JP2004108860A - Assay method using biochemistry analysis unit - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve a detection limit by improving the fluidity of a blocking buffer. <P>SOLUTION: Ligands or receptors are joined to adsorptive regions of a biochemistry analysis unit having porous adsorptive regions. Marked receptors or marked ligands marked by a marking material are peculiarly joined to the ligands or the receptors. The blocking buffer containing particles having diameters smaller than the diameters of holes for the adsorptive regions are used for forcibly fluidizing enzyme marked antibodies crossing the adsorptive regions to block the adsorptive regions. Subsequently, the enzyme marked antibodies are forcibly fluidized crossing the adsorptive regions to be peculiarly joined to the marked receptors or the marked ligands, a chemically chemiluminescent substrate is put in contact with the enzyme marked antibodies peculiarly joined to the marked receptors or the marked ligands, and chemiluminescence released from the adsorptive regions of the biochemistry analysis unit is photoelectrically detected. <P>COPYRIGHT: (C)2004,JPO

Description

【００７０】
表１から明らかなように、本発明の方法による実施例１では、ブロッキングバッファに含有される粒子の粒子径（０．２２μｍ）が、吸着性領域の孔径（０．４５μｍ）よりも小さいものを用いたので、ブロッキングバッファが多孔性の吸着性領域に詰まることなく流動し、効率的に吸着性領域をブロッキングすることができたので、吸着性領域の孔径よりも大きい粒子径を有するブロッキングバッファをそのまま用いた比較例１に比較して、バックグラウンドの発光量を半分に抑えることができた。これによって、検出対象が微量であっても、Ｓ／Ｎ比の大きい正確な検出を行うことが可能となった。
【００７１】
なお、本発明は、ブロッキングバッファとして、ブロッキングバッファに含有される粒子の粒子径が多孔性の吸着性領域の孔径よりも小さいものを用いることとしたので、ブロッキングバッファを多孔性の吸着性領域に詰まるせることなく流動させることが可能となるものであるが、生化学解析用ユニットを利用したアッセイ法のさらに別の態様として、リガンドまたはレセプタが結合された多孔性の吸着性領域を有する生化学解析用ユニットのリガンドまたはレセプタに、レセプタまたはリガンドを吸着性領域を横切るように強制的に流動させてレセプタまたはリガンドを特異的に結合させ、レセプタまたはリガンドを標識物質を利用して検出するものであって、この検出を行うにあたり吸着性領域を横切るように強制的に溶液を流動させる工程を含むアッセイ方法において、強制的に流動させる溶液として溶液中に含まれる粒子の粒子系が吸着性領域の孔径よりも小さい溶液を用いることとすれば、多孔性材料からなる吸着性領域の孔を閉塞させることなくアッセイを行うことを可能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. Further, in the case of the shaking method, there is a problem that the hybridization results vary depending on the experimenter and the reproducibility is lowered, and the reproducibility may be lowered even for the same experimenter.
[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 forcibly circulated inside the adsorptive region. However, for example, in the case of chemiluminescence, which is one of the analysis methods, an enzyme label is used to prevent the enzyme-labeled antibody from directly binding to the adsorptive region, not the labeled receptor or the antigen of the labeled ligand. It is common practice to block the adsorptive region with a blocking buffer prior to specifically binding the antibody to the labeled receptor or labeled ligand antigen, and the blocking buffer is forced due to the poor flowability of this blocking buffer. However, there is a problem that the blocking ability is lowered and the amount of light emitted from the background of the adsorptive region is greatly increased.
[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 unit for biochemical analysis capable of improving the flowability of a blocking buffer and improving the detection limit. Is.
