the solution circulation pipe 14. ing.
[0029]
FIG. 3 is a schematic sectional view showing another embodiment of a reactor used in the chemiluminescence method using the biochemical analysis unit of the present invention. This reactor is provided with a syringe 23 and a piston 24 instead of the pump shown in FIG. 2, and is configured such that the reaction solution reciprocates in the reaction vessel. Thus, the forced flow of the reaction solution may reciprocate through the adsorptive region 4 of the biochemical analysis unit 1.
[0030]
2 and 3 both show a reactor in which the reaction solution circulates through the biochemical analysis unit, but the reaction solution passes from the bottom to the top (or from top to bottom) of the biochemical analysis unit. It is also possible to use a reactor that does not circulate the reaction solution.
[0031]
Next, the chemiluminescence method using the biochemical analysis unit of the present invention will be described step by step.
The assay method using the biochemical analysis unit of the present invention comprises a labeled receptor labeled with a labeling substance on the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which a ligand or receptor is bound. Alternatively, an assay for detecting the labeled receptor or the labeled ligand by forcibly flowing the hybridization buffer added with the labeled ligand across the adsorptive region to specifically bind the labeled receptor or the labeled ligand to the ligand or the receptor. The method is characterized in that a hybridization buffer not containing Denhart's solution and Salmon palm DNA is used as the hybridization buffer.
[0032]
The ligand or receptor bound to the porous adsorptive region includes hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, cDNA, DNA, RNA, etc., characteristics, composition, structure Alternatively, the base sequence and base length are known.
[0033]
The labeled receptor or labeled ligand is a hormone or tumor marker, enzyme, antibody, antigen, abzyme, other protein, nucleic acid, DNA, mRNA, etc. that specifically binds to the ligand or receptor bound to the porous adsorptive region. It is collected from a living body by extraction, isolation, etc., or is subjected to chemical treatment after being collected and labeled with a labeling substance.
[0034]
Even if the labeling substance itself emits light, develops color, or emits fluorescence when irradiated with light, the chemical substance is decomposed or reacted with the labeling substance by bringing the chemical into contact with the labeling substance. Thus, it may emit light, color, or emit fluorescence. As the former labeling substance, acridinium ester or the like can be used as a luminescent substance, colloidal gold particles can be used as a coloring substance, and fluorescein can be used as a fluorescent substance. Moreover, as the latter labeling substance, an enzyme can be used. For example, enzymes such as alkaline phosphatase, peroxidase, luciferase, and beta galactosidase can be preferably used. When these enzymes are contacted with a chemiluminescent substrate, a dye substrate, or a fluorescent substrate, chemiluminescence, color development, and fluorescence are emitted, respectively.
[0035]
The chemiluminescent substrate is not particularly limited when the enzyme is alkaline phosphatase, peroxidase, or luciferase, but dioxetane, luminol, or luciferin can be used, respectively. In addition, as the chromogenic substrate, when the enzyme is alkaline phosphatase, paranitrophenol phosphate, when the enzyme is peroxidase, a combination of 4-aminoantipyrine and a Trinder reagent, diaminobenzidine, tetramethylbenzidine, enzyme is beta In the case of galactosidase, paranitrophenyl β-D-galactoside or the like can be used. As the fluorescent substrate, 4-methylumbelliphenyl phosphate is used when the enzyme is alkaline phosphatase, 3 (4-hydroxyphenyl) -propionic acid is used when the enzyme is peroxidase, and 4-methyl is used when the enzyme is beta-galactosidase. Umbellylphenyl β-D-galactoside and the like can be used.
[0036]
As another aspect of the assay method using the biochemical analysis unit of the present invention, the receptor or ligand is present in the ligand or receptor of the biochemical analysis unit having a porous adsorptive region bound with the ligand or receptor. The added hybridization buffer is forced to flow across the adsorptive region to specifically bind the receptor or ligand, and specifically bind the receptor or ligand to the label labeled with the labeling substance. Alternatively, in the assay method for detecting a ligand, a hybridization buffer containing neither Denhart's solution nor salmon palm DNA is used as a hybridization buffer.
[0037]
This is applied to a so-called sandwich method in which a receptor or ligand to be detected is sandwiched between a ligand or receptor in an adsorptive region and a label. As used herein, receptor or ligand refers to hormones, tumor markers, enzymes, antibodies, antigens, abzymes, other proteins, nucleic acids, DNA, which specifically bind to the ligand or receptor bound to the porous adsorptive region, It is a substance collected from a living body by extraction or isolation of mRNA or the like, or a substance subjected to chemical treatment after being collected.
[0038]
The labeled body labeled with the labeling substance is labeled with the above-described labeling substance, and in addition to an antigen and an antibody that can specifically bind to the reaction site of the receptor or ligand, hormones, tumor markers, enzymes, abzymes, It means other proteins, nucleic acids, cDNA, DNA, RNA, etc., whose characteristics, composition, structure, base sequence, base length, etc. are known.
[0039]
In still another embodiment of the assay method using the biochemical analysis unit of the present invention, an auxiliary substance is added to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound. The hybridization buffer to which the bound auxiliary substance-binding receptor or auxiliary substance-binding ligand is added is forced to flow across the adsorptive region to specifically bind to the auxiliary substance-binding receptor or the auxiliary substance-binding ligand. A binding substance that can be specifically bound to a substance is forced to flow across the adsorptive region to specifically bind to an auxiliary substance-binding receptor or an auxiliary substance-binding ligand, and binds to an auxiliary substance-binding receptor or an auxiliary substance. As a hybridization buffer in ligand detection assays Characterized by using the hybridization buffer containing no den Hearts solutions and salmon Sperm DNA.
[0040]
The auxiliary substance is a substance to which a bindable labeling substance binds, and preferred examples include antigens such as digoxigenin, biotin, avidin, and fluorescein, and antibodies to these antigens. It may also be a biological binding partner such as avidin for biotin. The bindable labeling substance is a substance that can specifically bind to the auxiliary substance and is labeled with the aforementioned labeling substance.
[0041]
Next, among the assay methods using the biochemical analysis unit of the present invention, an antigen-binding receptor or antigen labeled with an antigen (auxiliary substance) with a ligand or receptor immobilized on the adsorptive region of the biochemical analysis unit The binding ligand is specifically bound to the ligand or receptor immobilized on the adsorptive region, such as by hybridization, and then an antibody against the antigen labeled with the antigen-binding receptor or antigen-binding ligand is chemiluminescent. A chemiluminescent substrate that is labeled with an enzyme (hereinafter referred to as an enzyme-labeled antibody), specifically binds to the antigen of the antigen-binding receptor or antigen-binding ligand, and further specifically binds to the enzyme of the enzyme-labeled antibody. Photoelectric detection of chemiluminescence in the visible light wavelength region caused by contact between the chemiluminescent substrate and enzyme Examples assays chemiluminescent method that will be described in order.
[0042]
In the chemiluminescence method using the biochemical analysis unit of the present invention, first, a ligand or a receptor is bound to the adsorptive region of the biochemical analysis unit having a porous adsorptive region.
[0043]
The ligand or receptor bound to the porous adsorptive region is as described above, and the ligand or receptor can be fixed to the adsorptive region by being irradiated with ultraviolet rays after being dropped onto the adsorptive region. As described above, this step is omitted when a biochemical analysis unit in which a ligand or a receptor is already bound to a porous adsorptive region is used.
[0044]
Next, after the antigen-binding receptor or antigen-binding ligand is specifically bound to the ligand or receptor bound to the porous adsorptive region, the biochemical analysis unit is shown in FIG. 2 or FIG. The reaction solution is attached to a reaction vessel that can be forced to flow across the adsorptive region. The hybridization buffer to which the antigen-binding receptor or antigen-binding ligand has been added is forced to flow across the adsorptive region to specifically bind the antigen-binding receptor or antigen-binding ligand to the ligand or receptor.