[0012]
[Means for Solving the Problems]
The assay method using the biochemical analysis unit of the present invention is labeled with a labeling substance so as to cross the adsorptive region of the biochemical analysis unit having a porous adsorptive region to which a ligand or a receptor is bound. Blocking the adsorptive region by forcibly flowing a blocking buffer for the labeled receptor or labeled ligand, and forcing the labeled receptor or ligand to flow across the adsorptive region to the ligand or receptor. In the assay method in which the labeled receptor or labeled ligand is specifically bound to detect the labeled receptor or labeled ligand, the particle size of the particles contained in the blocking buffer is larger than the pore size of the adsorptive region as the blocking buffer. It is characterized by using a small one It is the law.
[0013]
When the labeled receptor or labeled ligand is forced to flow across the adsorptive region, it is preferably added to a blocking buffer for the labeled receptor or labeled ligand labeled with a labeling substance.
[0014]
As another aspect of the assay method using the biochemical analysis unit of the present invention, the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound may be a receptor or ligand. The binding body is forced to flow through a blocking buffer for the labeling substance labeled with the labeling substance so as to cross the adsorptive area, thereby blocking the adsorptive area, and the labeling substance is allowed to pass through the adsorptive area. Particles of particles contained in the blocking buffer as the blocking buffer in an assay for detecting the receptor or ligand by forcibly flowing across and binding the label to the receptor or ligand specifically It is preferable to use one having a diameter smaller than the hole diameter of the adsorptive region. It is a method to.
[0015]
When the labeling body is forced to flow across the adsorptive region, it is preferably added to a blocking buffer for the labeling body labeled with a labeling substance.
[0016]
As another aspect 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 a ligand or receptor is bound. The bound auxiliary substance-binding receptor or the auxiliary substance-binding ligand is specifically bound, and a blocking buffer for a bindable labeling substance that can specifically bind to the auxiliary substance is forced to flow across the adsorptive region. Blocking the adsorptive region and forcibly flowing the binding labeling substance across the adsorptive region to specifically bind the auxiliary substance-binding receptor or the auxiliary substance-binding ligand, and binding the auxiliary substance In an assay for detecting a receptor or an auxiliary substance binding ligand, A method of particle diameter of particles contained in the blocking buffer is characterized by using a smaller than the diameter of the adsorptive regions.
[0017]
When the bindable labeling substance is forced to flow across the adsorptive region, it is preferably added to a blocking buffer for the bindable labeling substance that can specifically bind to the auxiliary substance.
[0018]
In still another aspect of the assay method using the biochemical analysis unit of the present invention, the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which a ligand or receptor is bound may be a receptor or a receptor. In this detection, a ligand is forced to flow across the adsorptive region to specifically bind the receptor or ligand, and the receptor or ligand is detected using a labeling substance. In an assay method including a step of forcibly flowing a solution across the adsorptive region, a particle system of particles contained in the solution as the forcibly flowing solution is smaller than a pore size of the adsorptive region It is a method characterized by using a solution.
[0019]
【The invention's effect】
The assay using the biochemical analysis unit of the present invention can specifically bind to a labeling receptor labeled with a labeling substance or a blocking buffer for a labeled ligand, a blocking buffer labeled with a labeling substance, or an auxiliary substance. Since the blocking buffer for the binding labeling substance is such that the particle size of the particles contained in the blocking buffer is smaller than the pore size of the porous adsorptive region, the blocking buffer is clogged with the porous adsorptive region. The adsorbing region can be efficiently blocked and the amount of light emitted from the background of the adsorbing region can be kept low.
[0020]
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 chemiluminescence 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.
[0021]
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.
[0022]
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.
[0023]
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.
[0024]
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).
[0025]
In the present invention, a porous material or a fiber material is preferably used as the porous material forming the adsorptive region. Further, the adsorptive region can be formed by using a porous material and a fiber material together. In the present invention, the porous material used for forming the adsorptive region may be either an organic material or an inorganic material, or an organic / inorganic composite.
[0026]
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.
[0027]
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.
[0028]
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.
[0029]
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.
[0030]
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.
[0031]
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.
[0032]
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.
[0033]
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.
[0034]
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.