[0045]
Use a hybridization buffer that has a function of preventing non-specific binding of an antigen-binding receptor or antigen-binding ligand to the adsorptive region, but use a hybridization buffer that does not contain Denherz Solution and Salmon Palm DNA. . In hybridization, the fluid flows for a long time at a high temperature. When using a hybridization buffer containing Denhart's solution and Salmon's palm DNA, the adsorptive region is often clogged and the fluidity of the fluid decreases. And using a hybridization buffer that does not contain salmon palm DNA, the adsorbent region is not clogged, and in the subsequent washing step, the labeled receptor or labeled ligand that is non-specifically bound to the adsorbent region is sufficiently removed. It can be washed out and the amount of luminescence in the background of the adsorptive region can be reduced to enable detection of trace amounts of antigen-binding receptor or antigen-binding ligand.
[0046]
The biochemical analysis unit attached to the reaction vessel is used to remove the antigen-binding receptor or antigen-binding ligand that did not specifically bind to the ligand or receptor bound to the porous adsorptive region. It is preferable to perform washing by forcibly flowing a so-called cleaning solution. Since the washing solution is forced to flow through the adsorptive region, the antigen-binding receptor or the antigen-binding ligand that is not specifically bound to the ligand or receptor bound to the porous adsorptive region is efficiently separated, It becomes possible to remove, and cleaning efficiency can be improved significantly.
[0047]
In this washing step, an enzyme labeled antibody, which will be described later, was forced to flow across the adsorptive region and specifically bound to the antigen-binding receptor or antigen-binding ligand, and then the enzyme was not specifically bound. It is also preferable to remove the labeled antibody. As a result, the enzyme-labeled antibody not specifically bound to the antigen-binding receptor or the antigen-binding ligand can be efficiently peeled and removed, and the washing efficiency can be greatly improved.
[0048]
Force the blocking buffer against the enzyme-labeled antibody across the adsorptive region before binding the enzyme-labeled antibody to the antigen-bound receptor or antigen-binding ligand specifically bound to the ligand or receptor bound to the adsorptive region. It is preferable to block the adsorptive region. The blocking buffer is preferably used after being filtered with a filter having a pore size smaller than that of the adsorptive region.
[0049]
Next, the enzyme-labeled antibody is forced to flow across the adsorptive region to specifically bind to the antigen-binding receptor or antigen-binding ligand. As the enzyme of the enzyme-labeled antibody, enzymes such as alkaline phosphatase, peroxidase and luciferase described above can be preferably used.
[0050]
Subsequently, the biochemical analysis unit is taken out from the reaction container, and the chemiluminescent substrate is brought into contact with the enzyme-labeled antibody specifically bound to the antigen-binding receptor or the antigen-binding ligand. Since chemiluminescence in the visible light wavelength region is generated from each adsorptive region by contact with the chemiluminescent substrate and the enzyme, if this is detected photoelectrically and image data for biochemical analysis is generated, antigen binding receptor or antigen binding Ligand can be detected and measured.
Hereinafter, the present invention will be described more specifically with reference to examples.
[0051]
【Example】
(Example 1)
A SUS304 sheet (substrate material sheet) having a size of 80 mm × 80 mm and a thickness of 100 μm is formed into 10 × A total of 6400 pieces were formed with 10 pieces as one unit.
[0052]
Next, 14 parts of nylon 6 (manufactured by Polysciences), 66 parts of formic acid, and 20 parts of water were uniformly dissolved to prepare a solution of a porous adsorptive region forming material. The solution is cast in a stable state and casted onto a substrate material sheet by a casting coater so that the thickness of the dried film is 160 μm. Scraped off the solution. Subsequently, the substrate material sheet was immediately immersed in a coagulation tank filled with a 40% aqueous solution of formic acid to form a large number of micropores in the film in the through hole. Thereafter, it was washed with water and dried to produce a biochemical analysis unit composed of a stainless steel barrier and a polymer-filled region having a large number of holes.
[0053]
The molecular weight markers pBR328 / BgII and HinfI (250 nl / μg: manufactured by Roche) dissolved in TE buffer were boiled for 5 minutes and then ice-cooled for 1 minute to make pBR328 / BgII and HinfI single-stranded. This was spotted on the adsorptive region of the biochemical analysis unit produced above, and then irradiated with ultraviolet rays (254 nm, 33 mJ / cm ² ), and single-stranded pBR328 / BgII, HinfI was fixed to the adsorbent region. .
[0054]
Next, 10 pg of pBR328-DNA solution (manufactured by Roche) labeled with digoxigenin (DIG) was thermally denatured, and hybridization buffer A (0.5 M Church phosphate buffer (0.5 M Na ₂ HPO ₄ with H ₃ PO ₄₎ was used. pH adjusted to 7.2), 1 mM EDTA, 7% SDS).
[0055]
The biochemical analysis unit was fixed to the reaction vessel shown in FIG. 2, and 5 ml of prehybridization buffer (same as hybridization buffer A) was circulated for 1 hour under the condition of 65 ° C. (linear velocity: 0.2 cm / sec). ). Next, hybridization was carried out by circulating the hybridization buffer to which the DIG-labeled pBR328-DNA solution was added at 65 ° C. for 18 hours. When the flow rate in the system of the hybridization buffer after 18 hours was measured, the flow rate was 0.9 with respect to the set value. Subsequently, circulating washing was performed twice with washing buffer 1 (40 mM Church phosphate buffer, 1% SDS) for 5 minutes and twice with washing buffer 2 (0.1 × SSC, 0.1% SDS) (washing buffer). All temperatures are 65 ° C).
[0056]
A blocking buffer (DIG made by Roche, as described in Wash and Block buffer Set) was filtered through a PES filter having a pore size of 0.2 μm (manufactured by Nalgen), and the filtered blocking buffer was circulated at room temperature for 10 minutes. Circulation was stopped for 50 minutes.
[0057]
Next, alkaline phosphatase-labeled DIG antibody is filtered in advance with an ultra-free (Millipore) with a pore size of 0.22 μm, and a PES filter with a pore size of 0.2 μm (Nalgen) is used so that the concentration becomes 1/10000. The sample was diluted with the blocking buffer filtered in step 1), circulated at room temperature for 1 minute, and then stopped for 60 minutes.
[0058]
Subsequently, a chemirmi washing solution (DIG manufactured by Roche, as described in Wash and Block buffer Set) was circulated for 15 minutes at room temperature. This is repeated 3 times, and after adjusting pH with a detection buffer (DIG manufactured by Roche, described in Wash and Block buffer Set), CDP-star (CDP-star, read to use) which is a chemiluminescent substrate is included. After contacting with the solution for 1 hour, chemiluminescence emitted from the adsorptive region of the biochemical analysis unit was photoelectrically detected by a cooled CCD camera (LAS1000: manufactured by Fuji Photo Film Co., Ltd.).
[0059]
(Examples 2 to 4)
Chemiluminescence was detected in the same manner as in Example 1 except that the prehybridization, hybridization, and wash buffer temperatures were adjusted as described in Table 1.
[0060]
(Comparative Examples 1-4)
Prehybridization buffer and hybridization buffer were prehybridized using hybridization buffer B (6 × SSC, 0.01M EDTA, 5 × denhardt's solution, 0.5% SDS, 100 μg Sheard, Detected salmon sperm DNA). Chemiluminescence was detected in the same manner as in Example 1 except that the temperature of the hybridization and washing buffer was adjusted as described in Table 1.
[0061]
Pre-hybridization buffer, hybridization buffer and washing buffer set temperature of Examples 1 to 4 and Comparative Examples 1 to 4, set flow rate at the time of hybridization / in the system after 18 hours of hybridization (circulating pipe part) Table 1 shows the ratio of the flow rates of the light sources and the amount of luminescence in the background. In Comparative Examples 2 and 3, the reaction solution was not circulated at the hybridization stage, and it was not possible to proceed to the subsequent process, so the background light emission could not be measured.