[0035]
The assay method using the biochemical analysis unit of the present invention is labeled with a labeling substance so as to cross the adsorptive region of the biochemical analysis unit having a porous adsorbent region to which a ligand or a receptor is bound. Blocking the adsorptive region by forcibly flowing the blocking buffer for the labeled receptor or labeled ligand, and forcibly flowing the labeled receptor or labeled ligand across the adsorptive region to cause the ligand or receptor to be labeled receptor or labeled ligand In the assay method that specifically binds and detects a labeled receptor or a labeled ligand, a blocking buffer is used in which the particle size of particles contained in the blocking buffer is smaller than the pore size of the adsorptive region .
[0036]
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.
[0037]
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.
[0038]
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.
[0039]
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.
[0040]
As another aspect of the assay method using the biochemical analysis unit of the present invention, 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. The binding buffer is forced to flow by blocking the adsorbing region by forcing the blocking buffer against the labeling substance labeled with the labeling substance to specifically bind and cross the adsorbing region, and to force the labeling member to cross the adsorbing region. In the assay method in which the label is specifically bound to the receptor or the ligand by detecting the receptor or the ligand, the particle size of the particles contained in the blocking buffer is smaller than the pore size of the adsorptive region as a blocking buffer. It is characterized by using a thing.
[0041]
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.
[0042]
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.
[0043]
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. Adsorbing by binding the binding substance of the binding substance or the binding substance of the binding substance to the binding substance and forcing the blocking buffer against the binding substance capable of binding specifically to the auxiliary substance to flow across the adsorptive region. Blocking the binding region and forcing the binding labeling substance to flow across the adsorptive region to specifically bind to the auxiliary substance binding receptor or the auxiliary substance binding ligand, and to bind the auxiliary substance binding receptor or the auxiliary substance binding ligand. In the assay method to be detected, the blocking buffer contains Particle diameter of the particles is characterized by using a smaller than the hole diameter of the adsorptive regions.
[0044]
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.
[0045]
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.
[0046]
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.
[0047]
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.
[0048]
Next, the antigen-binding receptor or antigen-binding ligand is specifically bound to the ligand or receptor bound to the adsorptive region.
[0049]
After the antigen-binding receptor or antigen-binding ligand is specifically bound to the ligand or receptor bound to the porous adsorptive region, the reaction solution shown in FIG. It is attached to a reaction vessel that can be forced to flow across the adsorptive region. It should be noted that the antigen-binding receptor or antigen-binding ligand is not intended to block the adsorptive region. For example, in the case of antigen-binding DNA, the antigen-binding receptor or antigen-binding ligand is bound to the ligand or receptor bound to the adsorptive region. The biochemical analysis unit may be attached to the reaction vessel from the step of specifically binding the. In this case, the antigen-binding receptor or the antigen-binding ligand can be efficiently and uniformly brought into contact with the ligand or receptor bound to the adsorptive region, so that the detection limit of the antigen-binding receptor or the antigen-binding ligand is further increased. In addition, the detection can be performed with a higher S / N ratio even if the amount of the antigen-binding receptor or antigen-binding ligand is very small.
[0050]
In addition, from the step of specifically binding the ligand or receptor bound to the porous adsorptive region and the antigen-binding receptor or antigen-binding ligand, the enzyme-labeled antibody is specifically bound to the antigen-binding receptor or antigen-binding ligand described later. Since the reaction solution can be forced to flow through the adsorptive region up to the bonding stage, the process can be simplified and the detection can be performed with higher accuracy.
[0051]
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.
[0052]
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.
[0053]
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. To block the adsorptive region. As the blocking buffer, one having a particle size of particles contained in the blocking buffer smaller than the pore size of the adsorptive region is used. Both the pore size of the adsorptive region and the pore size of the filter mean the average pore size.