[0062]
[Table 1]

[0063]
As is clear from Table 1, in Examples 1 to 4 using a hybridization buffer containing no Denhart's solution and salmon palm DNA, the set flow rate and the hybridization buffer in the system after 18 hours of circulation were determined. Since the flow rate hardly changed and the fluidity was maintained, the labeled receptor or the labeled ligand non-specifically bound to the adsorptive region could be sufficiently washed away in the subsequent washing step. As a result, the amount of light emitted from the background of the adsorptive region could be reduced. On the other hand, in Comparative Example 1 and Comparative Example 4 using a hybridization buffer containing Denharz Solution and Salmon Palm DNA, the flow rate of the hybridization buffer in the system after 18 hours of circulation is less than half of the set flow rate. The background was about five times higher than that of Examples 1 to 4 using a hybridization buffer not containing Denhart's solution and Salmon palm DNA.
[0064]
As described above, the chemiluminescence method using the biochemical analysis unit of the present invention forces the hybridization buffer to which a labeled receptor or labeled ligand labeled with a labeling substance is added so as to cross the adsorptive region. When binding the labeled receptor or labeled ligand specifically by flowing, the hybridization buffer that does not contain Denhart's solution and salmon palm DNA is used as the hybridization buffer. In this washing step, the labeled receptor or labeled ligand non-specifically bound to the adsorptive region could be sufficiently washed away, and the amount of light emitted from the background of the adsorptive region could be reduced. Therefore, this enables detection of a trace amount of labeled receptor or labeled ligand.
[Brief description of the drawings]
FIG. 1 is a schematic perspective view of a biochemical analysis unit used in the chemiluminescence method of the present invention. FIG. 2 shows an embodiment of a reactor used in the chemiluminescence method using the biochemical analysis unit of the present invention. Schematic sectional view [FIG. 3] Schematic sectional view showing another embodiment of the reactor used in the chemiluminescence method utilizing the biochemical analysis unit of the present invention [Explanation of symbols]
1 Biochemical analysis unit 2 Substrate 3 Hole 4 Adsorbent region 5 Ligand or receptor

Claims (3)

A hybridization buffer in which a labeled receptor labeled with a labeling substance or a labeled ligand is added to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which a ligand or receptor is bound is attached to the adsorptive region. In an assay method in which the labeled receptor or labeled ligand is specifically bound to the ligand or receptor by forcibly flowing across the ligand, and the labeled receptor or labeled ligand is detected.
An assay method using a biochemical analysis unit, characterized in that a hybridization buffer not containing Denhart's solution and salmon palm DNA is used as the hybridization buffer. The ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound is forced to flow through the hybridization buffer to which the receptor or ligand is added so as to cross the adsorbent region. In the assay method in which the receptor or ligand is specifically bound, the receptor or ligand is specifically bound to a label labeled with a labeling substance, and the receptor or ligand is detected.
An assay method using a biochemical analysis unit, characterized in that a hybridization buffer not containing Denhart's solution and salmon palm DNA is used as the hybridization buffer. A hybridization buffer in which an auxiliary substance-binding receptor or an auxiliary substance-binding ligand to which an auxiliary substance is bound is added to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound. Forcibly flowing across the adsorptive region to specifically bind to the auxiliary substance-binding receptor or the auxiliary substance-binding ligand, and to bind the labeling substance capable of binding specifically to the auxiliary substance. In the assay method for detecting the auxiliary substance-binding receptor or the auxiliary substance-binding ligand by forcibly flowing across the substance and specifically binding to the auxiliary substance-binding receptor or the auxiliary substance-binding ligand,
An assay method using a biochemical analysis unit, characterized in that a hybridization buffer not containing Denhart's solution and salmon palm DNA is used as the hybridization buffer.

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