[0054]
The pore diameter of the adsorptive region can be measured by a porous material automatic pore measuring system manufactured by PMI (Porous Materials, Inc.). The measurement procedure is to gradually increase the pressure to the porous adsorptive region wetted with the test solution and send air to measure the air permeation flow rate (wetting flow rate curve). Measure the air permeation flow rate when there is no (dry flow curve). By comparing these two permeate flow rates (determining the pressure at the point where the curve of the half of the dry flow curve and the wetting flow curve intersect), the average pore diameter is calculated from the relationship between the pressure and the pore pore diameter It can ask for.
[0055]
The blocking buffer in which the particle size of the particles contained in the blocking buffer is smaller than the pore size of the adsorptive region is larger than the pore size of the adsorptive region measured as described above (in the case of being commercially available). It can be adjusted by filtering with a filter having a small pore size. In this case, the particle size of the particles contained in the blocking buffer is more preferably less than half the pore size of the adsorptive region.
[0056]
In addition, although the blocking buffer used for blocking is not specifically limited, For example, commercially available blocking, such as Block Ace (made by Dainippon Pharmaceutical), Blocker casein (made by Pierce), Liquid block (made by Amersham Pharmacia), etc. A buffer can be used.
[0057]
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. At this time, the enzyme-labeled antibody is preferably added to a blocking buffer for the enzyme-labeled antibody, and is forced to flow across the adsorptive region to specifically bind to the antigen-binding receptor or antigen-binding ligand. In this case as well, the enzyme-labeled antibody specifically binds to the antigen-binding receptor or antigen-binding ligand by using a blocking buffer whose particle size is smaller than the pore size of the adsorptive region. In this case, since the adsorptive region is blocked, the amount of light emitted from the background of the adsorptive region can be further reduced.
[0058]
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.
[0059]
【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.
[0060]
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. The average pore diameter of the adsorptive region was 0.45 μm (measured by a porous material automatic pore measurement system manufactured by PMI).
[0061]
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. .
[0062]
Next, 10 pg of pBR328-DNA solution (manufactured by Roche) labeled with digoxigenin (DIG) was thermally denatured, and hybridization buffer (6 × SSC, 0.01 MEDTA, 5 × denhardt's solution, 0.5% SDS, (100 μg Sheared, denatured salmon sperm DNA) was added to 5 ml.
[0063]
The biochemical analysis unit was fixed to the reaction vessel shown in FIG. 2, and 5 ml of a 65 ° C. prehybridization buffer (same as the above hybridization buffer) was circulated for 1 hour (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. Subsequently, the washing buffer 1 (2 × SSC, 0.1% SDS) was washed twice for 5 minutes, and the washing buffer 2 (0.1 × SSC, 0.1% SDS) was washed twice for 5 minutes (buffer temperature). Are all 65 ° C.).
[0064]
A blocking buffer (DIG manufactured by Roche, as described in Wash and Block buffer Set) was filtered through 0.22 μm Ultra Free (manufactured by Millipore), and the filtered blocking buffer was circulated at room temperature for 10 minutes, and then for 50 minutes. Circulation was stopped. In addition, when the blocking buffer before filtration was measured with a particle size distribution meter (FPIF-2100: manufactured by Sysmex Corporation), two particles having a particle size of 5 μm to 10 μm / 0.33 μl and a particle size of 0.6 μm to 5 μm 5722 particles / 0.33 μl of particles having a particle diameter of 0 μm and 3 particles / 0.33 μl of particles having a particle diameter of 0.6 μm to 5 μm. It was.
[0065]
Next, the alkaline phosphatase-labeled DIG antibody was previously filtered with an ultra-free pore size of 0.22 μm (manufactured by Millipore), and filtered with an ultra-free pore size of 0.22 μm so that the concentration would be 1/10000. After diluting with blocking buffer, this was circulated for 1 minute at room temperature and then stopped for 60 minutes.
[0066]
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 was repeated 3 times, and after immersion for 5 minutes in a detection buffer (DIG manufactured by Roche, described in Wash and Block buffer Set), finally, a chemiluminescent substrate CDP-star (CDP-star, read to use). ), And the 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.).
[0067]
(Comparative Example 1)
Except for using the blocking buffer before filtration, chemiluminescence was performed in the same manner as in Example 1 to detect the amount of luminescence.
[0068]
Table 1 shows the amount of light emitted in the background of Example 1 and Comparative Example 1.
[0069]
[Table 1]

[0070]
As is clear from Table 1, in Example 1 according to the method of the present invention, the particle size (0.22 μm) of the particles contained in the blocking buffer is smaller than the pore size (0.45 μm) of the adsorptive region. Since the blocking buffer flowed without clogging the porous adsorptive region and could effectively block the adsorbent region, a blocking buffer having a particle size larger than the pore size of the adsorptive region was used. Compared to Comparative Example 1 used as it was, the amount of light emitted from the background could be reduced to half. This makes it possible to perform accurate detection with a large S / N ratio even if the detection target is a very small amount.
[0071]
Since the present invention uses a blocking buffer having a particle size of particles contained in the blocking buffer smaller than the pore size of the porous adsorptive region, the blocking buffer is used as the porous adsorptive region. Although it is possible to flow without clogging, as yet another aspect of the assay method using a unit for biochemical analysis, biochemistry having a porous adsorptive region to which a ligand or a receptor is bound The receptor or ligand is forced to flow across the adsorption region to the ligand or receptor of the analysis unit to specifically bind the receptor or ligand, and the receptor or ligand is detected using a labeling substance. For this detection, the solution is forced to flow across the adsorptive region. In the assay method including the steps, if a solution in which the particle system of particles contained in the solution is smaller than the pore size of the adsorptive region is used as the solution to be forced to flow, the pores of the adsorbent region made of a porous material are used. It can be possible to carry out the assay without blocking.
[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 (4)

A blocking buffer for a labeled receptor or a labeled ligand labeled with a labeling substance is forced to flow across the adsorptive region of a biochemical analysis unit having a porous adsorptive region to which a ligand or receptor is bound. Blocking the adsorptive region, forcibly flowing the labeled receptor or labeled ligand across the adsorptive region to specifically bind the labeled receptor or labeled ligand to the ligand or receptor, In an assay to detect a receptor or labeled ligand,
An assay method using a biochemical analysis unit, wherein the blocking buffer has a particle size of particles contained in the blocking buffer smaller than the pore size of the adsorptive region. The receptor or ligand is specifically bound to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound, and is labeled with a labeling substance so as to cross the adsorptive region. Blocking the adsorptive region by forcibly flowing a blocking buffer for the labeled body, and forcing the label to flow across the adsorptive region to make the label specific to the receptor or ligand In an assay that binds to and detects the receptor or ligand,
An assay method using a biochemical analysis unit, wherein the blocking buffer has a particle size of particles contained in the blocking buffer smaller than the pore size of the adsorptive region. An auxiliary substance-binding receptor or an auxiliary substance-binding ligand to which an auxiliary substance is bound is specifically bound to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or the receptor is bound, Blocking the adsorptive region by forcibly flowing a blocking buffer for the bindable labeling substance that can specifically bind to the auxiliary substance across the adsorptive region, and allowing the bindable labeling substance to pass through the adsorptive region In an assay method for detecting the auxiliary substance-binding receptor or the auxiliary substance-binding ligand by forcibly flowing across and binding the auxiliary substance-binding receptor or the auxiliary substance-binding ligand specifically,
An assay method using a biochemical analysis unit, wherein the blocking buffer has a particle size of particles contained in the blocking buffer smaller than the pore size of the adsorptive region. The receptor or ligand is forced to flow across the adsorptive region to the ligand or receptor of the biochemical analysis unit having a porous adsorptive region to which the ligand or receptor is bound. In an assay method comprising specifically binding and detecting the receptor or ligand using a labeling substance, and forcing the solution to flow across the adsorptive region in performing this detection ,
An assay method using a biochemical analysis unit, wherein a solution having a particle system of particles contained in the solution smaller than the pore size of the adsorptive region is used as the solution forcibly flowing.

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